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Using ISSR Genomic Fingerprinting to Study the Genetic Differentiation of Artemia Leach, 1819 (Crustacea: Anostraca) from Iran and Neighbor Regions with the Focus on the Invasive American Artemia franciscana

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Due to the rapid developments in the aquaculture industry, Artemia franciscana, originally an American species, has been introduced to Eurasia, Africa and Australia. In the present study, we used a partial sequence of the mitochondrial DNA Cytochrome Oxidase subunit I (mt-DNA COI) gene and genomic fingerprinting by Inter-Simple Sequence Repeats (ISSRs) to determine the genetic variability and population structure of Artemia populations (indigenous and introduced) from 14 different geographical locations in Western Asia. Based on the haplotype spanning network, Artemia urmiana has exhibited higher genetic variation than native parthenogenetic populations. Although A. urmiana represented a completely private haplotype distribution, no apparent genetic structure was recognized among the native parthenogenetic and invasive A. franciscana populations. Our ISSR findings have documented that despite that invasive populations have lower variation than the source population in Great Salt Lake (Utah, USA), they have significantly revealed higher genetic variability compared to the native populations in Western Asia. According to the ISSR results, the native populations were not fully differentiated by the PCoA analysis, but the exotic A. franciscana populations were geographically divided into four genetic groups. We believe that during the colonization, invasive populations have experienced substantial genetic divergences, under new ecological conditions in the non-indigenous regions.
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diversity
Article
Using ISSR Genomic Fingerprinting to Study the
Genetic Dierentiation of Artemia Leach, 1819
(Crustacea: Anostraca) from Iran and Neighbor
Regions with the Focus on the Invasive American
Artemia franciscana
Amin Eimanifar 1, *, Alireza Asem 2, * , Pei-Zheng Wang 3, Weidong Li 4and Michael Wink 1
1Institute of Pharmacy and Molecular Biotechnology (IPMB), Heidelberg University, Im Neuenheimer Feld
364, 69120 Heidelberg, Germany; wink@uni-heidelberg.de
2College of Fisheries and Life Science, Hainan Tropical Ocean University, Sanya 572000, China
3College of Ecology and Environment, Hainan Tropical Ocean University, Sanya 572000, China;
condywpz@126.com
4College of Ecology and Environment, Hainan University, Haikou 570228, China; lwd542148880@163.com
*Correspondence: amineimanifar1979@gmail.com (A.E.); asem.alireza@gmail.com (A.A.)
Received: 4 March 2020; Accepted: 26 March 2020; Published: 31 March 2020


Abstract: Due to the rapid developments in the aquaculture industry, Artemia franciscana, originally
an American species, has been introduced to Eurasia, Africa and Australia. In the present study,
we used a partial sequence of the mitochondrial DNA Cytochrome Oxidase subunit I (mt-DNA COI)
gene and genomic fingerprinting by Inter-Simple Sequence Repeats (ISSRs) to determine the genetic
variability and population structure of Artemia populations (indigenous and introduced) from 14
dierent geographical locations in Western Asia. Based on the haplotype spanning network, Artemia
urmiana has exhibited higher genetic variation than native parthenogenetic populations. Although
A. urmiana represented a completely private haplotype distribution, no apparent genetic structure
was recognized among the native parthenogenetic and invasive A. franciscana populations. Our
ISSR findings have documented that despite that invasive populations have lower variation than the
source population in Great Salt Lake (Utah, USA), they have significantly revealed higher genetic
variability compared to the native populations in Western Asia. According to the ISSR results, the
native populations were not fully dierentiated by the PCoA analysis, but the exotic A. franciscana
populations were geographically divided into four genetic groups. We believe that during the
colonization, invasive populations have experienced substantial genetic divergences, under new
ecological conditions in the non-indigenous regions.
Keywords:
genetic variation; brine shrimp Artemia; invasive species; mt-DNA COI; Inter-Simple
Sequence Repeats (ISSRs) genomic fingerprinting; Western Asia
1. Introduction
The brine shrimp Artemia is a unique zooplankton that has a limited number of species, distributed
globally, except in Antarctica [
1
]. This tiny crustacean has potentially adapted to live in extreme
environmental conditions, such as hypersaline environments [2,3].
Artemia has been mainly used as live food in larviculture and fishery industries, especially
in Asia [
4
]. Artemia has been used to improve the quality of sodium chloride production in solar
salt-fields [
5
,
6
]. It was also introduced as a model organism in many bioscience studies, including
Diversity 2020,12, 132; doi:10.3390/d12040132 www.mdpi.com/journal/diversity
Diversity 2020,12, 132 2 of 21
cellular and molecular biology [
7
], phylogeography and asexual evolution [
8
], bioencapsulation [
9
]
and toxicity assessment [10].
The genus Artemia consists of seven bisexual species and a large number of parthenogenetic
populations with dierent ploidy levels [
3
,
11
]. It has been assumed that Asia is the origin of
Artemia urmiana Günther, 1899 (Lake Urmia, Iran), Artemia sinica Cai, 1989 (China), Artemia tibetiana
Abatzopoulos, Zhang and Sorgeloos, 1998 (Qinghai-Tibetan Plateau, China), and corresponding
parthenogenetic populations [
3
,
12
]. Recently, two new species, Artemia frameshifta and Artemia murae
have been described from Mongolia [
13
]. These species have been described using a single mitochondrial
DNA protein-coding Cytochrome Oxidase subunit I (COI) gene sequence without confirmation by any
morphometric and population genetic analyses [
11
]. A main problem in the submitted COI sequence
of A. frameshifta (LC195588) was detected in the protein sequence with several stop codon(s). In a
protein coding gene, this is an indication for a nuclear copy of this mtDNA gene. Sometimes, the
COI marker provides a sharp PCR amplified band on the agarose gel, but the sequence information
presents heterogeneities [
14
]. These kinds of results could mislead the interpretation of downstream
phylogenetic analyses. Therefore, the population would need more biosystematic evidences to
determine its taxonomical status. In A. murae, neither the existence of males nor the reproductive mode
has been revealed [
13
]. Based on the current information, we assume that A. murae is a parthenogenetic
population, which needs more investigations to confirm the biological status of the species.
Generally, the long-distance translocations of the American species Artemia franciscana to other
non-indigenous regions have occurred as a result of commercial activities, which have been fully
documented previously [
2
,
15
18
]. Artemia franciscana is a successful invader in saltwater ecosystems
due to its faster filter-feeding rate, a high potential of reproduction [
15
,
19
], and a better physiological
immune system, which is associated with nutritional behavior against cestode parasites [
15
] than the
native species. Asem et al. [
17
] have suggested that these biological characteristics could aord a
high level of adaptive potential of A. franciscana in the new non-indigenous habitats, which would
eventually result in the replacement with native species.
Lee [
20
] has documented that the genetic structure of introduced populations to the non-indigenous
habitats is one of the most determinative parameters in their successful establishment. Generally,
genetic diversity of the species could determine the potential of an exotic species to acclimatize in the
new environmental conditions [21].
Previous studies on A. franciscana have documented that invasive populations demonstrated
genetic variations relative to the native American source populations [
2
,
17
,
18
,
22
24
]. The low genetic
diversity in the non-indigenous populations has been attributed to the founder eect [
22
] or population
bottleneck due to the decreasing of population size in introduced populations during the process
of establishment [
17
]. Moreover, high genetic variation could be a result of adaptive capacity and
physiological flexibility as a special biological trait observed in invasive populations [2,6,2426].
Eimanifar et al. [
2
] have reported the existence of invasive A. franciscana in four sites from Iran
(three sites) and Iraq (one site) using the mitochondrial COI sequence marker. The aim of the present
study was to further perform an analysis based on population genetic approaches to determine the
intra- and inter-specific genetic variations of native and invasive Artemia populations from Iran and
neighboring regions (14 sites) using Inter-Simple Sequence Repeats (ISSRs) genomic fingerprinting.
Genomic fingerprinting by ISSR has been demonstrated to be a useful molecular tool to recognize
DNA polymorphisms among Artemia taxa [
27
30
]. We hypothesize that the establishment of an exotic
species in the new geographical habitats should be accompanied by intra-species genetic divergence to
better adapt to the new environmental conditions. Here, we utilized high-resolution ISSR genomic
fingerprinting to compare the genetic dierentiation in native and colonizing populations of American
A. franciscana in the indigenous environments.
Diversity 2020,12, 132 3 of 21
2. Material and Methods
2.1. Sample Collection and DNA Extraction
Artemia cyst specimens were collected from 14 geographical localities across Iran and neighbor
countries (Figure 1). All studied populations had been confirmed to be bisexual or parthenogenetic
according to Asem et al. [
3
]. The sample localities with their geographical coordinates, abbreviations and
IPMB voucher are summarized in Table 1. Total genomic DNA was extracted from part of the antenna
of adult males and females using the Chelex
®
100 Resin method (6%, Bio-Rad Laboratories, Hercules,
CA, U.S.A.) [16]. All extracted DNA was stored at 80 C for subsequent genetic characterization.
Figure 1.
Map of Artemia sampling sites (1 =URM, 2 =LAGW, 3 =LAGE, 4 =QOM, 5 =MIG, 6 =MSH,
7=MAH, 8 =NOG, 9 =INC, 10 =CAM, 11 =ABG, 12 =GAA, 13 =KBG, 14 =KOC; Abbreviations are
listed in Table 1).
2.2. Population Identification and Phylogenetic Analyses
A partial sequence of the mitochondrial marker cytochrome c oxidase subunit I (COI) was utilized to
identify the taxonomical status of the studied populations using phylogenetic analyses as implemented
in the MEGA X program (Temple University, Philadelphia, USA) [
2
,
17
,
18
]. To identify the taxonomical
status of the studied populations, the COI reference sequences from the recognized bisexual species and
parthenogenetic populations were downloaded from GenBank (Table 2). Sequences were aligned using
MEGA X with default settings [
31
]. Phylogenetic trees were reconstructed based on the Maximum
Likelihood approach included in the MEGA X program. To reveal the genealogical and geographical
relationships, a median haplotype network was established, following the median-joining algorithm in
the Network program ver. 5.0.1.1 (Universität Hamburg, Hamburg, Germany) [32].
Diversity 2020,12, 132 4 of 21
Table 1.
Origin of Artemia samples from Iran and neighbor regions. (IPMB =Institute of Pharmacy and Molecular Biotechnology, Heidelberg University,
Abb. =Abbreviation).
No. Voucher Number
(IPMB) Abb. Species/Population Locality Country Geographic
Coordinates
COI
Accession Numbers
1 57211 URM A. urmiana Urmia Lake Iran 37200E–45400NJX512748-808 [28]
2 57223 LAGW Parthenogenetic
Western Lagoon
around Urmia
Lake
Iran 37150E–45850NKF691338-342 [2]
3 57224 LAGE Parthenogenetic
Eastern Lagoon
around
Urmia Lake
Iran 37500E–46400NKF691343-345 [2]
4 57225 QOM Parthenogenetic Qom Salt Lake Iran 34400E–51800NKF691367-372 [2]
5 57226 MIG Parthenogenetic Mighan Salt Lake Iran 34200E–49800NKF691357-361 [2]
6 57230 MSH A. franciscana Mahshar port Iran 49110E–30330NKF691351-356 [2]
7 57228 MAHR A. franciscana Maharlu Lake Iran 29570E–52140NKF691347, 349-350 [2]
8 57229 NOG A. franciscana Nough Catchment Iran 30600E–56500NKF691362-366 [2]
9 57227 INC Parthenogenetic Incheh Lake Iran 37240E–54360NKF691333-337 [2]
10 57292 CAM Parthenogenetic Camalti Lake Turkey 27080E–38250NKF691520-525; 527-529 [2]
11 57255 ABG Parthenogenetic Abu-Ghraib Iraq 44300E–33200NKF691373-375 [2]
12 57256 GAA A. franciscana Garmat Ali Iraq 47490E–30300NKF691376-383 [2]
13 57258 KBG Parthenogenetic Kara Bogaz Gol Turkmenistan 53330E–41170NKF691530-532,534 [2]
14 57257 KOC Parthenogenetic Korangi Creek
(Karachi coast) Pakistan 67100E–24480NKF691442-445; 447-448
JX512748 [2]
[28] Eimanifar and Wink (2013); [2] Eimanifar et al. (2014).
Diversity 2020,12, 132 5 of 21
Table 2. Species information and GenBank accession numbers for COI reference sequences.
Species/Population Abbreviation Individual Accession
Numbers Ref.
A.urmiana URM 4 JX512748-751 [28]
A.sinica SIN 4 KF691298-301 [2]
A.tibetiana TIB 4 KF691215-218 [2]
A.salina SAL 4 KF691512-515 [2]
A.persimilis PER 4
DQ119647
HM998992
EF615594
EF615593
[27]
[33]
[14]
[14]
A.franciscana FRA 4 KJ863440-443 [2]
Diploid Pop. DI 4 KU183949-952 [3]
Triploid Pop. TRE 3 HM998997-999 [33]
Tetraploid Pop. TETR 4 KU183954-957 [3]
Pentaploid Pop. PEN 4 KU183968-971 [3]
Ref: [
28
] Eimanifar and Wink 2013; [
2
] Eimanifar et al. 2014; [
27
] Hou et al. 2006; [
33
] Maniatsi et al. 2011; [
14
] Wang
et al. 2008; [3] Asem et al. 2016.
2.3. Genomic Fingerprinting by ISSR-PCR
Genomic variability was examined by inter simple sequence repeat ISSR-PCR using the same
DNA template used for phylogenetic analyses. Initially, 15 ISSR primers were analyzed to distinguish
the intra- and inter-specific genetic variability within and among 83 randomly selected individuals,
belonging to 14 geographically dierent localities of Artemia. Out of 15 tested ISSR primers, five were
selected because of unambiguous banding patterns of the PCR products (Table 3).
Table 3. List of primers screened for Inter-Simple Sequence Repeats (ISSR) analysis.
Primer Sequence GC (%) Annealing
Temperature (C)
Amplification
Pattern
ISSR1 (AC)8T 47.1 48–54 Smear
ISSR2 (CAC)5 66.7 48–54 Smear
ISSR3 (GACA)4 50 48–54 Smear
ISSR4 (AG)12 50 48–54 Poor
ISSR5 (TC)9 50 48–54 Poor
ISSR6 (GT)10 50 48–54 Smear
ISSR7 (CA)10A 47.6 48–54 Poor
ISSR8 (GAA)5 33.3 48–54 No amplification
ISSR9 (CAG)6 66.7 48–54 No amplification
ISSR10 (GCCG)4 100 48–54 No amplification
ISSR11 (AG)8C 52.9 48 Good and sharp
ISSR12 (AG)8YTa 50 48 Good and sharp
ISSR13 (GA)9T 47.4 50 Good and sharp
ISSR14 (TG)8G 52.9 50 Good and sharp
ISSR15 (AC)8C 52.9 49 Good and sharp
Diversity 2020,12, 132 6 of 21
PCR was carried out in a 25
µ
l volume consisting of 40 ng template DNA, 2.5
µ
l 10
×
PCR buer
(160 mM (NH
4
)
2
SO
4
, 670 mM Tris-HCl pH 8.8, 0.1% Tween-20, 25 mM MgCl
2
), 10 pmol primer, 2
µ
g/
µ
l
bovine serum albumin (BSA), 0.5 units Taq DNA polymerase (Bioron), 0.1 mM dGTP, dCTP, and dTTP,
0.045 mM dATP, 1
µ
Ci [
α
-33P]-dATP (Perkin Elmer, LAS, Rodgau, Germany). PCR amplifications were
executed in a thermal cycler based on the following conditions: 94
C denaturation for 1 min, 35 cycles
of 46–54
C annealing for 50 s and 72
C extension for 2 min. The final cycle was continued for 7-min
at 72
C. Final PCR products were mixed with 8
µ
l bromophenol blue and run on high-resolution
denaturing polyacrylamide gels 6% (0.2 mm) for 3 h at 65 W (size 45
×
30 cm) including 1
×
TBE
buer. The gels were dried and exposed for 2 days to X-ray hyperfilm (Kodak, Taufkirchen, Germany)
and subsequently developed. Finally, the autoradiograms were scanned to identify the polymorphic
bands [25].
2.4. ISSR Statistics
The quality and quantity of ISSR bands were inspected visually. Ambiguous and smeared bands
were excluded from the analysis, and only unequivocally reproducible bands were scored for each
individual as present (1) or absent (0). The binary data matrix (presence =1; absence =0) was
formulated in MS Excel v.2016 and used for subsequent genetic analyses.
ISSR data were analyzed via the Bayesian model-based clustering algorithm as implemented
in the STRUCTURE v. 2.3 program (University of Oxford, Oxford, United Kingdom) [
34
,
35
]. We
analyzed the genetic structure among populations by assigning individuals into potential numbers
of clusters (K=1
10). ISSR genotypes were processed with a period of burn-in 50,000 and 100,000
MCMC repetitions [
34
]. The CLUMPAK online program was employed to identify the pattern of
clustering modes and packaging population structure [
35
]. The online programs, CLUMPAK [
36
]
and STRUCTURE HARVESTER [
37
] were implemented to assess and visualize the most appropriate
number of Kby calculating the likelihood of the posterior probability [38].
The binary data matrix was employed to calculate the genetic diversity parameters of each
population using GenAlex ver. 6.5 (Australian National University, Acton, Australia) [
39
]. The
population genetic parameters were as follows: Na (number of dierent alleles), Ne (number of eective
alleles), I(Shannon’s information index), He (expected heterozygosity), uHe (unbiased expected
heterozygosity), PPL (percentage of polymorphic loci), NB (number of bands) and NPB (number of
private bands) and pairwise population matrix of Nei genetic distance [28,29].
Intra- and inter-specific molecular variations and genetic relationships among populations were
implemented by Principal Coordinate Analysis (PCoA) and Analysis of Molecular Variance (AMOVA)
as utilized by GenAlex ver. 6.5, respectively [39].
To better understand the population genetic variations, ISSR analyses were performed on three
platforms separately, as follows: whole populations, native populations and invasive American
A. franciscana.
3. Results
3.1. Phylogenetic Analyses and Haplotype Distribution
Our phylogenetic analyses provide evidence that the studied bisexual specimens from three
localities of Iran, Nough Catchment (NOG), Mahshar port (MSH) and Maharlu Lake (MAHR), and a
locality from Iraq, Garmat Ali (GAA), clustered in the clade of A.franciscana (Figure 2). In addition, all
parthenogenetic populations clustered in two separated clades that shared a common ancestor with
urmiana. Although most of the parthenogenetic populations were located in clade P1, the majority of
CAM specimens (eight out of nine). The clade P1 contained two sub-clades, consisting of diploids and
triploid populations.
Diversity 2020,12, 132 7 of 21
Figure 2.
Phylogenetic tree of Artemia using COI sequences based on the ML approach. The number
behind major nodes denotes bootstrap confidential values. Daphnia tenebrosa (HQ972028) was used as
an outgroup. (URM: Artemia urmiana, TIB: Artemia tibetiana, SIN: Artemia sinica, FRA: Artemia franciscana,
PER: Artemia prersimilis, SAL: Artemia salina, DI: Diploid parthenogenetic population, TRI: Triploid
parthenogenetic population, TETRA: Tetraploid parthenogenetic population, PENTA: Pentaploid
parthenogenetic population; abbreviations listed in Table 1).
Figure 3represents the haplotype spanning network of COI among native A. urmiana and
parthenogenetic populations. Results demonstrated that A. urmiana has wider genetic variation
compared to parthenogenetic ones. Genetic dierentiation and a close relationship of Camalti Lake
(CAM) population (Turkey) with A. urmiana was clearly revealed in the tree. While COI sequences
of nine parthenogenetic populations were distributed in five Haplotypes (H2–6), A. urmiana showed
private haplotypes without a shared haplotype in other populations. The COI sequences of invasive A.
franciscana were grouped into six distinct haplotypes. No population with private haplotypes was
observed (Figure 4). The numbers of individuals and population composition were calculated for each
haplotype (Appendix A, Tables A1 and A2).
Diversity 2020,12, 132 8 of 21
Figure 3.
The relationship of COI haplotypes distribution among native populations (abbreviations
listed in Table 1).
Figure 4.
The relationship of COI haplotypes distribution among invasive A. franciscana populations
(Abbreviations listed in Table 1).
Diversity 2020,12, 132 9 of 21
3.2. ISSR Profiling
We have shown previously that ISSR can detect substantial genetic variation in the genus
Artemia [
28
,
29
]. ISSR fingerprints can dier within and among populations. Altogether, 152 observed
and unambiguously identified ISSR bands were analyzed. The total number of polymorphic loci
showed a mean value of 25.42
±
2.28%; the lowest number was seen in Camalti Lake (CAM) (7.24%)
and the highest in Nough Catchment (NOG) (38.82%). Generally, the lowest values of genetic indices
belonged to the parthenogenetic CAM, while the highest values were shared between the native
parthenogenetic population from Eastern lagoon around Urmia Lake (LAGE) (Ne =1.256
±
0.031,
I=0.208
±
0.024, He =0.143
±
0.017, uHe =0.171
±
0.020) and invasive A. franciscana from NOG
(Na =1.013
±
0.071, I=0.208
±
0.023) (Table 4). In summary, 134 and 126 distinguished ISSR bands were
examined for ten native and four American A. franciscana populations, respectively (Tables 5and 6).
According to the results of the Nei genetic matrix, parthenogenetic CAM from Turkey and invasive
GAA from Iraq showed the high genetic distance with parthenogenetic and invasive populations,
respectively (Tables 7and 8).
Table 4. Genetic indices among examined populations according to ISSR markers.
Pop. N Na Ne I He uHe PPL NB NPB
KOC 70.645
(0.062)
1.102
(0.021)
0.090
(0.017)
0.059
(0.012)
0.064
(0.012) 17.76 71 0
ABG 70.743
(0.066)
1.149
(0.024)
0.131
(0.020)
0.088
(0.013)
0.095
(0.014) 23.68 77 0
LAGE 30.914
(0.072)
1.256
(0.031)
0.208
(0.024)
0.143
(0.017)
0.171
(0.020) 34.87 86 0
LAGW 70.829
(0.066)
1.157
(0.024)
0.140
(0.020)
0.093
(0.014)
0.100
(0.015) 25.66 87 0
KBG 70.743
(0.068)
1.161
(0.025)
0.139
(0.020)
0.094
(0.014)
0.101
(0.015) 25.00 75 0
QOM 70.638
(0.062)
1.097
(0.020)
0.087
(0.016)
0.058
(0.011)
0.062
(0.012) 17.11 71 0
MIG 50.678
(0.067)
1.150
(0.025)
0.128
(0.020)
0.087
(0.014)
0.096
(0.015) 23.03 68 0
CAM 70.559
(0.051)
1.041
(0.014)
0.036
(0.011)
0.024
(0.008)
0.026
(0.008) 7.24 74 0
INC 70.658
(0.064)
1.125
(0.022)
0.110
(0.018)
0.074
(0.013)
0.080
(0.014) 19.74 70 0
URM 50.783
(0.072)
1.200
(0.027)
0.173
(0.022)
0.117
(0.015)
0.130
(0.017) 30.26 73 0
MAH 20.822
(0.066)
1.181
(0.025)
0.155
(0.021)
0.106
(0.015)
0.142
(0.020) 25.66 86 1
NOG 71.013
(0.071)
1.244
(0.030)
0.208
(0.023)
0.140
(0.016)
0.151
(0.017) 38.82 95 0
MSH 71.066
(0.066)
1.223
(0.028)
0.193
(0.022)
0.129
(0.015)
0.139
(0.016) 36.84 106 0
GAA 50.921
(0.067)
1.206
(0.028)
0.172
(0.022)
0.117
(0.015)
0.130
(0.017) 30.26 94 0
Mean 5.9
(0.035)
0.787
(0.018)
1.164
(0.007)
0.141
(0.005)
0.095
(0.004)
0.106
(0.004)
25.42
(2.28%) - -
Pop. =Population, N =number of individuals, Na =number of Dierent Alleles, Ne =number of Eective
Alleles, I=Shannon’s Information Index, He =Expected Heterozygosity, uHe =Unbiased Expected Heterozygosity,
PPL =Percentage of Polymorphic Loci, NB =number of bands, NPB =number of private bands.
Diversity 2020,12, 132 10 of 21
Table 5. Genetic indices among native populations according to ISSR markers.
Population N Na Ne I He uHe PPL NB NPB
KOC 7.000 0.731
(0.067)
1.115
(0.023)
0.102
(0.019)
0.067
(0.013)
0.073
(0.014) 20.15 71 2
ABG 7.000 0.843
(0.071)
1.169
(0.027)
0.148
(0.022)
0.100
(0.015)
0.107
(0.016) 26.87 77 4
LAGE 3.000 1.037
(0.075)
1.291
(0.034)
0.236
(0.026)
0.162
(0.018)
0.195
(0.022) 39.55 86 1
LAGW 7.000 0.940
(0.069)
1.178
(0.027)
0.158
(0.022)
0.106
(0.015)
0.114
(0.016) 29.10 87 4
KBG 7.000 0.843
(0.073)
1.183
(0.028)
0.158
(0.023)
0.107
(0.015)
0.115
(0.017) 28.36 75 0
QOM 7.000 0.724
(0.066)
1.110
(0.023)
0.099
(0.018)
0.065
(0.013)
0.070
(0.014) 19.40 71 0
MIG 5.000 0.769
(0.073)
1.170
(0.027)
0.145
(0.022)
0.098
(0.015)
0.109
(0.017) 26.12 68 3
CAM 7.000 0.634
(0.055)
1.046
(0.016)
0.041
(0.013)
0.027
(0.009)
0.029
(0.009) 8.21 74 0
INC 7.000 0.746
(0.069)
1.142
(0.025)
0.125
(0.021)
0.084
(0.014)
0.091
(0.015) 22.39 70 1
URM 5.000 0.888
(0.077)
1.227
(0.029)
0.196
(0.024)
0.133
(0.017)
0.148
(0.018) 34.33 73 3
Mean 6.200
(0.036)
0.816
(0.022)
1.163
(0.008)
0.141
(0.007)
0.095
(0.005)
0.105
(0.005)
25.45
(2.74) - -
Pop. =Population, N =number of individuals, Na =number of Dierent Alleles, Ne =number of Eective
Alleles, I=Shannon’s Information Index, He =Expected Heterozygosity, uHe =Unbiased Expected Heterozygosity,
PPL =Percentage of Polymorphic Loci, NB =number of bands, NPB =number of private bands.
Table 6. Genetic indices among invasive A. franciscana populations according to ISSR markers.
Population N Na Ne I He uHe PPL NB NPB
MAH 2.000 0.992
(0.071)
1.219
(0.029)
0.187
(0.025)
0.128
(0.017)
0.171
(0.023) 30.95 86 3
NOG 7.000 1.222
(0.073)
1.295
(0.034)
0.251
(0.026)
0.169
(0.018)
0.182
(0.019) 46.83 95 6
MSH 7.000 1.286
(0.065)
1.269
(0.033)
0.233
(0.025)
0.156
(0.018)
0.168
(0.019) 44.44 106 8
GAA 5.000 1.111
(0.070)
1.249
(0.033)
0.207
(0.025)
0.141
(0.018)
0.157
(0.020) 36.51 94 5
Mean 5.250
(0.091)
1.153
(0.035)
1.258
(0.016)
0.220
(0.013)
0.149
(0.009)
0.169
(0.010)
39.68
(3.65) - -
Pop. =Population, N =number of individuals, Na =number of Dierent Alleles, Ne =number of Eective
Alleles, I=Shannon’s Information Index, He =Expected Heterozygosity, uHe =Unbiased Expected Heterozygosity,
PPL =Percentage of Polymorphic Loci, NB =number of bands, NPB =number of private bands.
Diversity 2020,12, 132 11 of 21
Table 7.
Pairwise Population Matrix of Nei Genetic Distance among invasive A. franciscana populations.
Population KOC ABG LAGE LAGW KBG QOM MIG CAM INC
ABG 0.162 - - - - - - - -
LAGE 0.167 0.175 - - - - - - -
LAGW 0.193 0.127 0.190 - - - - - -
KBG1 0.156 0.117 0.142 0.111 - - - - -
QOM 0.182 0.131 0.171 0.135 0.099 - - - -
MIG 0.217 0.185 0.171 0.212 0.124 0.148 - - -
CAM 0.362 0.346 0.249 0.358 0.279 0.354 0.331 - -
INC 0.233 0.165 0.184 0.223 0.149 0.176 0.091 0.351 -
URM 0.219 0.194 0.165 0.190 0.141 0.174 0.194 0.351 0.211
Table 8. Pairwise Population Matrix of Nei Genetic Distance among native populations.
Population MAH NOG MAH
NOG 0.143 - -
MSH 0.145 0.144 -
GAA 0.198 0.170 0.152
Based on the total results of ISSR, the AMOVA analysis documented that most of the genetic
variations were attributed among native and invasive populations more than within populations (69%
vs. 31%). There was no indicative genetic variability observed in the midst of among- and within
variation (55% vs. 45%) in native populations. In contrast, the high dierentiation was represented
within populations (71% vs. 29%) of non-indigenous A. franciscana in Asia (Table 9, Figure 5A–C).
Table 9.
Molecular variation (within and among populations) for examined populations by AMOVA
based on ISSR markers.
Whole Populations
Source df SS MS Est. Var. %
Among Pops 13 1673.318 128.717 20.265 69%
Within Pops 69 639.405 9.267 9.267 31%
Total 82 2312.723 - 29.532 100%
Source df SS MS Est. Var. %
Native Populations
Among Pops 9 627.912 69.768 10.006 55%
Within Pops 52 418.362 8.045 8.045 45%
Total 61 1046.274 - 18.052 100%
A. franciscana
Source df SS MS Est. Var. %
Among Pops 3 117.338 39.113 5.239 29%
Within Pops 17 221.043 13.003 13.003 71%
Total 20 338.381 - 18.241 100%
Diversity 2020,12, 132 12 of 21
Figure 5.
Contribution of genetic variation within and among populations for the examined populations
by AMOVA, based on ISSR markers (
A
=whole examined populations,
B
=native populations,
C=invasive A. franciscana).
Bayesian clustering analysis using STRUCTURE was performed to investigate the genetic patterns
of the studied populations. The optimum Kwas obtained at K=2 for the whole 14 populations
and ten native populations, and K=9 for four invasive A. franciscana, respectively. Figure 6showed
the clustering of genetic structures, where the first highest posterior probability (K) was represented
by dierent colors for each population. With regard to the genetic patterns of ISSR, native and
exotic populations could be completely divided into two groups (Figure 6A). The results of the
analysis for native populations documented that parthenogenetic CAM is a distinct population with
a diering clustering pattern. The STRUCTURE analysis could not fully distinguish A. urmiana and
parthenogenetic populations (Figure 6B). The high value of optimum K(K=9) was prominent in
the clustering analysis for A. franciscana populations, which generally revealed a complex pattern
(Figure 6C). Proportions of genetic clusters (percentage) for each locality were summarized in Figure 7
(see also Tables A3A5).
Figure 6.
Clustering of genetic structures based on ISSR markers (
A
=whole examined populations,
B=native populations, C=invasive A. franciscana; abbreviations listed in Table 1).
Diversity 2020,12, 132 13 of 21
Figure 7.
Proportion of genetic clusters for each locality in the STRUCTURE analysis (
A
=whole
examined populations,
B
=native populations,
C
=invasive A. franciscana; abbreviations listed in
Table 1).
The first and second PCoA coordinates explained 64.66% and 7.00% of the variance, respectively
(overall, 71.66% of total variation). The results showed all populations clustered into three groups,
where invasive A. franciscana has been significantly separated from the native populations based on
the first coordinate. Ten native populations (including A. urmiana and parthenogenetics) were divided
into two distinct groups, G2 and G3. G2 included all of the CAM population and a single individual
of Eastern lagoon around Urmia Lake (LAGE). Bisexual A. urmiana and other parthenogenetics
were placed in G3 (Figure 8). The results of the separate analyses of PCoA for native and invasive
populations are shown in Figures 9and 10, which include overall 42.55% and 41.55% of the total
variation, respectively. Although native populations produced almost the same result with the “whole
populations” analysis (Figure 9), the separated analyses of PCoA for invasive A. franciscana could
separate all four populations in isolated groups (Figure 10).
Figure 8.
Principal Coordinates Analysis (PCoA) showing dierentiation patterns among whole
examined populations based on ISSR markers (abbreviations listed in Table 1).
Diversity 2020,12, 132 14 of 21
Figure 9.
Principal Coordinates Analysis (PCoA) showing dierentiation patterns among native
populations based on ISSR markers (abbreviations listed in Table 1).
Figure 10.
Principal Coordinates Analysis (PCoA) showing dierentiation patterns among invasive A.
franciscana populations based on ISSR markers (abbreviations listed in Table 1).
Similar to the phylogeny based on COI sequences, clustering analysis of ISSR sequences by
STRUCTURE has revealed a distinguished structure for non-indigenous A. franciscana populations. In
this analysis, all native populations (bisexual A. urmiana and parthenogenetic ones) have displayed
almost similar patterns, but a separated analysis for native populations has revealed a non-identical
structure for the CAM population. Additionally, an inconsistent pattern of the Eastern lagoon around
Urmia Lake (LAGE) was also observed. A separated analysis for invasive populations could not
reveal dierent patterns among examined populations by STRUCTURE (Figure 6A–C). On the other
hand, PCoA has divided A. franciscana from native populations. Although PCoAs could not branch o
natives by localities, the separated PCoA has divided invasive populations based on localities in the
Diversity 2020,12, 132 15 of 21
four groups. Contrary to COI haplotype distribution, the ISSR marker was unable to reveal a private
pattern for bisexual A. urmiana in comparison to native parthenogenetic populations.
4. Discussion
The present study was performed to compare the population structure and genetic dierentiation
of native and invasive Artemia populations. The mitochondrial COI gene has been established as a
useful molecular marker to determine the intra- and inter-specific evolutionary associations [
2
,
3
,
17
,
18
].
Asem et al. [
2
] have documented that di- and triploid parthenogenetic brine shrimps are maternally
related to A. urmiana, while tetra- and pentaploid lineages shared a common maternal ancestor with A.
sinica. Based on the mitochondrial COI dataset, all examined parthenogenetic individuals have grouped
in a close evolutionary relationship with A. urmiana (Figure 2). Our results have demonstrated that
they should include di- and/or triploids. This observation has also been confirmed by the phylogenetic
tree. Although individuals of eight Artemia populations have been exactly located in sub-clades of di-
and/or triploid, eight out of nine specimens of CAM from Turkey have been placed in a particular
clade (P2), in a close connection with A. urmiana (Figure 2). This finding has also been confirmed by
haplotype distribution (Figure 3). Previously, Sayg [
40
] has confirmed that triploid and pentaploid
parthenogenetic populations coexisted in Camalti Lake (CAM). Our observation has confirmed that
Camalti Lake (CAM) populations had a parthenogenetic reproductive mode and those examined
specimens should be considered as a diploid and/or triploid population (see [
3
]). Despite the fact that A.
urmiana shared a common ancestor with di- and triploids, it has presented an unexpectedly high level
of haplotype diversity of the COI marker. These results have also been documented by Eimanifar and
Wink [
28
]. The high level of haplotype variation might be attributed to the evolutionary life history of
A. urmiana [
30
] and/or its large population size [
28
]. Urmia Lake has undergone considerable changes
in environmental conditions, such as salinity and temperature [
41
,
42
], which could have influenced
genetic variation and population size during evolution.
Although COI sequences should reveal a phylogeographic structure in the closely related
species [
43
], our results could not determine a level of geographical dierentiation among native as
well as among invasive populations. Contrary to the results of the mitochondrial marker, genomic
fingerprinting ISSR could not reveal a significant high level of genetic variation in A. urmiana. This
result might be due to dierences in the rate of variation of mitochondrial and nuclear genes and
potential hybridization events in the past.
The ISSR method had been utilized to study ten diploid parthenogenetic Artemia populations from
China by Hou et al. [
27
]. Their genetic variation was significantly higher than our examined
parthenogenetic populations. For example, the percentage of polymorphic loci ranged from
54.12–87.06% vs. 8.21–39.55%. Western Asia is the origin of di- and triploid parthenogenetic
populations [
3
], so high genetic diversity of Chinese populations could be the result of their adaptation
during colonization through biological dispersal to the new environments in East Asia under historical
evolutionary progress.
In general, it was reported that the invasive populations have lower genetic variation in the
new environments as compared with their origin populations [
44
]. The colonized population of
A.franciscana in Vietnam has a lower genetic diversity than its native source population from San
Francisco Bay (SFB) [
22
]. In contrast, Eimanifar et al. [
2
] have documented that Asian invasive
A.franciscana populations had higher genetic variation than the American Great Salt Lake (GSL)
population and native Asian species. Similar results have been reported for some invasive populations
from Mediterranean regions [
23
,
24
]. A recent study assumed that invasive populations of A. franciscana
show a wide degree of genetic dierentiation in Australia [17].
Overall, our ISSR results have documented that invasive A. franciscana populations had distinctly
higher genetic variation than Western Asian native parthenogenetic populations. On the other hand,
native A. franciscana from Great Salt Lake (GSL) have represented higher variation than examined
invasive populations in this study, as the percentage of polymorphic loci diered from 67–81% vs.
Diversity 2020,12, 132 16 of 21
30.95–46.83% (see [
29
]). Additionally, all four invasive A. franciscana populations clearly revealed
a dierent genetic structure. Observation of low genetic diversity in native populations might be
attributed to the eect of asexual reproduction in parthenogenetic populations and/or critical climatic
conditions in West Asia, especially Urmia Lake in the last two decades (see [
3
,
30
]). We believe
that interactions between dierent ecological conditions in the new environments and the high
potential of physiological plasticity and genetic adaptation of A. franciscana could exert dierent
evolutionary pathways during the introduction of exotic populations, which would have ultimately
caused intra-specific variations and genetic divergence in the examined invasive populations.
In conclusion, it is expected that the non-indigenous species should have a lower genetic variation
than their source populations [
25
,
44
,
45
]. However, non-indigenous A. franciscana populations gave
opposite results in comparison with native populations from GSL and SFB. Since there is neither a
taxonomical identification key nor morphological identifications to distinguish bisexual species and
parthenogenetic populations [
11
,
46
], it would not be possible to identify the exotic population at the
earliest time of invasion. Therefore, there is a lack of information to regularly determine the colonization
progress and evolutionary development of A. franciscana in the new habitats. We assume that dierences
in the genetic variation of non-indigenous populations could be due to the study on dierent invasion
periods consisting of i) introduced, ii) establishing/colonizing, iii) established/colonized populations.
Author Contributions:
Research design, material preparation, and data collection were performed by A.E. Data
analysis was carried out by A.A. The first draft of the manuscript was written by A.A. and A.E. P.-Z.W., W.L. and
M.W. reviewed the draft. All authors have read and agreed to the published version of the manuscript.
Funding:
This study was carried out at IPMB, Department of Biology, University Heidelberg and A/10/97179.
Amin Eimanifar was supported by a Ph.D. fellowship from the Deutscher Akademischer Austauschdienst (DAAD,
German Academic Exchange Service).
Acknowledgments:
We would like to express our appreciations to the Dr. Razia Sultana from the Food and
Marine Resources Research Center, PCSIR Laboratories Complex, Pakistan and Prof. Gilbert Van Stappen from
the Artemia Reference Center, Belgium for providing Artemia samples to this research project.
Conflicts of Interest: The authors declare no conflict of interest. M.W. is Editor-in-Chief of Diversity.
Appendix A
Table A1. Information of network haplotype composition of native populations.
Haplotype Ind. Pop. (Ind.) Haplotype Ind. Pop. (Ind.)
H1 18 URM (18) H24 1 URM (1)
H2 7 CAM (7) H25 1 URM (1)
H3 16 KOC (6), LAGW (5)ABG (3), LAGE
(1), KBG (1) H26 1 URM (1)
H4 15 ING (5), MIG (4), KBG (3), LAGE (2) H27 1 URM (1)
H5 7 QOM (6), MIG (1) H28 1 URM (1)
H6 1 CAM (1) H29 1 URM (1)
H7 1 URM (1) H30 1 URM (1)
H8 1 URM (1) H31 2 URM (2)
H9 2 URM (2) H32 1 URM (1)
H10 1 URM (1) H33 1 URM (1)
H11 1 URM (1) H34 1 URM (1)
H12 3 URM (3) H35 1 URM (1)
Diversity 2020,12, 132 17 of 21
Table A1. Cont.
Haplotype Ind. Pop. (Ind.) Haplotype Ind. Pop. (Ind.)
H13 1 URM (1) H36 1 URM (1)
H14 1 URM (1) H37 1 URM (1)
H15 1 URM (1) H38 1 URM (1)
H16 1 URM (1) H39 1 URM (1)
H17 1 URM (1) H40 1 URM (1)
H18 1 URM (1) H41 1 URM (1)
H19 1 URM (1) H42 1 URM (1)
H20 1 URM (1) H43 1 URM (1)
H21 1 URM (1) H44 1 URM (1)
H22 1 URM (1) H45 1 URM (1)
H23 1 URM (1) - - -
Ind. =Individual, Pop. =Population.
Table A2. Information of network haplotype composition of invasive A. franciscana populations.
Haplotype Ind. Pop. (Ind.)
H1 8 GAA (4), MAH (2), MSH (2)
H2 7 NOG (4), MSH (3)
H3 4 GAA (4)
H4 1 NOG (1)
H5 1 MAH (1)
H6 1 MSH (1)
Ind. =Individual, Pop. =Population.
Table A3.
Proportion of genetic clusters for each locality in STRUCTURE analysis among whole
examined populations.
Population K1 (%) K2 (%)
KOC 0.1 99.9
ABG 0.2 99.8
LAGE 0.7 99.3
LAGW 0.3 99.7
KBG 0.1 99.9
QOM 0.1 99.9
MIG 1.9 98.1
CAM 0.1 99.9
INC 0.2 99.8
URM 3.6 96.4
MAH 98.7 1.3
Diversity 2020,12, 132 18 of 21
Table A3. Cont.
Population K1 (%) K2 (%)
NOG 99.9 0.1
MSH 99.9 0.1
GAA 99.9 0.1
Table A4.
Proportion of genetic clusters for each locality in STRUCTURE analysis among
native populations.
Population K1 (%) K2 (%)
KOC 000099.7 0.3
ABG 99.8 0.2
LAGE 72.1 27.9
LAGW 99.6 0.4
KBG 99.5 0.5
QOM 99.8 0.2
MIG 99.6 0.4
CAM 0.2 99.8
INC 99.7 0.3
URM 93.1 6.9
Table A5.
Proportion of genetic clusters for each locality in STRUCTURE analysis among invasive A.
franciscana populations.
Population K1 (%) K2 (%) K3 (%) K4 (%) K5 (%) K6 (%) K7 (%) K8 (%) K9 (%)
MAH 1.1 0.4 0.3 1.7 0.4 1.1 46 1.8 47.1
NOG 0.6 0.5 0.4 57 0.6 33.6 3.6 0.3 3.4
MSH 0.5 28.6 0.3 0.4 27.9 0.4 3.4 33.9 4.6
GAA 38.8 0.2 42.3 0.3 0.4 0.2 7 1.1 9.7
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... 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]. ...
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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.
... Unintentional escapes caused by normal use in hatcheries and/or transmission by migratory waterfowl should be considered as a secondary factor in the distribution of A. franciscana in new habitats. At present, A. franciscana has been colonized in numerous regions across Eurasia, especially in the Mediterranean (Amat et al., 2005;Mura et al., 2006;Van Stappen, 2008;Muñoz, 2009;Ben Naceur et al., 2010, Eimanifar et al., 2014Scalone and Rabet, 2013;Horvath et al., 2018;Saji et al., 2019;Eimanifar et al., 2020) and Australia (Asem et al., 2018). ...
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Artemia franciscana, native to America, has recently colonized as non-indigenous population in Asia, Europe, North Africa, and Australia. We evaluated the effects of the colonization of A. franciscana on genetic differentiation in new environments in the United Arab Emirates (UAE). We used the COI marker to determine the genetic structure and origins of exotic populations in the UAE. Results confirmed the colonization of A. franciscana in two localities. Invasive populations of A. franciscana had significantly lower genetic variation than native populations in the Great Salt Lake and San Francisco Bay. Results showed that the studied populations could not have colonized directly from natural American habitats, and they possibly were from secondary introduction events of other non-indigenous populations. Genetic analysis yielded different demographic patterns for the studied invasive populations. The population in Al Wathba Wetland Reserve (AWWR) demonstrated demographic expansion, whereas in Godolphin Lakes (GL), it reached a demographic equilibrium. Neutrality tests showed an excess of recent and historical mutations in the COI gene pool of invasive AWWR Artemia in the new environment. The results suggest that different ecological conditions in new environments can exert selective pressures during the introduction of an exotic population, which can affect genetic variation.
... Unintentional escapes caused by normal use in hatcheries and/or transmission by migratory waterfowl should be considered as a secondary factor in the distribution of A. franciscana in new habitats. At present, A. franciscana has been colonized in numerous regions across Eurasia, especially in the Mediterranean (Amat et al., 2005;Mura et al., 2006;Van Stappen, 2008;Muñoz, 2009;Ben Naceur et al., 2010, Eimanifar et al., 2014Scalone and Rabet, 2013;Horvath et al., 2018;Saji et al., 2019;Eimanifar et al., 2020) and Australia (Asem et al., 2018). ...
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Artemia franciscana, native to America, has recently colonized as non-indigenous population in Asia, Europe, North Africa, and Australia. We evaluated the effects of the colonization of A. franciscana on genetic differentiation in new environments in the United Arab Emirates (UAE). We used the COI marker to determine the genetic structure and origins of exotic populations in the UAE. Results confirmed the colonization of A. franciscana in two localities. Invasive populations of A. franciscana had significantly lower genetic variation than native populations in the Great Salt Lake and San Francisco Bay. Results showed that the studied populations could not have colonized directly from natural American habitats, and they possibly were from secondary introduction events of other non-indigenous populations. Genetic analysis yielded different demographic patterns for the studied invasive populations. The population in Al Wathba Wetland Reserve (AWWR) demonstrated demographic expansion, whereas in Godolphin Lakes (GL), it reached a demographic equilibrium. Neutrality tests showed an excess of recent and historical mutations in the COI gene pool of invasive AWWR Artemia in the new environment. The results suggest that different ecological conditions in new environments can exert selective pressures during the introduction of an exotic population, which can affect genetic variation.
... 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]. ...
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Full-text available
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 parthenogenetic 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 sequence 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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Artemia is an industrially important genus used in aquaculture as a nutritious diet for fish and as an aquatic model organism for toxicity tests. However, despite the significance of Artemia, genomic research remains incomplete and knowledge on its genomic characteristics is insufficient. In particular, A. franciscana of North America has been widely used in fisheries of other continents, resulting in invasion to native species. Therefore, studies on population genetics and molecular marker development as well as morphological analyses are required to investigate its population structure and to discriminate closely related species. Here, we used the Illumina Hi-Seq platform to estimate the genomic characteristics of A. franciscana through genome survey sequencing. Further, simple sequence repeat (SSR) loci were identified for microsatellite marker development. The predicted genome size was ~867 Mb using K-mer analysis (K=17), and heterozygosity and duplication rates were 0.655% and 0.809%, respectively. A total of 421,467 SSRs were identified from the genome survey assembly, most of which were dinucleotide motifs with a frequency of 77.22%. This study will be a useful basis in genomic and genetic research for A. franciscana.
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Domestication of animal species is often associated with a reduction in genetic diversity. The honey bee, Apis mellifera Linnaeus, 1758, has been managed by beekeepers for millennia for both honey and wax production and for crop pollination. Here we use both microsatellite markers and sequence data from the mitochondrial COI gene to evaluate genetic variation of managed A. mellifera in Thailand, where the species is introduced. Microsatellite analysis revealed high average genetic diversity with expected heterozygosities ranging from 0.620 ± 0.184 to 0.734 ± 0.071 per locus per province. Observed heterozygosities were generally lower than those expected under Hardy-Weinberg equilibrium, both locally and across the population as a whole. Mitochondrial sequencing revealed that the frequency of two evolutionary linages (C-Eastern European and O-Middle Eastern) are similar to those observed in a previous survey 10 yr ago. Our results suggest that Thai beekeepers are managing their A. mellifera in ways that retain overall genetic diversity, but reduce genetic diversity between apiaries.
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The genus Artemia Leach, 1819 is a cosmopolitan halophilic crustacean, consisting of bisexual species and obligate parthenogenetic populations. Asia is rich in Artemia biodiversity. More than 530 Artemia sites have been recorded from this area and more than 20 species/subspecies/variety names have been used for them. There exist various problems in the nomenclature, identification, and phylogenetic status of Artemia native to Asia, which are discussed in this paper.
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Urmia Lake, the largest natural habitat of the brine shrimp Artemia urmiana, has progressively desiccated over the last two decades, resulting in a loss of 80% of its surface area and producing thousands of hectares of arid salty land. This ecological crisis has seriously affected the lake's native biodiversity. Artemia urmiana has lost more than 90% of its population during the decade from 1994 (rainy period) to 2004 (drought period) due to salinity increasing to saturation levels (∼300 g/l). We studied the influence of this ecological crisis on the genetic diversity of A. urmiana in Urmia Lake, based on one cyst collections in 1994 and 2004. AMOVA analysis on ISSR data demonstrated a 21% genetic variation and there was a 5.5% reduction of polymorphic loci between samples. PCoA showed that 77.42% and 68.75% of specimens clustered separately in 1994 and 2004, respectively. Our analyses of four marker genes revealed different genetic diversity patterns with a decrease of diversity at ITS1 and an increase for Na + /K + ATPase. There was no notable difference in genetic variation detected for COI and 16S genes between the two periods. However, they represented distinctly different haplotypes. ITS1 and COI followed a population expansion model, whereas Na + /K + ATPase and 16S were under demographic equilibrium without selective pressure in the 1994 samples. Neutrality tests confirmed the excess of rare historical and recent mutations present in COI and ITS1 in both samples. It is evident that a short-term ecological disturbance has impacted the genetic diversity and structure of A. urmiana.
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The taxonomic identity of an unknown Artemia population inhabiting the Al Wathba Wetland Reserve in Abu Dhabi, U.A.E., was determined using phylogenetic analysis of the mitochondrial marker Cytochrome Oxidase Subunit 1 ( COI ). The results showed that the examined population belongs to an exotic invasive species, Artemia franciscana . Based on the distribution pattern of haplotypes, the studied population could possibly have originated from that inhabiting the Great Salt Lake (Utah, U.S.A.).
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Among each of the two different bisexual clades of the brine shrimp Artemia in Asia, at least two cryptic species of Artemia (one in each clade) were found in Mongolia in the present study. As in other, similar cases, once the discreteness of such units at species level is known, meticulous observations using light microscopy will reveal distinguishable morphological differences with other congeners, slight as those features may be. In such classifications, the emphasis usually lies on structures of the copulatory organs, as the small differences present in most cases are best expressed in those organs that primarily ensure the species’ reproductive isolation. Since physical differences in the reproductive organs will tend to (partly) obstruct effective mating, it may be presumed this is likely to result in (some degree of) reproductive isolation, the prime criterion for recognizing biological species. Two cryptic species from Mongolia are described herein, and our results also showed for one of them an extremely rapid individual growth and very early maturity: in fact it makes the most extreme r-strategist documented, at least, as far as we know. One of the consequences of this observation could be, that where such Artemia cysts (i.e., resting eggs) are used as inoculations in aquaculture activities, these should be monitored closely and, as required, be subjected to adequate, perhaps more stringent regulations. The present authors are not aquaculture scientists but researchers of the genus Artemia, so for us, inoculation under natural conditions in the field is our direct concern. We report a highly reproductive Artemia species as new to science, but at the same time, we worry that this new species could escape from the aquaculture industry into the natural environment. This is because in this type of aquaculture no isolated basins are used but only incompletely separated, i.e., only partially screened off, parts of natural water bodies. Artemia franciscana Kellogg, 1906, as well as parthenogenetic populations of Artemia could be disturbed by escapees of that extremely prolific new species.
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The sex of relatively primitive animals such as invertebrates is mostly determined by environmental factors and chromosome ploidy. Heteromorphic chromosomes may also play an important role, as in the ZW system in lepidopterans. However, the mechanisms of these various sex determination systems are still largely undefined. In the present study, a Masculinizer gene (Ar-Masc) was identified in the crustacean Artemia franciscana Kellogg 1906. Sequence analysis revealed that the 1140-bp full-length open reading frame of Ar-Masc encodes a 380-aa protein containing two CCCH-type zinc finger domains having a high degree of shared identities with the MASC protein characterized in the silkworm Bombyx mori, which has been determined to participate in the production of male-specific splice variants. Furthermore, although Ar-Masc could be detected in almost all stages in both sexual and parthenogenetic Artemia, there were significant variations in expression between these two reproductive modes. Firstly, qRT-PCR and Western blot analysis showed that levels of both Ar-Masc mRNA and protein in sexual nauplii were much higher than in parthenogenetic nauplii throughout the hatching process. Secondly, both sexual and parthenogenetic Artemia had decreased levels of Ar-Masc along with the embryonic developmental stages, while the sexual ones had a relatively higher and more stable expression than those of parthenogenetic ones. Thirdly, immunofluorescence analysis determined that sexual individuals had higher levels of Ar-MASC protein than parthenogenetic individuals during embryonic development. Lastly, RNA interference with dsRNA showed that gene silencing of Ar-Masc in sexual A. franciscana caused the female-male ratio of progeny to be 2.19:1. These data suggest that Ar-Masc participates in the process of sex determination in A. franciscana, and provide insight into the evolution of sex determination in sexual organisms.