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Phylogeography and population genetic structure of an exotic invasive brine shrimp, Artemia Leach, 1819 (Crustacea : Anostraca), in Australia

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Native American Artemia franciscana has become an introduced species in the Old World due to the rapid development of the aquaculture industry in Eurasia. The recent colonisation of A. franciscana in Mediterranean regions and Asia has been well documented, but Australia is a continent where the dispersal of this species is not well understood. In the present study, we sequenced the cytochrome oxidase subunit I (COI) and examined the phylogenetic relationships, haplotype network and population genetic structure of Artemia from four geographical localities in Australia and two American native localities. Our results confirmed the colonisation of Australia in all four localities by A. franciscana. First, we document the occurrence of Artemia in Mulgundawa and St Kilda localities in Australia. The Dampier population is a monomorphic population, but there is high genetic variation and a degree of demographic expansion observed in other introduced A. franciscana populations in Australia. This observation suggests an interaction between environmental conditions and adaptive potentials of A. franciscana. Our findings imply that populations from St Kilda and Port Hedland might have originated from a San Francisco Bay source, while the two other locations resulted from admixture between Great Salt Lake and San Francisco Bay sources, perhaps resulting from secondary introduction events.
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Phylogeography and population genetic structure of an
exotic invasive brine shrimp, Artemia Leach, 1819
(Crustacea : Anostraca), in Australia
Alireza Asem
A,D,E
, Amin Eimanifar
B,D,E
, Weidong Li
A
, Pei-Zheng Wang
A
,
Samantha A. Brooks
C
and Michael Wink
B
A
College of Life Sciences and Ecology, Hainan Tropical Ocean University, Yucai Road, Sanya 572000, China.
B
Institute of Pharmacy and Molecular Biotechnology (IPMB), Heidelberg University, Im Neuenheimer Feld 364,
69120 Heidelberg, Germany.
C
Department of Animal Sciences, University of Florida, Gainesville, FL 32611, USA.
D
These two authors contributed equally to this paper.
E
Corresponding authors. Email: asem.alireza@gmail.com; amineimanifar1979@gmail.com
Abstract. Native American Artemia franciscana has become an introduced species in the Old World due to the rapid
development of the aquaculture industry in Eurasia. The recent colonisation of A. franciscana in Mediterranean regions and
Asia has been well documented, but Australia is a continent where the dispersal of this species is not well understood. In the
present study, we sequenced the cytochrome oxidase subunit I (COI) and examined the phylogenetic relationships,
haplotype network and population genetic structure of Artemia from four geographical localities in Australia and two
American native localities. Our results conrmed the colonisation of Australia in all four localities by A. franciscana. First,
we document the occurrence of Artemia in Mulgundawa and St Kilda localities in Australia. The Dampier population is a
monomorphic population, but there is high genetic variation and a degree of demographic expansion observed in other
introduced A. franciscana populations in Australia. This observation suggests an interaction between environmental
conditions and adaptive potentials of A. franciscana. Our ndings imply that populations from St Kilda and Port Hedland
might have originated from a San Francisco Bay source, while the two other locations resulted from admixture between
Great Salt Lake and San Francisco Bay sources, perhaps resulting from secondary introduction events.
Additional keywords: Australian Artemia, biodiversity, introduced species, mtDNA-COI.
Received 23 November 2018, accepted 3 April 2019, published online 26 April 2019
Introduction
The brine shrimp Artemia is a primitive microcrustacean
inhabiting many hypersaline habitats worldwide such as inland
salt lakes, coastal saltworks, salt ponds and lagoons (Van
Stappen 2002). Artemia can withstand extreme environmental
conditions such as high salinity (7.0 340 g/L) and ionic
compositions in the natural environment due to its unique
osmoregulation mechanism (Post and Youssef 1977; Bowen
et al.1985; Lenz 1987; Browne et al.1988; Liu and Zheng 1990).
The genus Artemia consists of seven bisexual species and
numerous parthenogenetic populations with different ploidy
levels (Asem et al.2010; Asem et al.2016). Three bisexual
species occur naturally in the New World: Artemia monica
Verrill, 1869 (Mono Lake, USA), Artemia franciscana Kellogg,
1906 (North America, Central America and South America) and
Artemia persimilis Piccinelli & Prosdocimi, 1968 (Argentina and
Chile). The other four bisexual species are native to the Old
World, namely Artemia salina (Linnaeus, 1758) (Mediterranean
basin), Artemia urmiana Gunther, 1899 (Lake Urmia, Iran, and
the Crimean salt lakes, Russia), Artemia sinica Cai, 1989 (China
and Mongolia) and Artemia tibetiana Abatzopoulos, Zhang &
Sorgeloos 1998 (QinghaiTibetan Plateau, China). Previous
studies have documented that the Tibetan populations were
placed in two different groups in the phylogenetic trees of the
mitochondrial COI marker, while all of them represented a single
clade when analysed with the nuclear marker ITS1 (Maccari et al.
2013; Eimanifar et al.2014). This contradiction could be
attributed to a hybridisation event that occurred between two
ancestors. Two new species were recently described from
Mongolia (Asia), namely Artemia frameshifta and Artemia
murae (Naganawa and Mura, 2017), although the taxonomic
status of these taxa requires conrmation. The biosystematics
of these two new species were determined by single individual
differentiation from the cytochrome oxidase subunit I (COI)
sequence combined with morphological parameters, whereas
morphometric study and population genetic analysis have
Journal compilation CSIRO 2018 www.publish.csiro.au/journals/ajz
CSIRO PUBLISHING
Australian Journal of Zoology, 2018, 66, 307316
https://doi.org/10.1071/ZO18077
not previously been examined. Additionally, the existence
of males has not been investigated in A. frameshifta so that
the reproductive mode of that population is still doubtful.
Parthenogenetic populations are obligate clones containing di-,
tri-, tetra-, penta- and heteroploids, and inhabit the Old World and
Oceania (Sun et al.1999; Abatzopoulos et al. 2003).
Artemia has been broadly used as live food in the shery and
aquaculture industry, especially in the coastal areas of eastern
Asia (Van Stappen 2008). It is also used to improve the quality of
sodium chloride production in solar salt-elds by aiding in the
control of phytoplankton blooms and increasing the density of
red-pigmented bacteria to accelerate evaporation (Jones et al.
1981; Ruebhart et al.2008). Artemia is a model organism in many
biological elds, including phylogeography and population
genetics (Kappas et al.2011), molecular and cellular biology (Li
et al.2017), bioassay toxicity (Rajabi et al.2015) and
bioencapsulation (Vázquez-Silva et al.2017).
Since 1950, cysts of A. franciscana have been exported
overseas from the USA for applications in shery markets.
Genetic studies have documented that these exports originated
primarily from two major natural sources in the USA, namely the
Great Salt Lake, Utah (GSL), and San Francisco Bay, California
(SFB) (Van Stappen 2008; Muñoz 2009; Eimanifar et al.2014).
Phylogeographic analysis revealed that the expansion of
A. franciscana to non-native regions has resulted in rapid
colonisation of numerous regions across Eurasia (Amat et al.
2005; Mura et al.2006; Van Stappen 2008; Muñoz 2009; Ben
Naceur et al.2010; Scalone and Rabet 2013; Eimanifar et al.
2014; Horvath et al.2018). Phylogenetic analysis of Artemia has
not previously been conducted in Australia because of difculties
in obtaining adequate samples.
Previous studies have suggested the introduction of
A. franciscana into Australia (Clark and Bowen 1976; Geddes
1979,1981; Abreu-Grobois and Beardmore 1982; Geddes and
Williams 1987; Vanhaecke et al.1987; Pinder et al.2002;
McMaster et al.2007) but there was no evidence using genetic
barcoding to support the regional colonisation of A. franciscana
in Australia. The aim of the present study was to perform a
phylogenetic analysis of Artemia populations from Dampier,
Mulgundawa, Port Hedland and St Kilda in Australia to conrm
the taxonomical status of Artemia in these localities. Here we
sequenced the mitochondrial COI gene and determined the
genetic diversity, population genetic structure and the genetic
source of Artemia populations as compared with two American
native populations of A. franciscana from GSL and SFB.
Materials and methods
Origin of samples and sample analysis
In total, 67 individuals of bisexual Artemia were collected from
four geographical sites in Australia in summer 2011 (Fig. 1).
The sampling sites, with their abbreviations, geographical
coordinates, IPMB code numbers and number of individuals
analysed, are summarised in Table 1.
Total DNA was separately extracted from part of the antenna
of male and female shrimps (1 : 1) following the Chelex
®
100
Resin method (Bio-Rad Laboratories, USA). The samples were
crushed, incubated for 2.53hat60
C (tubes were vortexed
every 30 min) and then a nal 10 min at 80C. Then the tubes were
centrifuged at 10 000 rpm for 1 min and the supernatant phase
was directly used in the PCR reaction (Montero-Pau et al.2008;
Eimanifar and Wink 2013; Asem et al.2016). All extracted DNA
was stored at 80C for further genetic analyses.
A fragment of the mitochondrial cytochrome oxidase subunit I
(COI) was amplied. PCR was performed in a nal reaction
volume of 50 mL in a thermocycler (Biometra, Tgradient,
Germany) with Taq DNA polymerase (Bioron, GmbH, Germany)
according to conditions published previously (Eimanifar and
Wink 2013). The COI partial fragment (~588 bp) was amplied
using the metazoan invertebratesuniversal primers LCOI490/
HC02198 (Folmeret al.1994). PCR amplication was carried out
under the following conditions: a cycle of 3 min at 94C, followed
by 35 cycles of 45 s at 94C, 60 s at 45C, and 60 s at 72C, with a
nal step of 5 min at 72C. Before sequencing, PCR products were
puried using standard procedures (Eimanifar and Wink 2013).
Sequence alignment and phylogenetic analyses
Sequences were aligned using MEGA 7.0.26 with default
parameters (Kumar et al. 2016). A lack of pseudogenes enabled
utilisation of the protein-coding sequence; additionally, multiple
mutations or deletion(s) and duplication(s) were not observed.
To estimate the phylogenetic relationship among samples
collected from Australia and other species, COI reference
sequences of bisexual species and parthenogenetic populations
including di-, tri-, tetra- and pentaploidy were downloaded from
GenBank (Table 2). The phylogenetic tree was generated using
Bayesian Inference (BI) (Huelsenbeck and Ronquist 2001), as
implemented in MrBayes 3.2.2 on XSEDE (Miller et al.2010).
For BI the best-tting nucleotide substitution model was
calculated based on MrModelltest 2.2 (Nylander 2004) and HKY
+G was chosen as the best-t model. Additionally, for posterior
probabilities, the values <0.94 and 0.95 were considered to be
low and high, respectively (Alfaro et al.2003).
To nd the origin and genealogical relationships among
haplotypes of Australian samples and A. franciscana (more
information in Results), a median network was performed using
the median-joining algorithm in Network 5.0.0.3 (Bandelt et al.
1999). The sequences of A. franciscana were chosen from
two natural habitats in the USA: GSL and SFB (Table S1,
Supplementary Material).
For each population,the number of polymorphic sites (S), total
number of mutations (Eta), number of haplotypes (h), haplotype
diversity (Hd), haplotype ratio (Hr), nucleotide diversity (Pi) and
average number of nucleotide differences (k) were calculated
using DnaSP 5.10 (Librado and Rozas 2009). Expected
heterozygosity, F
ST
(an overall population differentiation index),
mismatch distribution, Harpendings Raggedness index (Hri) and
sum of squared deviations (SSD) were computed in Arlequin 3.5
(Excofer and Lischer 2010).
Results
All COI sequences of A. franciscana from Australia had 18
variable sites, of which four sites were parsimony informative
and 14 sites were singletons. The total COI sequences of native
American A. franciscana displayed 12 variable sites, of which
ve sites were parsimony informative and seven sites were
singletons.
308 Australian Journal of Zoology A. Asem et al.
(a)
(b)(c)
(d)(e)
Fig. 1. (a) Map of Artemia sampling sites in Australia, (b) DAM: Dampier, (c) HED: Port Hedland, (d) SKI:
St Kilda, (e) MUL: Mulgundawa. Map data Google 2018.
Phylogeography of invasive Artemia in Australia Australian Journal of Zoology 309
The phylogenetic tree revealed that all examined Artemia
individuals from Australia clustered in the clade of
A. franciscana (Fig. 2). The haplotype distribution network
analysis of the A. franciscana complex was performed to
determine the population structure of individuals, but a
geographically unique haplotype could not be distinguished
(Fig. 3). Most sequences belonged to H1 (31.3%) and H2
(28.5%). The haplotype frequency of A. franciscana from SFB
for H1, H2 and H3 haplotypes were 70.3%, 16.2% and 10.8%,
respectively. A. franciscana from GSL grouped in the H2 and H4
haplotypes with frequencies of 62.3% and 26.2%, respectively.
Two localities from Australia had the greatest H1 haplotype
frequency: HED (80.96%: 17 individuals out of 21) and SKI
(70%: 7 individuals out of 10). There were seven haplotypes
around H1 with a frequency of 1.0 (Fig. 3). All A. franciscana
sequences from the DAM locality were recovered in H3. The
MUL locality consisted of 73.1% H3 and 11.5% H2 haplotype
frequencies (Fig. 3, Tables S2, S3 in the Supplementary
Material).
The population differentiation test (F
ST
) among the
examined populations suggested that there is no signicant
differentiation between DAM and MUL (4.8%), SKI and HED
(1.3%) and SKI and SFB localities (7.8%), respectively.
A signicant population differentiation was observed between
the GSL and SFB localities (60.9%). The highest variation was
revealed between DAM and SKI (90.9%) and DAM and HED
(91.9%), and the lowest was observed between HED and SFB
(10.3%), respectively (Table 3).
All estimated genetic indices for the examined localities are
summarised in Table 4. The lowest genetic variation was
observed in the DAM location, which had only a single
haplotype. Two localities, SKI and MUL, exhibited the highest
genetic variation. The highest-ranking levels of Hd (0.533
0.180) and Hr (0.4) were found in SKI, whereas the MUL locality
had the highest values for Pi (0.00349 0.0023), k (1.554) and
H
exp
(0.0013 0.016) (Table 4). Between the native American
populations, the highest values for Hd (0.550 0.058) and Hr
(0.15) were found in GSL, whereas the other genetic indices were
highest in the SFB locality. In total, among all American and
Australian populations, the highest values of Pi (0.00349
0.0023), expected heterozygosity (0.0013 0.016) and k (1.554)
were recorded in MUL. The highest values for Hr (0.4) and Hd
(0.550 0.058) were observed in SKI and GSL, respectively
(Table 4).
We calculated mismatch distributions for pairwise
differences from exotic and native populations of A. franciscana.
These revealed that the SKI, HED and GSL localities had a
unimodal pattern, whereas MUL and SFB localities showed a
pattern likely to be multimodal. Additionally, the indices of SSD
and Hri for all examined localities were non-signicant, except
Table 1. Origin of Artemia samples used for this study
IPMB, Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Germany; WA, Western Australia; SA, South Australia
Site Abbreviation Geographic coordinates IPMB voucher
no.
No. of
individuals
Reference
Dampier, WA DAM 2042019.3800S, 1164202.0900E 66843 10 Ruebhart et al.(2008); McMaster et al.(2007)
Mulgundawa, SA MUL 3517039.8400S, 13912037.7600 E 66844 26 This study
Port Hedland, WA HED 2020026.5700S, 11839020.9800E 77593 21 Ruebhart et al.(2008)
St Kilda, SA SKI 3444009.4900S, 13832020.3400 E 66849 10 This study
Table 2. Species information and GenBank accession numbers
Pop., population
Species/population Abbreviation No. of individuals Accession nos Reference
A. urmiana URM 4 JX512748751 Eimanifar and Wink (2013)
A. sinica SIN 4 KF691298301 Eimanifar et al.(2014)
A. tibetiana TIB 4 KF691215218 Eimanifar et al.(2014)
A. salina SAL 4 KF691512515 Eimanifar et al.(2014)
A. persimilis PER 4 DQ119647 Hou et al.(2006)
HM998992 Maniatsi et al.(2011)
EF615594 Wang et al.(2008)
EF615593 Wang et al.(2008)
A. franciscana FRA 4 KJ863440443 Eimanifar et al.(2014)
Diploid Pop. DI 4 KU183949952 Asem et al.(2016)
Triploid Pop. TRE 3 HM998997999 Maniatsi et al.(2011)
Tetraploid Pop. TETR 4 KU183954957 Asem et al.(2016)
Pentaploid Pop. PEN 4 KU183968971 Asem et al.(2016)
Unidentied
A
DAM 10 MK613273282 This study
MUL
B
26 MK613283308 This study
HED 21 MK613309329 This study
SKI
B
10 MK613330339 This study
A
Australian samples
B
New record site.
310 Australian Journal of Zoology A. Asem et al.
for MUL, where we observed a signicant SSD value (P<0.001)
(Fig. 4).
Discussion
The occurrence of American A. franciscana in Australia had been
suggested for three geographical regions (11 localities) in
Australia, including western (seven sites), southern (one site) and
Queensland (three sites) (Ruebhart et al.2008). Our ndings
conrmed the colonisation of Australia with A. franciscana.
Previously, parthenogenetic populations have been reported
from two localities in Australia Port Hedland and Dry Creek
(Van Stappen 2002)but our ndings could not support the
existence of parthenogenetic populations in these localities. In
the present study, we have documented the rst scientic record
of Artemia in Mulgundawa and St Kilda localities in southern
Australia, which are clearly colonised by A. franciscana. Brine
shrimps from GSL and SFB have often been introduced to other
regions of the world for aquaculture production of Artemia cysts
and biomass (Sorgeloos et al.2001; Amat et al.2005; Eimanifar
et al.2014; Muñoz et al.2014; Saji et al.2019) therefore these
populations were included in this analysis.
Mitochondrial markers reect the maternal evolutionary
pathway and are important for understanding the passageways
0.04
1
0.99
0.97
0.95
10.96
0.99
1
1
Diploid P.P.
URM
Triploid P.P.
TIB
SIN
Tetraploid P.P.
Pentaploid P.P.
FRA
MU MUDAM HED SKI
Daphnia
SAL
PER
Fig. 2. COI phylogeny of Artemia based on a Bayesian inference approach. The numbers behind major nodes denote posterior
probabilities. Daphnia tenebrosa (HQ972028) was used as an outgroup. Red dots show the position of reference sequences of
A. franciscana. P.P., parthenogenetic population; URM, Artemia urmiana; TIB, Artemia tibetiana; SIN, Artemia sinica; FRA,
Artemia franciscana; PER, Artemia persimilis; SAL, Artemia salina; DAM, Dampier; MUL, Mulgundawa; HED, Port Hedland; SKI,
St Kilda.
Phylogeography of invasive Artemia in Australia Australian Journal of Zoology 311
and elucidating the source of invasive species in non-indigenous
habitats (Ashton et al.2008; Ficetola et al.2008; Mabuchi et al.
2008; Gaubert et al.2009). The genetic structure of
mitochondrial COI in the Mediterranean Artemia populations
have clearly documented an invasion of A. franciscana from the
GSL and SFB (Muñoz et al.2014; Horvath et al.2018).
Phylogeographical analysis of Asian populations has
documented colonisation by A. franciscana from multiple
origins in both America and Europe (Eimanifar et al.2014).
Haplotype distribution revealed A. franciscana in Al Wathba
Wetland Reserve (United Arab Republic; Abu Dhabi), likely
originating from the GSL (Saji et al.2019). Our results strongly
support the idea that A. franciscana found in the HED and SKI
H17
H16
H15
H18
MUL
SKI
DAM
HED
SFB
GSL
H19
H20
H21
H22
H23 H13
H12 H11
H10
H9
H8
H2
H3
H14 H4
H6
H7
[3]
[3]
H1
H5
Fig. 3. The relationship of COI haplotype distribution among Artemia franciscana individuals from Great Salt
Lake (GSL), San Francisco Bay (SFB) and Australian populations (MUL, Mulgundawa; SKI, St Kilda; HED, Port
Hedland; DAM, Dampier).
Table 3. Pairwise population matrix of F
ST
values from COI loci
Results are shown as percentages. ns, non-signicant; *, P<0.05; **,
P<0.001
Site DAM MUL ADE HED GSL
MUL 4.8
ns
SKI 90.9** 63.1**
HED 91.9** 68.6** 1.3
ns
GSL 84.1** 68.1** 75.7** 77.5**
SFB 69.7** 52.2** 7.8
ns
10.3** 60.9**
Table 4. Population genetic indices for Australian and native American A. franciscana based on COI loci
N, no. of sequences; S, no. of polymorphic (segregating) sites; Eta, total no. of mutations; h, no. of haplotypes;
Hd, haplotype (gene) diversity; Hr, no. of haplotypes/no. of sequences; Pi, nucleotide diversity; k, average no.
of nucleotide differences; Exp. Het., expected heterozygosity
Genetic indices DAM MUL SKI HED GSL SFB
N102610216137
S0103485
Eta 0 10 3 4 8 5
h164594
Hd
(s.d.)
0 0.465
(0.116)
0.533
(0.180)
0.352
(0.131)
0.550
(0.058)
0.480
(0.087)
Hr 0.1 0.23 0.4 0.24 0.15 0.11
Pi
(s.d.)
0 0.00349
(0.0023)
0.00135
(0.00131)
0.00085
(0.0009)
0.0015
(0.0012)
0.0025
(0.0018)
k 0 1.554 0.600 0.381 0.666 1.135
Exp. Het.
(s.d.)
0 0.0034
(0.029)
0.0013
(0.016)
0.0008
(0.008)
0.0014
(0.021)
0.0025
(0.026)
312 Australian Journal of Zoology A. Asem et al.
localities is derived from commercialised SFB populations in the
USA, based on similar haplotypes (Fig. 3). These two groups also
possessed the lowest values for the population differentiation
index (F
ST
) between SFB and HED/SKI (Table 3).
Interestingly, we found a single haplotype (H3) connected to
the main haplotype H2 from GSL, consisting of SFB (12.12%),
DAM (30.03%) and MUL (57.58%) localities. All sequences of
DAM and 73.06% of MUL individuals belonged to this
haplogroup, while it held only 10.82% of total SFB sequences.
Generally, the observed haplotype pattern of DAM could suggest
an origin in the SFB, while MUL possessed an intermediate
structure between GSL and SFB (Fig. 3, Tables S2, S3,
Supplementary Material). In contrast, the high values of F
ST
among GSL/SFB and DAM/MUL, and in particular between
SFB and DAM (69.7%) are considerable and cannot corroborate
the suggested network distribution (Table 3). Ordinarily,
introduction from the multiple sources (both GSL and SFB)
could explain this observation. As an alternative hypothesis, the
origin of MUL may be secondary introduction from other
sources, in particular eastern Asia, where A. franciscana cysts
from the Mekong Delta (Vietnam) and Bohai Bay (China) are
easily obtainable in aquaculture markets (Van Stappen et al.
2007; Muñoz et al.2014;Leet al.2018).
The F
ST
value was strongly signicant between the two
American populations, GSL and SFB (60.9%) (see Table 3). This
result is similar to previous calculations (59.3%) performed by
Muñoz et al.(2014). The pattern of haplotype frequencies
(Fig. 3), as well as the value of F
ST
, strongly suggest that there is a
high degree of genetic separation between the GSL and SFB
populations.
Typically, the invasive populations have lower genetic
variation in their non-native locations compared with the original
population (Golani et al.2007). Reduction of haplotype variation
and low intraspecic genetic differentiation has also been
0
350
300
250
200
150
100
50
0
25
20
15
10
5
0
0123
MUL SKI
HED
GSL SFB
SSD: 0.283, P = 0.000
Hri: 0.29, P = 0954
SSD: 0.017, P = 0.463
Hri: 0.164, P = 0.703
SSD: 0.002, P = 0.625
Hri: 0.192, P = 0.500
SSD: 0.074, P = 0.164
Hri: 0.308, P = 0.253
SSD: 0.009, P = 0.098
Hri: 0.124, P = 0.109
4
160
140
120
100
80
60
40
20
0
900
800
700
600
500
400
300
200
100
0
400
350
300
250
200
150
100
50
0
5678
0
01
Observed Simulated
23 4 0 1 2 3 4 5
12 3
90123
Observed Simulated
Observed Simulated
Observed Simulated Observed Simulated
Fig. 4. Observed mismatch distributions and their curve t to simulated models of demographic expansion. MUL, Mulgundawa;
SKI, St Kilda; HED, Port Hedland; DAM, Dampier; GSL, Great Salt Lake; SFB, San Francisco Bay.
Phylogeography of invasive Artemia in Australia Australian Journal of Zoology 313
observed for introduced A. franciscana in Vinh Chau (Vietnam)
as compared with its source population from SFB, likely due to
founder effects (Kappas et al. 2009). In contrast, Eimanifar et al.
(2014) showed that the genetic diversity of invasive Asian
A. franciscana is higher than in GSL and native Asian species.
Similar results have been recorded in some invasive
Mediterranean populations (Hontoria et al.2012; Muñoz et al.
2014). Our results indicate that DAM is a static population with
no genetic variation, which may be the result of a founder effect
and population bottleneck during the process of colonisation. In
general, MUL had the highest genetic diversity among the
populations examined in this work. This nding might be due to
multiple introductions by human-mediated dispersal events or
secondary introductions, although, higher genetic diversity can
be the result of adaptive pressure and/or physiological plasticity
of the exotic population in a non-native region (Dlugosch and
Parker 2008; Ruebhart et al.2008; Vikas et al.2012; Muñoz et al.
2014; Eimanifar et al.2014). We propose that environmental
conditions in new habitats could also have exerted selective
pressure during development of the invasive population.
Two locations from Australia (SKI and HED) and GSL
showed a unimodal structure of mismatch distribution, as well as
a low and non-signicant value of SSD and Hri, which indicate a
recent demographic expansion. These ndings suggest high
adaptive potential and physiological plasticity of exotic
Australian populations in the new habitats. The demographic
history of MUL and SFB presented a complex structure indicated
by multimodal patterns of mismatch distribution due to
demographic equilibrium. A signicant value of SSD (P<0.001)
in MUL conrmed these results, but a non-signicant value of
SSD in SFB and a non-signicant value of Hri in both
populations highlights the existence of demographic expansion.
The results of this study indicate that the native American and
Australian A. franciscana populations have undergone some
degree of demographic expansion, with the exception of the
DAM population.
In conclusion, our results verify the previous observations of
the colonisation of Australia by invasive A. franciscana
populations. Yet these populations harbour higher levels of
genetic variation than the native America population, in contrast
to other previously studied taxa (see Golani et al.2007).
A. franciscana possesses a faster lter-feeding rate and higher
reproductive rate than the native species (Amat et al.2007;
Sanchez et al.2016). It is also immune to the reduced feeding rate
caused by cestode parasites, contrary to the native populations
(Sanchez et al.2016). These characteristics provide a high
adaptive potential for A. franciscana in new non-indigenous
regions, ultimately resulting in replacement of native species.
Although there are many more populations of A. franciscana in
America, the ndings conrmed that Great Salt Lake, Utah, and/
or San Francisco Bay, California, should be the most likely
source for all the current invasion populations outside America.
The utilisation of Artemia in aquaculture production without
regard to potential environmental hazards threatens the
biodiversity of Artemia worldwide.
Conicts of interest
The authors declare no conicts of interest.
Acknowledgements
This work was nancially supported by the German Academic Exchange
Service (Grant No. A/10/97179). The authors thank Mark Coleman and Brian
Timms for collecting Artemia cyst specimens from Australia.
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Handling Editor: Steven Cooper
316 Australian Journal of Zoology A. Asem et al.
www.publish.csiro.au/journals/ajz
... 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). ...
... Our results also confirmed the colonization of the same species in GL. SFB and GSL are the two major sources of Artemia that are usually used to culture in saline ecosystems for industrial aquaculture and fishery activities to produce Artemia cysts and biomass (Eimanifar et al., 2014;Muñoz et al., 2014;Asem et al., 2018;Saji et al., 2019). Thus, these populations were considered in this analysis to determine the genetic alterations of colonized populations in new nonnative environments. ...
... Mitochondrial DNA presents some exceptional characteristics, including rapid evolutionary rates, maternal origin, and lack of recombination (Boore, 1999;Miller et al., 2009). Therefore, mitochondrial markers are important for the apprehension of tracing and the explanation of the source of non-indigenous species in new habitats (Ashton et al., 2008;Ficetola et al., 2008;Mabuchi et al., 2008;Gaubert et al., 2009;Asem et al., 2018;Saji et al., 2019). ...
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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.
... The genus Artemia comprises seven (Rogers 2013) or possibly nine (Naganawa and Mura 2017) species. Although Artemia is widespread and common in hypersaline lakes in most continents, only Artemia franciscana Kellogg and Artemia parthenogenetica Bowen & Sterling are present in Australia (McMaster et al. 2007;Asem et al. 2018). A. franciscana was introduced to Australia by humans to aid in salt production and is overwhelmingly restricted to constructed evaporative ponds (salt works; Timms and Hudson 2009;Asem et al. 2018). ...
... Although Artemia is widespread and common in hypersaline lakes in most continents, only Artemia franciscana Kellogg and Artemia parthenogenetica Bowen & Sterling are present in Australia (McMaster et al. 2007;Asem et al. 2018). A. franciscana was introduced to Australia by humans to aid in salt production and is overwhelmingly restricted to constructed evaporative ponds (salt works; Timms and Hudson 2009;Asem et al. 2018). A. parthenogenetica may have also been introduced into salt works by humans (McMaster et al. 2007), but its presence in a range of lakes in south-western Australia could be the result of intercontinental bird-mediated dispersal followed by local dispersal (McMaster et al. 2007). ...
... All Artemia species, including the two species found in Australia, have high dispersal capacity because their cysts float and are effectively transported by animals and wind (Timms and Hudson 2009). Despite this, A. franciscana does not seem to be spreading (Timms and Hudson 2009;Asem et al. 2018), but the distribution of A. parthenogenetica is increasing in southwestern Australia, where it is mostly colonising degraded salt lakes (McMaster et al. 2007). It is unclear how the further spread of A. parthenogenetica may affect Parartemia species, but currently A. parthenogenetica appears limited to lakes not already occupied by Parartemia (McMaster et al. 2007). ...
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... 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). ...
... Our results also confirmed the colonization of the same species in GL. SFB and GSL are the two major sources of Artemia that are usually used to culture in saline ecosystems for industrial aquaculture and fishery activities to produce Artemia cysts and biomass (Eimanifar et al., 2014;Muñoz et al., 2014;Asem et al., 2018;Saji et al., 2019). Thus, these populations were considered in this analysis to determine the genetic alterations of colonized populations in new nonnative environments. ...
... Mitochondrial DNA presents some exceptional characteristics, including rapid evolutionary rates, maternal origin, and lack of recombination (Boore, 1999;Miller et al., 2009). Therefore, mitochondrial markers are important for the apprehension of tracing and the explanation of the source of non-indigenous species in new habitats (Ashton et al., 2008;Ficetola et al., 2008;Mabuchi et al., 2008;Gaubert et al., 2009;Asem et al., 2018;Saji et al., 2019). ...
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Full-text available
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.
... These findings have evidenced that these populations were at demographic equilibrium. In contrast, Asem et al. 21 found that GSL represented a unimodal mismatch distribution and have recorded this population was under demographic expansion. This difference can be attributed using sequences in different period from GSL. Asem et al. 56 have proved ecological variation could alter genetic structure of Artemia from Urmia Lake. ...
... Our results have also documented the colonization of same species in Godolphin Lakes locality.The San Francisco Bay (SFB) and Great Salt Lake (GSL) are the two main sources of Artemia that have usually been used to culture in other saline ecosystems for industrial aquaculture and fishery activates to produce Artemia cysts and biomass13,20,21,34 , for this reason these populations were considered in this analysis to find out the genetic alterations of the colonized populations in new non-native environments.Mitochondrial DNA represented some exceptional characteristics consisting rapid evolutionary rates, maternal origin, and lack of recombination35,36 . Then mitochondrial markers are important for apprehension the tracing and explanation the source of non-indigenous species in new habitats 20,21,37-40 . ...
... . The paradox between our result and previous study on Al Wathba Wetland Reserve can be referred to technical error using short sequences of COI in the previous study (446 bp vs 604 bp). Regarding to results of the current study, the geographical origin of both UAE populations might be secondary introduction from other Artemia production sources, especially Eastern Asia including Mekong Delta (Vietnam) and Bohai Bay (China) where these are commercially available in aquaculture markets21,34,41,42 .Golani et al.43 showed that the invasive populations generally possess lower genetic variation in non-indigenous new environments in comparison with the source populations. An introduced population of A. franciscana in Vinh Chau (Vietnam) has displayed low intraspecific genetic variation and reduced haplotype diversity as compared with its original population from SFB44 . ...
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Artemia franciscana, native to America, has recently colonized non-indigenous populations in Eurasia, Mediterranean regions and Australia. In present we sought to evaluate the potential effects of colonization of A. franciscana on genetic differentiation in the new environments in UAE. We used the COI marker to determine population genetic structure and identify the origins of exotic populations in UAE. Our findings have confirmed the colonization of both localities by A. franciscana. Genetic variation of invasive A. franciscana were exclusively lower than native population in Great Salt Lake and San Francisco Bay. Results have showed the studied population could not possibly have colonized directly from natural American localities, perhaps resulting from secondary introduction events from other non-indigenous populations. Genetic analysis have yielded different demographic patterns for invasive studied populations. Al Wathba Wetland Reserve (AWWR) population have represented demographic expansion. In contrast, Godolphin Lakes (GL) population was at demographic equilibrium. Neutrality tests have documented the excess of both recent and historical mutations in the COI gene pool of invasive AWWR Artemia throughout establishment in the new environment.
... 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][16][17][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. ...
... 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 afford 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. ...
... Previous studies on A. franciscana have documented that invasive populations demonstrated genetic variations relative to the native American source populations [2,17,18,[22][23][24]. The low genetic diversity in the non-indigenous populations has been attributed to the founder effect [22] or population bottleneck due to the decreasing of population size in introduced populations during the process of establishment [17]. ...
Article
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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.
... Eventually, this had led to the invasion of A. franciscana into various countries (Muñoz et al., 2009;Ruebhart et al., 2008) including in Asia, Africa, and Europe, resulting in the elimination of native Artemia species (Ben Naceur et al., 2010;Scalone and Rabet, 2013;Muñoz et al., 2014;Amat et al., 2007). With the evidence of morphology, morphometric and a mitochondrial DNA (COXI) marker, recent studies confirm the invasion of A. franciscana in Egyptian and Australian hypersaline habitats (Sheir et al., 2018;Asem et al., 2020). Similarly, invasion of A. franciscana and replacement of the native population of Artemia parthenogenetica was confirmed in Indian salterns by using ITS-I gene sequences (Vikas et al., 2012). ...
... KBM haplotypes never shared their genetic character with other sampling sites while VDM, TUT, and NGC showed strong genetic dispersal between population. This observation can be supported by an earlier report on invasion and occurrence of A. franciscana from Australian salterns demonstrated by the COX1 gene (Hd = 0.533) (Asem et al., 2020). The deep-sea decapod crustacean species Aristeus antennatus also showed a high level of haplotype diversity based on the mtDNA control region analysis (H = 0.884 to 0.989) (Maggio et al., 2009;Eimanifar et al., 2014). ...
Article
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The invader brine shrimp Artemia franciscana is a micro-crustacean with diverse morphology and morphometric traits that are found in hypersaline habitats of Asian countries. In this study, we used the mtDNA control region to assess the impact of colonization of A. franciscana on genetic diversity and population structure encountered in different sampling sites of the southeast coast of India. Samples were collected from four hypersaline habitats includes Kelambakkam (KBM); Vedaranyam (VDM); Tuticorin (TUT) and Nagarcoil (NGC). Genetic diversity of the populations were assessed based on the sequence of a conserved region of the mtDNA control region (CR). For this study, a primer based on the conserved region of a closely related crustacean group was used to sequence the 493 bp of the mtDNA CR. Results indicated a clear demarcation with the existence of sequence divergence between the populations studied. Maximum likelihood and Bayesian phylogenetic studies revealed that the population of TUT, VDM and NGC formed a single cluster whereas the KBM formed a separate cluster with distinct genetic characteristics. The mean haplotype diversity and nucleotide diversity of the four populations were found to be 0.742 ± 0.060 and 0.012 ± 0.016, respectively. Interestingly, we observed that these different populations had unique haplotypes varying with their habitats, demonstrating sequence divergence in the homogenous population of A. franciscana despite them being in a similar geographic zone.
... Samples L. Sainz-Escudero et al. Additionally, to our data, all the available Artemia cox1 sequences available in GenBank (Valsala et al. 2005;Hou et al. 2006;Tizol-Correa et al. 2009;Muñoz et al. 2008Muñoz et al. , 2010Muñoz et al. , 2013Maniatsi et al. 2009Maniatsi et al. , 2011Maccari et al. 2013b;Eimanifar and Wink 2013;Eimanifar et al. 2014Eimanifar et al. , 2015Eimanifar et al. , 2016Asem et al. 2016Asem et al. , 2019Asem et al. , 2020Naganawa and Mura 2017;Horváth et al. 2018) and one of Branchinecta ferox used as outgroup (LT821334 [Rodríguez-Flores et al. 2017]) were retrieved in order to build a dataset represented by 1505 sequences, that allowed us to depict the structuring of the genus through the Neighbour Joining analysis and to perform phylogeographic analyses. Some dissimilar sequences that featured stop codons when traduced to amynoacids were removed from the analyses due to the existence of pseudogenes according to Rode et al. (2021). ...
Article
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Brine shrimps ( Artemia ) have undergone geographic range and demographic expansions as a result of their interaction with humans since the beginning of salt harvesting. This interaction has favoured the expansion of some species but compromising the survival of others. Mediterranean native populations of Artemia salina from coastal salterns and lagoons are facing the presence and expansion of the introduced and invasive American species Artemia monica (= A . franciscana ). However, this species could not be the only threat. Parthenogenetic populations of the Asian species A . urmiana and A . sinica are widespread along the Mediterranean and other areas of the world. In this work, with the use of large cox1 and mitogenomic datasets, phylogenetic and phylogeographic inferences, and a time calibrated tree, we confirmed the Asian origin and recent arrival of the current Western Mediterranean parthenogenetic populations of Artemia . In addition, the replacement of Iberian populations of A . salina by Asiatic parthenogenetic populations lead us to recognize parthenogens as invasive. Current salterns development and commercial importance of Artemia make human-mediated introduction probable. These results demonstrate again the impact that changing human interests have on population expansion or decline of species adapted to anthropogenic habitats. Artemia salina decline makes urgent the implementation of conservation measures such as its use in fish farming and salt production or its inoculation in inland salterns.
... The mitochondrial markers, COX1 and 16S, have been successfully used in phylogeny of branchiopods [60][61][62][63][64][65]. To date only COX1 has been utilized for phylogenetic studies on Artemia, nevertheless mitogenomic results demonstrated significant difference in the nucleotide composition of ATP8, ATP6, ND3, ND6, ND1 and COX3. ...
Article
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 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.
... Moreover, specific genetic patterns were confirmed using p26 gene, which revealed that changes in the genetic patterns can alter the morphology (Maniatsi et al., 2011). Similarly, recent studies have also shown that A. franciscana invasion was documented with exact morphology and genetic evidence [using mitochondrial cytochrome oxidase subunit I (COX1)] in Egyptian and Australian salterns (Sheir et al., 2018;Asem et al., 2020). ...
Article
The brine shrimp Artemia franciscana consists of a complex of phenotypes within a population confined to various salterns in southeast coast of India. Studies are scarce for their occurrence based on morphology and morphometric traits, while seldom any on their genetic variability. Therefore, the present study investigates the genetic variability among the populations of Artemia franciscana samples collected from four different hypersaline habitats Kelambakkam (KBM), Vedaranyam (VDM), Tuticorin (TCN) and Nagarcoil (NGC) from southeast coast of India. Since the genetic variability could be anywhere in their genomes the study was analyzed using two different Random Amplified Polymorphic DNA (RAPD) markers such as (i) the Operon series (OPK) and (ii) Enterobacterial Repetitive Intergenic Consensus (ERIC). The OPK primers generated 367 fragments which indicated polymorphism of 85.29%, Nei's diversity index of 0.25 ± 0.16 and Shannon's index as 0.39 ± 0.22 among the population. Furthermore, ERIC-PCR generated 138 fragments with 20.83% of polymorphism while the Nei's diversity and Shannon's index was 0.06 ± 0.13 and 0.10 ± 0.20 respectively. Interestingly, operon primers of OPK3 and OPK17 revealed the presence of genetic variations among the populations, and the results were consistent with ERIC markers. Scatter plot and cluster analyses revealed the existence of two major groups in the population collected from the study sites; one with KBM and VDM, while other with TUT and NGC. Hence, the present investigation attributes the genetic variations in a panmictic population of A. franciscana which exist as two distinct groups.
... The mitochondrial markers, COX1 and 16S, have been successfully used in phylogeny of branchiopods [60][61][62][63][64][65]. To date only COX1 has been utilized for phylogenetic studies on Artemia, nevertheless mitogenomic results demonstrated significant difference in the nucleotide composition of ATP8, ATP6, ND3, ND6, ND1 and COX3. ...
Article
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.
Article
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We present the latest version of the Molecular Evolutionary Genetics Analysis (MEGA) software, which contains many sophisticated methods and tools for phylogenomics and phylomedicine. In this major upgrade, MEGA has been optimized for use on 64-bit computing systems for analyzing bigger datasets. Researchers can now explore and analyze tens of thousands of sequences in MEGA. The new version also provides an advanced wizard for building timetrees and includes a new functionality to automatically predict gene duplication events in gene family trees. The 64-bit MEGA is made available in two interfaces: graphical and command line. The graphical user interface (GUI) is a native Microsoft Windows application that can also be used on Mac OSX. The command line MEGA is available as native applications for Windows, Linux, and Mac OSX. They are intended for use in high-throughput and scripted analysis. Both versions are available from www.megasoftware.net free of charge.
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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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In the last 30 years since its first appearance in Portugal, the North-American Artemia franciscana has successfully invaded hypersaline habitats in several Mediterranean countries. Here, we review its spread in the Mediterranean Basin since its first occurrence in the 1980s and report its first occurrence in Croatia, based on both morphological identification (adults) and genetic evidence (cysts). The haplotypes we found in the population from this new locality (two of which were new to both the native and invaded ranges of A. franciscana) suggest either direct or secondary introduction from the main harvested cyst sources (Great Salt Lake or San Francisco Bay, USA) and indicate that some genetic native diversity in the species has not yet been captured by existing studies. Our finding means that the species has reached the eastern shores of the Adriatic Sea and therefore is now present on the Balkan Peninsula. We detected that its eastward spread is still continuing, posing a fundamental threat to remaining populations of native Artemia species in Eastern Europe, which highlights the need for preventive measures.
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The shortfin silverside (Chirostoma humboldtianum) is a native fish of central Mexico with high value for artisanal fisheries. So far, attempts aimed to establish intensive culturing have failed. In this study, we evaluated the effect of probiotic strains; Bifidobacterium animalis subsp. lactis BB-12, Lactobacillus johnsonii C4, and Bacillus sp. B2 bio-encapsulated into Artemia franciscana on Chirostoma humboldtianum weight and length. Their influence on the fish intestinal bacterial communities was also assessed. The final weight and final length of the fishes fed with bio-encapsulated Bifidobacterium animalis BB-12, and L. johnsonii C4 were statistically different and higher than the control group. According to PCR-DGGE fingerprints of 16S rRNA gene, the intestinal content bacterial community associated with the shortfin silverside seems to be molded in early larval stages and only slight changes could be induced by the use of bio-encapsulated bacterial. An increase in fish survival rate and an improvement in weight and length were detected using L. johnsonii C4 bio-encapsulated into A. franciscana, in spite of its small impact on the structure of the bacterial community associated with the intestinal content of shortfin silverside. The use of L. johnsonii C4 bio-encapsulated into A. franciscana could be an excellent option to improve the yield during intensive culturing of the shortfin silverside.
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Using two nuclear (ITS1 and Na+/K+ ATPase) and three mitochondrial (COI, 16S and 12S) markers, we determined the genetic variation and evolutionary relationship of parthenogenetic and bisexual Artemia. Our analyses revealed that mitochondrial genes had higher genetic variation than nuclear genes and that the 16S showed more variety than the other mitochondrial genes in parthenogenetic populations. Triploid parthenogens showed lower genetic variation than diploid ones, whereas the tetra- and pentaploids had greater genetic distance than diploid parthenogens. No shared haplotype was found between individuals of parthenogenetic populations and Asian bisexual species with the exception of Na+/K+ ATPase (Artemia tibetiana). Only mitochondrial markers can demonstrate phylogenetic relationships, and showed that the parthenogenetic Artemia is a polyphyletic group in which the diploid lineages share a common ancestor with Artemia urmiana while tetraploids are closely related to Artemia sinica. The triploid and pentaploid linages are likely to be directly derived from diploid and tetraploid parthenogens, respectively. Subsequently, west Asia is origin for di-/triploids, and tetra-/pentaploids rose from East Asia.
Article
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.
Article
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.
Thesis
The brine shrimp Artemia is a small crustacean occurring worldwide in hypersaline biotopes. Its cysts, produced in stressful environmental conditions, can be stored for several years; the emerging larva is a convenient substitute for the natural plankton diet of fish and shrimp larvae and is an indispensable live food item in marine finfish and shellfish hatchery operations worldwide, thanks to its availability, nutritional quality, and easy use. According to present knowledge the genus Artemia groups a few bisexual species and numerous parthenogenetic forms. This research work contributes to the knowledge of the biodiversity of the genus Artemia in Central and Eastern Asia. Firstly the existing literature on Artemia sites in this area is reviewed, with focus on Southwest Siberia (Russia) and the Qinghai-Tibet Plateau (P.R. China), and anthropogenic threats to Artemia biodiversity in the area are sketched. This work further reports on a field survey conducted in salt lakes in Southwest Siberia and presents data on their topography, abiotic conditions, primary production and Artemia population dynamics. Data are presented on a study of Artemia samples from the Qinghai-Tibet Plateau. Laboratory culture tests showed these samples to contain mixtures of parthenogenetic and bisexual individuals. PCR-RFLP analysis of individual cysts using a 1500 bp mtDNA I fragment and digestion with four restriction endonucleases revealed that the 13 populations analysed could be classified in three distinct groups, each with its characteristic set of haplotypes. This work also presents a study assessing how the original parthenogenetic populations from the Bohai Bay, China, an area with intensive aquaculture activities, has evolved since 1989, using PCR-RFLP analysis These findings are discussed in the light of similar observations elsewhere in the world, and of possible bioconservation measures. Next this work describes tests to assess the usefulness of the PCR-DGGE technique as a tool for rapid and dependable screening of the species/strain composition of an Artemia sample, by analysing both artificial and natural samples of mixed species status. Finally, the overall results of this work are discussed in the framework of its objectives, and in the light of natural or man-generated heterogeneity of Artemia samples, and its repercussions for data interpretation. The importance of the isolation of representative biological study material from nature is emphasized, and the possible implications of analysing cysts or active life stages.