The impact of one-decade ecological disturbance on genetic changes: a study on the brine shrimp Artemia urmiana from Urmia Lake, Iran

Abstract

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.

Full text

Submitted 23 December 2018
Accepted 24 May 2019
Published 2 July 2019
Corresponding author
Shi-Chun Sun, sunsc@ouc.edu.cn
Academic editor
Scott Edwards
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Declarations can be found on
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DOI 10.7717/peerj.7190
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2019 Asem et al.
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OPEN ACCESS
The impact of one-decade ecological
disturbance on genetic changes: a study
on the brine shrimp Artemia urmiana
from Urmia Lake, Iran
Alireza Asem1,2, Amin Eimanifar3, Gilbert van Stappen4and Shi-Chun Sun1
1Institute of Evolution and Marine Biodiversity, Ocean University of China, Qingdao, China
2College of Life Sciences and Ecology, Hainan Tropical Ocean University, Sanya, China
3Easton, MD, USA
4Laboratory of Aquaculture and Artemia Reference Center, Faculty of Bioscience Engineering, Ghent
University, Gent, Belgium
ABSTRACT
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.
Subjects Biodiversity, Ecology
Keywords Climate change, Urmia lake, Artemia, Genetic variation, Historical mutations, Recent
mutations, Demographic history
INTRODUCTION
Urmia Lake (37◦420N, 45◦190E) is a landlocked thalassohaline lake with oligotrophic
characteristics located in Northwest Iran. Its historical water surface area has ranged from
4,750 to 6,100 km2with the average and greatest recorded depths being 6 and 16 m,
respectively (Azari Takami, 1993;Van Stappen, Fayazi & Sorgeloos, 2001). It is among the
largest hypersaline lakes in the world, like Great Salt Lake, USA, which has an average
How to cite this article Asem A, Eimanifar A, van Stappen G, Sun S-C. 2019. The impact of one-decade ecological disturbance on genetic
changes: a study on the brine shrimp Artemia urmiana from Urmia Lake, Iran. PeerJ 7:e7190 http://doi.org/10.7717/peerj.7190

surface area from 4,400 to 8,500 km2(Abatzopoulos et al., 2006;USGS, 2013), and it is
inhabited by the brine shrimp Artemia urmiana Günther, 1899.
Contrary to widespread opinions, Urmia Lake and its adjacent wetlands are the habitat
of various organisms. Based on its unique biodiversity, environmental gradients, socio-
economic importance and existence of indigenous communities, Urmia Lake has been
registered as a protected area since 1967 and as a national park since 1975. Because of
its importance for migratory birds, it was also registered in the Ramsar Convention on
Wetlands as a wetland of international importance in 1975 and considered as one of the
59 biosphere reserves by UNESCO in 1976 (Eimanifar & Mohebbi, 2007;Asem et al., 2014;
Asem, Eimanifar & Sun, 2016;Asem, Eimanifar & Wink, 2016).
In recent years, many aquatic ecosystems have been subject to severe ecological changes.
These alterations are imposing a considerable threat to local human societies in general
(Biemans et al., 2011;Fernandes et al., 2011;Haddeland et al., 2014;Santos et al., 2014;
Farokhnia, 2015). A progressive drought has increased the salinity of Urmia Lake from 170
g/l in 1994–1996 to more than 350 g/l (supersaturated) (Sorgeloos, 1997;Ahmadi, 2005;
Ahmadi, 2007;Asem, Mohebbi & Ahmadi, 2012). The persistence of these conditions has
caused the lake to lose 80% of its surface area (Aghakouchak et al., 2015). The desiccation
of Urmia Lake is due to the interaction of reduced rainfall and consequent increased
evaporation from the lake, human activity (uncontrolled construction of dams and
overuse of surface water resources), and environmental mismanagement (Farajzadeh,
Fakheri Fard & Lotfi, 2014;Fathian, Morid & Kahya, 2014;Merufinia, Aram & Esmaeili,
2014;Aghakouchak et al., 2015;Jalili, Hamidi & Ghanbari, 2016;Hamzekhani, Saghafian &
Araghinejad, 2016;Shadkam et al., 2016).
Historical records document that Urmia Lake has grappled with drought crises over
centuries to the extent that locals were able to walk across the lake (about 20 km) via a
paved road (Morier, 1818;Curzon, 1892). Additionally, it was reported that due to the
lack of freshwater and food, herbivorous animals, inhabiting the islands within the lake,
deserted the islands by swimming and migrated into the surrounding mountains (Binder,
1887).
Urmia Lake had the highest water-level elevation in 1994–1996 (1277.8 m a.s.l.) over
the past six decades (from 1955 to 2015). Based on the first resource assessment of Artemia
cysts and biomass in 1994–1995, Artemia urmiana cyst production in the upper 50 cm of
the lake’s water column ranged from 4,200 to 4,500 tonnes/year (dry weight) (Sorgeloos,
1997). Analysis estimated that the cyst concentration was 399 cysts/l in that period (Asem,
Mohebbi & Ahmadi, 2012). The water level of the lake fell below the ‘‘minimum ecological
water level’’ after 2001 (1274.1 m a.s.l.; Abbaspour & Nazaridoust, 2007) (Fig. 1). Later
estimates of Artemia cyst production declined to 27 and 25 cysts/l in 2003 and 2004,
respectively, when salinity increased to saturated levels (∼300 g/l) (Ahmadi, 2005). In the
following years cyst production dropped from an estimated 11 cysts/l in 2005 to 3 cysts/l in
2007 (Ahmadi, 2007). The lake lost most of its area after 2007 and no further assessments
were performed, but some estimations indicated that the cyst concentration decreased
to below 1 cyst/l (Asem, Mohebbi & Ahmadi, 2012). No live Artemia were observed in the
main body of the lake during the summer of 2016, but did occur in the surrounding lagoons
Asem et al. (2019), PeerJ , DOI 10.7717/peerj.7190 2/20

Figure 1 Annual average water level (elevation above the sea level) of Urmia Lake during 1965–2015
(AGRW, 2016).
Full-size DOI: 10.7717/peerj.7190/fig-1
and estuaries (IARC, 2016). Now Urmia Lake has become an ecological disaster, receiving
international attention. Iran’s Department of the Environment and the United Nations
Development Programme (UNDP) ratified a project to save the lake and the surrounding
wetlands (UNDP, 2014), at an estimated restoration cost of $1.3 billion (Hecht, 2014).
Critical environmental conditions can affect biodiversity and species distribution
(Menendez et al., 2006;Barrett & Schluter, 2008;Jump, Marchant & Penuelas, 2009;
Berkhout et al., 2014). Genetic diversity plays a decisive role in evolutionary history and
future evolutionary directions of taxa (May, 1994;Forest et al., 2007;Jump, Marchant
& Penuelas, 2009). While there have been many studies that focused on the effect of
environmental changes on biodiversity, few studies have focused on intraspecific genetic
variation (Pauls et al., 2013). Li et al. (1999),Li et al. (2000) and Li et al. (2001) documented
significant genetic differentiation of wild emmer wheat (Triticum dicoccoides) in response to
ecological change. A similar pattern was observed in slender oat (Avena barbata), followed
by environmental variations (Jump, Marchant & Penuelas, 2009). A comparable pattern
of genetic variation was observed in European white birch populations (Betula pendula),
which showed different genotypes in warm and cool years (Kelly et al., 2003). An additional
example was the observed significant negative correlation between Gly-3 allele frequency
and increasing summer precipitation in pinon pine (Pinus edulis) (Mitton & Duran, 2004).
Most studies on rapid evolutionary responses focus on morphological, physiological
and nutritional variation. There are few studies that consider altered environmental
conditions, especially short-term crises, on genetic variability (Thompson, 2009). Based on
cyst collections and assessments in 1994 and 2004, Artemia has lost more than 90% of its
reproductive potential and population size, and reproduction has stopped in the main body
of Urmia Lake (Asem, Mohebbi & Ahmadi, 2012). We hypothesized that the tremendous
changes in the environmental conditions of Urmia Lake and the reduction of the Artemia
population size may have affected the genetic diversity. And, if the short-term ecological
disturbance has influence on genetic variation, this change would be able to document in
the intraspecific genomic dissimilarity. The specific objective of this study was to determine
Asem et al. (2019), PeerJ , DOI 10.7717/peerj.7190 3/20

the independent impressionability of mitochondrial and nuclear genes through ecological
crises.
MATERIALS AND METHODS
Sampling strategy and DNA extraction
To assess intraspecific variation and population structure, we examined quiescent Artemia
embryos collected in 1994 and in 2004 from Urmia Lake (Kholman-khaneh station;
45◦290E, 37◦640N). The samples were obtained from the upper 50 cm of the water column
(see Sorgeloos, 1997). Because the bisexual A. urmiana coexists with a low ratio of a
parthenogenetic population (Azari Takami, 1989;Agh et al., 2007) every specimen studied
was first identified as belonging to the bisexual A. urmiana using SNP polymorphism in the
Na+/K +ATPase α-1 subunit (Manaffar et al., 2011), and re-certified by the phylogenetic
analyses using the COI mitochondrial marker.
Total DNA of each specimen decapsulated embryo (number of specimens for each
experiment below) were extracted following the Chelex R
100 Resin method (Bio-Rad
Laboratories, Hercules, CA, USA). The embryos were crushed via a sterilized pipette
tip, incubated for 2.5–3 h at 60 ◦C (tubes were shaken by vortex every 30 min) and
eventually for 10 min at 80 ◦C. The tubes were centrifuged at 10,000 rpm for 1 min and the
supernatant phase was directly used in the PCR reaction (Montero-Pau, Gómez & Muñoz,
2008;Eimanifar & Wink, 2013;Asem, Eimanifar & Sun, 2016;Asem, Eimanifar & Wink,
2016). The extracted DNA was stored at −80 ◦C for further genetic analyses.
Genomic fingerprinting by ISSR-PCR
ISSR amplification
Nuclear genotype variation between A. urmiana samples collected in 1994 (31 specimens)
and 2004 (32 specimens) was evaluated using inter-simple sequence repeats (ISSRs). ISSRs
were amplified from genomic DNAs with two universal primers (GA)8T (Tulchinsky,
Norenburg & Turbeville, 2012) and (AG)8YT (Eimanifar & Wink, 2013). PCR was carried
out in a total volume of 20 µl containing 8 µl of ddH2O, 10 µlTaq polymerase (2 ×
TsingKeTM Master Mix, Cat.# TSE004, TsingKe CO., CN), 1 µl template DNA and 1 µl
of primer. The PCRs were carried out separately using the following conditions: 94 ◦C
denaturation for 1 min, 35 cycles of 46–48 ◦C annealing for 50 s and 72 ◦C extension for 2
min. The final cycle was followed by a 7-min extension at 72 ◦C (Eimanifar & Wink, 2013).
The final PCR products were visualized on 1.5% agarose gel (Cat.# 75510-019; Invitrogen,
Carlsbad, CA, USA), run at 50 V for 3.5 h (for more information see Tulchinsky, Norenburg
& Turbeville, 2012;Tiwari et al., 2015;Liew et al., 2015;Sharma et al., 2015).
ISSR statistics
The binary matrix (1 =presence; 0 =absence of a band) was determined for each year and
population genetic information was computed separately. Genetic relationships among
ISSR genotypes were established by principal coordinate analysis (PCoA) using GenAlex
version 6.5 (Peakall & Smouse, 2012). The partition of genetic variation within and between
1994 and 2004 was determined using the Analysis of Molecular Variance implemented in
GenAlex ver. 6.5 program (Peakall & Smouse, 2012).
Asem et al. (2019), PeerJ , DOI 10.7717/peerj.7190 4/20

DNA sequencing
PCR amplification
Two fragments of nuclear markers (Na+/K +ATPase and ITS1) and two mitochondrial
markers (COI and 16S) were amplified. PCRs were carried out in a total volume of 15
µl containing 6 µl of ddH2O, 7.5 µl Taq polymerase (2 ×TsingKeTM Master Mix, Cat.#
TSE004; TSINGKE Biotechnology Co., Ltd., Chengdu, China), 0.3 µl template DNA and
0.6 µl of each primer. Sequencing was performed by TsingKe CO. (China).
A partial fragment of the nuclear gene, Na+/K +ATPase α-1 subunit, was amplified
using the primers of Manaffar et al. (2011). PCR amplification was performed under the
following conditions: 94 ◦C for 2 min, 32 cycles of 94 ◦C for 25 s and 56 ◦C for 25 s and
72 ◦C for 1 min, and final extension with 72 ◦C for 3 min.
A fragment of the nuclear DNA containing a partial sequence of the 18S ribosomal
RNA (18S), the complete sequence of internal transcribed spacer 1 (ITS1) and a partial
sequence of the 5.8S ribosomal RNA (5.8S) genes, was PCR-amplified using the primers
18d-50/R58 (Baxevanis, Kappas & Abatzopoulos, 2006). The thermal cycler PCR conditions
were as follows: 4 min at 93 ◦C, 32 cycles of 40 s at 93 ◦C, 40 s at 62 ◦C, 1 min at 72 ◦C,
and a final extension of 5 min at 72 ◦C.
Amplification of a partial fragment of the mitochondrial cytochrome oxidase subunit 1
(COI) gene was performed using the invertebrate universal primers LCOI 490/HC02198
(Folmer et al., 1994). PCR amplification was carried out using the following program: a
cycle of 3 min at 95 ◦C, followed by 35 cycles of one min at 95 ◦C, one min at 40 ◦C and
one and half min at 72 ◦C, with a final step of 7 min at 72 ◦C.
The fragment of 16S ribosomal RNA (16S) was amplified using the primers 16S-SP/12S-
SP (Bossier et al., 2004). PCR amplification was carried out under the following conditions:
1 cycle of 94 ◦C for 2 min, 34 cycles of 1 min 15 s at 94 ◦C, 45 s at 52 ◦C, 2 min at 72 ◦C
and a final extension cycle of 72 ◦C for 4 min.
Our DNA dataset consisted of 248 sequences including 70 specimens sampled for
Na+/K +ATPase, 60 specimens for ITS1 and COI, 58 specimens for 16S genes. The list of
genetic markers and GenBank accession numbers is presented in Table 1.
Sequence alignment and population genetic diversity
Sequences were aligned using MEGA ver. 6.00 with MUSCLE tool and default parameters
(Tamura et al., 2013). Alignment lengths were 198, 1150, 665 and 875 bp for Na+/K +
ATPase, ITS1,COI and 16S, respectively. Between-group mean distances (year 1994/2004)
were computed using p-distance in MEGA ver. 6.00. To estimate the genealogical
relationships among haplotypes for each gene, a maximum-parsimony haplotype network
was inferred using the software TCS version 1.21 (Clement, Posada & Crandall, 2000).
For each marker, the number of polymorphic (segregating) sites (S), total number of
mutations (M), number of haplotypes (H), haplotype (gene) diversity (Hd), differentiation
of haplotype frequencies (DHF), nucleotide diversity (π), average number of nucleotide
differences (K) and neutrality tests (i.e., Tajima D, Fu and Li’s D*, Fu’s Fs) were computed
using DnaSP v.5.10 program (Librado & Rozas, 2009). Fixation index FST(an overall
Asem et al. (2019), PeerJ , DOI 10.7717/peerj.7190 5/20

Table 1 Population genetic indices for two Artemia urmiana samples collected in 1994 and 2004.
Markers Na+/K +ATPase ITS1 COI 16S
Sampling year 1994 2004 1994 2004 1994 2004 1994 2004
N 35 35 30 30 30 30 28 30
GB MK697598–
MK697632
MK697633–
MK697667
MK691705–
MK691734
MK691735–
MK691764
MK682320–
MK682349
MK682350–
MK682379
MK691599–
MK691626
MK691627–
MK691656
NS 198 198 1,150 1,150 647 647 875 875
S 1 4 93 59 41 38 62 67
Eta 1 4 96 65 41 39 64 70
H 2 4 29 24 24 22 28 29
Hd (±sd) 0.057
(±0.053)
0.166
(±0.084)
0.998
(±0.009)
0.963
(±0.027)
0.972
(±0.021)
0.961
(±0.023)
1.000
(±0.010)
0.998
(±0.009)
DHF 0.610ns (±0.002) 0.475ns (±0.021) 0.191ns (±0.020) 0.760ns (±0.013)
π(±SD) 0.00029
(±0.0007)
0.00115
(±0.0839)
0.00751
(±0.0039)
0.00508
(±0.0027)
0.00525
(±0.0030)
0.00593
(±0.0009)
0.01041
(±0.0054)
0.01014
(±0.0053)
K 0.057 0.229 8.637 5.844 3.398 3.834 9.108 8.871
Exp. Het 0.057
(±0.000)
0.057
(±0.000)
0.092
(±0.070)
0.099
(±0.072)
0.083
(±0.037)
0.101
(±0.057)
0.146
(±0.126)
0.132
(±0.108)
Tajima’s D−1.13ns −1.88*−2.45** −2.42** −2.47** −2.24** −1.70ns −1.88*
Fu and Li’s D*−1.732ns −3.123*−4.104** −3.957** −3.734** −2.595*−2.368ns −2.534*
Fu’s Fs −1.33ns −3.12ns −23.18*** −15.43*** −23.29*** −16.61*** −23.41*** −22.77***
BD 0.001 0.007 0.006 0.011
FST (Pd) 0.000ns 0.011ns 0.001ns 0.016ns
Notes.
N, number of sequences; GB, GenBank accession numbers; NS, Total number of sites (excluding sites with gaps/missing data); S, Number of polymorphic (segregating) sites;
Eta, Total number of mutations; H, Number of haplotypes; Hd, Haplotype (gene) diversity; DHF, Differentiation of Haplotype Frequencies; π, Nucleotide diversity; K, Av-
erage number of nucleotide differences; Exp. Het, Expected heterozygosity; BD, Between group mean distance; Pd, Pairwise difference; sd, standard deviation.
*P<0.05
**P<0.02
***P<0.001
ns, non-significant (Fs should be regarded as significant if P<0.02; Ashfaq et al., 2014).
population differentiation index) was calculated using Arlequin v.3.5 (Excoffier & Lischer,
2010).
RESULTS
Species identification
The specimens analyzed in this study had a homozygous pattern (T-T) in the last valine
codon using the Na+/K +ATPase α-1 subunit with the exception of a single specimen
collected in 1994 which showed a heterozygous pattern (T-G). Our phylogenetic trees (ML
and BI) for COI showed that all analyzed specimens clustered with the reference sequence
of A. urmiana (Maniatsi et al., 2011:HM998991) (Fig. S1). Only one specimen from 1994
placed in the diploid parthenogenetic clade also revealed a heterozygous pattern in valine
codon; this sample was removed from the dataset.
ISSR Profiling
A summary of population genetic indices of A. urmiana for all observed ISSRs loci between
1994 (rainy period) and 2004 (drought period) is listed in Table 2. ISSR profiling generated
Asem et al. (2019), PeerJ , DOI 10.7717/peerj.7190 6/20

Table 2 Summary of the genetic variation of all ISSRs loci observed for two Artemia urmiana samples
collected in 1994 and 2004.
Sample (year) 1994 2004
Number of specimens 31 32
Number of bands 17 17
Number of private bands 1 1
Polymorphic loci (%) 83.33 77.78
Table 3 Molecular variation (within and among populations) for two Artemia urmiana samples col-
lected in 1994 and 2004 (by AMOVA).
Source df SS MS Est. Var. Molecular variance (%)
Among populations 1 20.116 20.116 0.572 21
Within population 61 128.837 2.112 2.112 79
Total 62 148.952 22.228 2.684 100
17 bands with a single private band for each sample. The samples collected in 1994 and
2004 contributed 83.33% and 77.78% of polymorphic loci, respectively. AMOVA analysis
demonstrated that 21% of genetic variation resided between the rainy and drought periods
of A. urmiana (df =1, SS =20.116, P-value =0.00001, Table 3). The first and second
PCoA coordinates contained 18.94% and 13.80% of the variance, respectively (overall
32.74% of total variation). PCoA demonstrated that 1994 and 2004 were distinct groups,
since there was only a narrow overlap between them (Fig. 2), with 77.42% and 68.75%
specimens from the rainy and dry year being distinguished, respectively (Table 4).
Haplotype distribution
The Na+/K +ATPase of 70 sequences produced five distinct haplotypes (H1–H5) for the
1994 and 2004 samples (Fig. 3). Among them, H1 was found in 94.3% (66/70) of specimens
analyzed, including 51.5% (34/66) of specimens from 1994 and 48.5% (32/66) of specimens
from 2004. In the four other haplotypes, one (H2) belonged to the 1994 sample and three
(H3, H4 and H5) were only found in the 2004 sample.
The ITS1 sequences of 60 specimens showed 50 haplotypes (H1–H50). There were three
groups of haplotypes (H1, H2 and H3) which were shared by 13.4% (8/60), 1.6% (1/60)
and 1.6% (1/60) of specimens, respectively. H1 included two specimens from 1994 and six
from 2004. With the exception of four haplotypes (H1, H21, H29 and H32) that shared
genotypes between sampling years, other haplotypes were only found in one sampling year
(Fig. 4).
The COI sequences for 60 specimens contained 43 haplotypes (H1–H43). Haplotype
1 was the major haplotype that was found in ten (16.7%) specimens. H12 and H23 were
found in two and four specimens from 2004, respectively. H31 and H43 were shared by
two specimens belonging to the two sampling years, while all other haplotypes came from
a single sampling year (Fig. 5).
The 16S sequences for 58 specimens revealed 56 haplotypes (H1–H56), which showed
high variation in comparison with the other markers. The central haplotype (H1) covered
Asem et al. (2019), PeerJ , DOI 10.7717/peerj.7190 7/20

Figure 2 Principal coordinates analysis (PCoA) showing differentiation between Artemia urmiana
samples collected in 1994 and 2004 using ISSR fingerprint genomic results.
Full-size DOI: 10.7717/peerj.7190/fig-2
Table 4 PCoA results for two Artemia urmiana samples collected in 1994 and 2004, data shown as
original count (percentage).
Year Sample size Unique area Overlap
1994 31 24 (77.42) 7 (22.58)
2004 32 22 (68.75) 10 (31.25)
only two specimens (3.5%) including a single specimen from each year. With the exception
of H39 that was shared by two specimens from 2004, other haplotypes were unique to a
single specimen (Fig. 6).
Genetic variation and neutrality tests
Genetic indices and allele frequency estimated for the aforementioned four markers are
presented in Table 1.Na+/K +ATPase showed the lowest population genetic indices in
1994. The haplotype diversity of ITS1 had no remarkable difference between the 1994 and
2004 samples, but the other indices (S,Eta,H,πand K) demonstrated lower values in 2004
(drought period). The mitochondrial COI marker revealed a reduction of polymorphic
sites, total number of mutations and number of haplotypes in 2004. In contrast, the number
Asem et al. (2019), PeerJ , DOI 10.7717/peerj.7190 8/20

Figure 3 Maximum parsimony haplotype network of Na+/ K+ATPase sequences. The size of each
square/circle is proportional to the frequency of specimens. Each joining line between haplotypes is equal
with single nucleotide substitutions. B.
Full-size DOI: 10.7717/peerj.7190/fig-3
of polymorphic sites, total number of mutations and number of haplotypes of the 16S
marker showed lower values in 1994. Similar to COI there was no notable dissimilarity for
the haplotype diversity, nucleotide diversity and average number of nucleotide differences
in the 16S marker between the two periods. Haplotype frequencies of all mitochondrial
and nuclear markers represented non-significant difference between rainy and drought
periods. Though there was no significant difference in the amount of genetic variation of
COI and 16S markers between rainy and drought periods, each marker presented different
distinct of haplotypes. 16S presented higher expected heterozygosity in 1994 while ITS1
and COI showed higher values in 2004. The values of the pairwise genetic differentiation
index (FST) were not significant. The minimum and maximum between-group distances
were detected in Na+/K +ATPase (0.001) and 16S (0.011), respectively. Neutrality tests
yielded negative values with different significant and non-significant levels.
DISCUSSION
The effect of ecological disturbance, especially short-term regional climate changes, on
genetic diversity is not well understood (Bálint et al., 2011;Banks et al., 2013). Ruediger
et al. (2012) demonstrated that inter-annual and seasonal changes in water temperature
produced significant variation in the genetic structure of Daphnia populations. The impact
of salinity changes on genetic variation has been considered less frequently in aquatic
organisms (Stoks, Geerts & De Meester, 2014).
Genetic variation and genetic diversity are important parameters to conserve biodiversity
at all levels including population variabilities, individual fitness and adaptability of
species to the environmental conditions (Amos et al., 2001;Hughes et al., 2008). Recently,
Avolio, Beaulieu & Smith (2013) showed that the genotypic diversity of Andropogon
Asem et al. (2019), PeerJ , DOI 10.7717/peerj.7190 9/20

Figure 4 Maximum parsimony haplotype network of ITS1 sequences. The size of each square/circle is
proportional to the frequency of specimens. Each joining line between haplotypes is equal with single nu-
cleotide substitutions. Black dots between haplotypes.
Full-size DOI: 10.7717/peerj.7190/fig-4
gerardii (big bluestem grass) was significantly reduced after 10 years of an increase of
experimentally-driven intra-annual precipitation variation. Brown et al. (2013) suggested
habitat conflagration as a major critical process to reduce allelic richness of the mallee
emu-wren Stipiturus mallee by reducing population size. Similar studies in zooplankton
species such as Artemia have not been done. Environmental instability directly affects the
FST (genetic differentiation among populations) through its impact on immigration and
genetic drift combined with population reduction (Banks et al., 2013). Genetic diversity has
been indicated to be important to population fitness since low levels of genetic variation
may decrease the ability of population to adapt to the environmental crisis (Chapman et
al., 2009;Pauls et al., 2013).
Although Urmia Lake is a wetland of international importance and is facing an acute
ecological threat, few studies have assessed risks to its biodiversity. Asem et al. (2010)
reported that in a rainy period (1994) the Artemia of this lake had a higher cysts size
variation, significantly larger average egg size, and a thinner chorion than in a dry period
(2004). The smaller cysts and thicker chorion produced during the dry period were
attributed to decreasing food availability and to an acclimation mechanism, respectively,
to increase the survivorship of the diapausing embryo under ecological crisis (Asem et al.,
Asem et al. (2019), PeerJ , DOI 10.7717/peerj.7190 10/20

Figure 5 Maximum parsimony haplotype network of COI sequences. The size of each square/circle is
proportional to the frequency of specimens. Each joining line between haplotypes is equal with single nu-
cleotide substitutions. Black dots between haplotypes r.
Full-size DOI: 10.7717/peerj.7190/fig-5
2010). Sankian, Heydari & Manaffar (2011) showed that A. urmiana hatching from cysts
collected in 1998 (salinity =180 g/l) had lower mortality but higher RNA content than
those from 2003 (salinity approximately 300 g/l; saturated). Our ISSR fingerprint analysis
on samples collected in 1994 and 2004 showed that each group had a single unique ISSR
band. AMOVA analysis showed that 21% genetic variation occurred between the two
periods; the drought period had lost 5.5% of polymorphic loci in comparison with the
rainy period (Tables 2 and 3). Furthermore, the 1994 and 2004 collections were divided as
two distinct groups, with 77.42% of the 1994 specimens and 68.75% of the 2004 specimens
separated by PCoA. These results suggest that one decade of environmental changes has
caused genetic structure and biometrical variation of cyst (see Asem et al., 2010) in this
population.
Theoritically a decreasing population size in response to unfavorable ecological changes
is expected to lead to a reduction of genetic diversity (Bálint et al., 2011;Cobben et al.,
2011;Pauls et al., 2013). But in our study, the population genetic indices generated different
patterns of genetic variation. Generally, results have demonstrated that the genetic variation
of ITS1 in the drought period was reduced when the salinity of the lake was increased near
saturation (300 g/l). In contrast, the genetic diversity of COI and 16S was not significantly
different between the two periods. Additionally Na+/K +ATPase revealed a remarkable
Asem et al. (2019), PeerJ , DOI 10.7717/peerj.7190 11/20

Figure 6 Maximum parsimony haplotype network of 16S sequences. The size of each square/circle is
proportional to the frequency of specimens. Each joining line between haplotypes is equal with single nu-
cleotide substitutions. Black dots between haplotypes r.
Full-size DOI: 10.7717/peerj.7190/fig-6
increase in variation in the drought period. These conflicting results might be attributed
to the difference in the potential of gene variability that might be confirmed by further
experimental evidence.
Our study showed a negative and significant Tajima’s Dvalue for both examined periods
in COI and ITS1 (Table 1), which indicated an excess of rare haplotypes resulting from
population expansion or from selective sweeps (Nei & Kumar, 2000;Swanson, Aquadro
& Vacquier, 2001;Akey et al., 2004;Cruciani et al., 2008;Levitan & Stapper, 2009). The
negative values of Tajima’s Dshould be referred to the demographic expansion of these
markers in both ecological periods with regard to developed haplotype networks of ITS1
and COI markers. Additionally, Fu and Li’s D* and Fu’s Fs tests showed a negative
departure from the neutrality test. Given that Fu and Li’s D* test and Fu’s Fs test recognize
an excess of rare historical mutations (Fu & Li, 1993;Fu, 1996;Zhao et al., 2008), and rare
recent mutations (Fu, 1997;Ramos-Onsins & Rozas, 2002;Zhao et al., 2008), respectively.
Therefore the excess of both old and novel mutations were confirmed in the gene pool of
COI and ITS1 during the rainy and drought periods. Consequently, the results of neutrality
tests of Fu and Li’s D* and Fu’s Fs could explain the patterns of haplotype networks and
population expansion of ITS1 and COI.
The major influence of short-term ecological disturbance was observed in the
demographic history of Na+/K +ATPase and 16S markers. Neutrality tests resulted in
a non-significant value for Na+/K +ATPase in the rainy period, which supported the
demographic equilibrium. While significantly negative Tajima’s D, Fu and Li’s D* strongly
supported a demographic expansion and an excess of rare historical mutations in the
Asem et al. (2019), PeerJ , DOI 10.7717/peerj.7190 12/20

drought period. These results are consistent with the pattern of haplotype distributions
developed during this period. In addition, the non-significant value of Fu’s Fs suggested
the absence of recent mutations in Na+/K +ATPase in both periods.
Another major alteration was observed in the 16S structure. Tajima’s Dand Fu and
Li’s D* were non-significant in the rainy period which could indicate 16S marker was at
demographic equilibrium without selection in the rainy period. In contrast, a negative and
significant neutrality value and expanded haplotype network indicated that 16S is involved
in recent expansion in the drought period (Fig. 6). The negative and significant value of Fu
Fs test suggested the excess of new mutations in the gene pool of 16S in both normal and
drought periods.
Overall, our results have demonstrated that ecological disturbance should be considered
in hypotheses about effects of short-term environmental changes on genetic variation. The
rapid genetic changes that we found has also been demonstrated in some other species
of animals and plants experiencing environmental crises (Avolio, Beaulieu & Smith, 2013;
Brown et al., 2013;Banks et al., 2013). This could be attributed to the hereditary potential
of populations respond to immediate ecological changes (Thompson, 2009). Although
previous studies have shown a decreasing genetic variation in response to ecological
disturbance, we found that Artemia urmiana shows dissimilar responses to environmental
changes. Consequently, changes in genetic diversity and the pathway of variation are
controlled by interaction between ecological conditions and the ability of genes to vary.
Rogers (2015) suggested phenotypic patterns can be affected by ecological conditions which
may cause genetic variation within an anostracan population during different periods. It is
evident that the ecological crisis at Urmia Lake has had a meaningful influence on Artemia
urmiana genetic structure, especially reducing genetic diversity, which ultimately could
risk the survival of this crustacean.
ACKNOWLEDGEMENTS
The help of Prof. William Shepard (University of California, USA) and Dr. Christopher
Rogers (Kansas University, USA) with the English text and scientific suggestions was highly
appreciated.
ADDITIONAL INFORMATION AND DECLARATIONS
Funding
This study was financed by the Fundamental Research Funds (201762017; 201562029) for
the Central Universities (China). The funders had no role in study design, data collection
and analysis, decision to publish, or preparation of the manuscript.
Grant Disclosures
The following grant information was disclosed by the authors:
Fundamental Research Funds: 201762017, 201562029.
Asem et al. (2019), PeerJ , DOI 10.7717/peerj.7190 13/20

Competing Interests
The authors declare there are no competing interests.
Author Contributions
•Alireza Asem conceived and designed the experiments, performed the experiments,
analyzed the data, prepared figures and/or tables, approved the final draft.
•Amin Eimanifar conceived and designed the experiments.
•Gilbert van Stappen contributed reagents/materials/analysis tools, authored or reviewed
drafts of the paper.
•Shi-Chun Sun contributed reagents/materials/analysis tools, authored or reviewed drafts
of the paper.
Data Availability
The following information was supplied regarding data availability:
Na+/K+ ATPase data is available at GenBank: MK697598 to MK697667.
ITS1 data is available at GenBank: MK691705 to MK691764.
COI data is available at GenBank: MK682320 to MK682379.
16S data is also available at GenBank: MK691599 to MK691656.
Supplemental Information
Supplemental information for this article can be found online at http://dx.doi.org/10.7717/
peerj.7190#supplemental-information.
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