Geographical genetic structure of Schistosoma japonicum revealed by analysis of mitochondrial DNA and microsatellite markers
Yin et al. Parasites & Vectors
Geographical genetic structure of Schistosoma japonicum revealed by analysis of mitochondrial DNA and microsatellite markers
Mingbo Yin 0 1
Hongyan Li 1
Donald P McManus
Jing Su 1
Zhong Yang 1
Bin Xu 0
Zheng Feng 0
Wei Hu 0 1
0 National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention , 207 Rui Jin Er Road, Shanghai 200025 , China
1 School of Life Science, Fudan University , Handan Road 220, Shanghai 200433 , China
Background: Schistosoma japonicum is a significant public health risk in parts of China and elsewhere in Southeast Asia. To gain an insight into the epidemiology of schistosomiasis japonica, a detailed investigation of S. japonicum genetic population structure is needed. Methods: Using three mitochondrial DNA fragments and ten microsatellite loci, we investigated the genetic diversity within and structure among twelve populations of S. japonicum sampled on a geographical scale covering most major endemic areas. Results: Schistosoma japonicum lineages from Indonesia, the Philippines and Chinese Taiwan were clearly distinct from each other and from those in mainland China. Within mainland China, there was some evidence for genetic divergence between populations from the mountain and lake regions. However, the analysis inferred no clear sub-population structure in the lake region of mainland China. High genetic diversity was found among S. japonicum populations of mainland China and this was significantly higher than those from island regions. Conclusions: High genetic diversity within and substantial differentiation among populations were demonstrated in S. japonicum.
Schistosoma japonicum; Genetic diversity; Genetic structure; Population differentiation; Mitochondrial DNA; Microsatellite
Schistosomiasis is one of the most serious of human
parasitic diseases, infecting an estimated 200 million
people in 76 tropical and subtropical countries . Three
major species, Schistosoma japonicum, S. mansoni and S.
haematobium, can infect humans . Of these, S. mansoni
is endemic in Africa, the Middle East and South America
and S. haematobium is found in Africa and the Middle
East, whereas S. japonicum occurs in China, Indonesia
and the Philippines. At present, this parasite is endemic in
China in the lake/marshland regions of Anhui, Hubei,
Hunan and Jiangxi provinces and in the mountainous
areas of Sichuan and Yunnan provinces . Although
tremendous efforts over the last 2030 years have reduced
the prevalence of S. japonicum in China by more than
90% , recent indications are that schistosomiasis has
re-emerged in certain areas over the last decade . To
gain an insight into the transmission and epidemiology
of Asian schistosomiasis, a detailed investigation of S.
japonicum population genetic structure is required.
Several classes of molecular markers have been applied to
investigate the genetic variability of S. japonicum
populations. Earlier studies using isoenzymes  and
mitochondrial (mt) DNA sequences  reported remarkable genetic
similarity between Chinese and Philippine S. japonicum
lineages. Later, differences among, but not within, Chinese
S. japonicum populations were detected using mt DNA
sequences (nad1) . Similarly, Gasser et al.  applied
random amplified polymorphism DNA (RAPD) analysis,
and found genetic variability among Chinese S. japonicum
populations. Subsequently, a set of high-resolution
microsatellite markers was used to investigate the genetic
structure of S. japonicum populations from eight locations
across China, and a high level of polymorphism was
detected both between and within populations .
Moreover, S. japonicum populations from the lake and
mountainous regions were genetically distinct . Similar
results from mt DNA data suggested that geographical
separation was the major factor accounting for the
population divergence of S. japonicum between the lake and
mountainous regions in China [3,11]. Previous studies
sampled S. japonicum either from mainland China, or a
few locations in mainland China and other Asian
countries [12,13], but did not explore the genetic population
structure of S. japonicum across its entire range.
Most phylogeographical studies have relied on the
analysis of mitochondrial DNA variation, because of its rapid
evolution and maternal transmission without
intermolecular recombination . In contrast, microsatellites can
provide information on relatively recent evolutionary
processes . Assessing the congruence among independent
genetic markers has become an important issue .
Indeed, more and more studies have inferred genetic
relationships among populations based on the combined
analysis of different classes of markers [16-18]. Consistent
results provide a robust perspective on species
evolutionary history , while findings of incongruent patterns
from different markers are also valuable to demonstrate
important evolutionary processes, such as introgressive
hybridization . To date, no study has been undertaken
on S. japonicum to investigate the genetic relationships
among populations using different classes of genetic
markers simultaneously on the same samples.
In this work, we analyzed S. japonicum samples from
eight locations in China mainland (covering the lake and
mountainous regions) and Taiwan, Indonesia, Japan and
the Philippines. We explored the genetic relationships
among these populations based on the analysis of three
mt genes (nad1, nad4 and 16 s-12 s) and ten microsatellite
loci from the same individual worms.
All procedures involving animals were carried out based
on the guidelines of the Association for Assessment and
Accreditation of Laboratory Animal Care International.
The study protocol followed institutional ethical
guidelines that were approved by the ethics committee at the
National Institute of Parasitic Diseases, Chinese Center
for Disease Control and Prevention (NIPD, China CDC;
Permit No: IPD2008-4).
Adult individuals of S. japonicum were obtained from
twelve different geographical locations (Figure 1), including
eight locations across six provinces in mainland China
(Anhui, Hunan, Hubei and Jiangxi Provinces in the Yangtze
River Basin/lake region, and Sichuan and Yunnan
Provinces in the mountainous area of Southwest China) and
four other island locations from Asia (Taiwan, Indonesia,
Japan and the Philippines). Infected snails (Oncomelania
hupensis) from each mainland Chinese location were
transported to the laboratory of NIPD, China CDC, Shanghai.
Cercariae were released from pooled infected snails from
each site and used to infect laboratory-raised rabbits.
Finally, 45 days later, the adult schistosomes were
perfused from the mesenteric veins of the infected rabbits
and washed in saline before being preserved in 95% (v/v)
ethanol at 4C. The S. japonicum isolate from Leyte, the
Philippines, was taken into culture originally in 1969 by
Dr. Scholice. The material sent to us, as lyophilized
adult worms, was provided by Dr. John Bruce, Centre
for Tropical Diseases, University of Lowell, USA. The
worms from Japan (Kofu) were gifted by Dr. Hiroshi
Yamasaki, National Institute of Infectious Diseases, Tokyo,
Japan. The other geographical samples (lyophilized adult
worms) from Indonesia (Lindu Lake, Sulawesi) and Taiwan
(Changhua) were provided by Dr. John Cross, Uniformed
Services University of the Health Sciences, Bethesda, USA.
These preserved samples were from populations that had
been maintained in laboratory animals.
In total, 42 worms were subjected to mitochondrial
DNA analysis and 486 individuals were used for the
microsatellite analysis (Table 1). Microsatellite data for
401 of the worms came from our previous work .
Genomic DNA was extracted from individual adult
schistosomes using the DNeasy Blood & Tissue Kit and
Animal Tissues (Spin-Column) protocol from QIAGEN
(Hilden, Germany), and stored at 20C until use.
Mitochondrial gene amplification and sequencing
We amplified three fragments of the mitochondrial
genome: 891 bp of the gene encoding NADH
dehydrogenase subunit 1 (nad1), 1275 bp of NADH
dehydrogenase subunit 4 (nad4) and 1748 bp of 16S-12S
mitochondrial ribosomal DNA (16S-12S rRNA). Three
pairs of primers were designed based on the complete
reference mitochondrial sequence for S. japonicum (ID in
GenBank: AF215860) as follows:
16S-12S rRNA-F (5-ATAATGTTGCGTCTAAGGTC)
Figure 1 Map showing the sampling locations of S. japonicum for this study. Abbreviations: AHGC: Guichi Country, Anhui Province; AHTL:
Tongling Country, Anhui Province; HBSH: Shashi City, Hubei Province; HNCD: Changde City, Hunan Province; HNYY: Yueyang City Hunan Province;
JXDC: Duchang Country, Jiangxi Province; SCXC: Xichang City, Sichuan Province; YNEY: Dali City, Eryuan Country, Yunnan Province; CTW: Chinese
Taiwan; IN: Indonesia JP: Japan; PH: The Philippines.
and 16S-12S rRNA-R (5-TAAACACTACCCATCAAA
The amplifications were performed in 20 l reactions
containing 0.5 M of each primer, ~ 50 ng of genomic
DNA from an individual worm, Has Taq polymerase
(1.25 U, TaKaRa), 1.25 mM MgCl2, 2 l 10 reaction
buffer and 1 l dNTPs (2.5 mM, TaKaRa). The PCR
protocol used a hot-start activation at 94C for 10 min, followed
by 35 cycles of denaturation for 30 s at 94C, 30 s
annealing (55C for nad1 and nad4, 49C for 16S-12S rRNA)
and extension (90 s for nad1, 150 s for nad4 and 16S-12S
rRNA) at 72C, followed by a final extension at 72C for
10 min. PCR products were examined using agarose
gel electrophoresis (1% w/v) to validate amplification
efficiency. Amplification products were then sent to BGI
(Beijing Genome Institute; Shanghai, China) for
sequencing using an ABI 3730 DNA Analyzer.
The DNA of each individual S. japonicum worm was
genotyped at ten microsatellite loci (i.e. Sjp1, 4, 5, 6, 8, 9,
10, 14, 15, and 17), which were characterized previously
. The PCRs were performed based on the protocol
described in Yin et al. . The PCR products were
Table 1 Localities, sample information and genetic diversity of S. japonicum based on three mtDNA genes and
Population location (and abbreviation used)
Latitude Longitude mtDNA genes
Anhui Province, Tongling Country, Laozhou
Island, Guanghui Village (AHTN)
Sichuan Province, Xichang City, Daxing Township, 27.50
Shian and Jianxin Village combined (SCXC)
Yunnan Province, Dali City, Eryuan Country (YNEY) 26.12
Chinese Taiwan (CTW)
The Philippines (PH)
70(66) 54 52 22 0.79 0.88 ***
34(32) 27 25 23 0.82 0.89 ***
47(43) 27 25 25 0.84 0.85 *
44(40) 32 31 22 0.86 0.87 **
77(73) 55 54 45 0.79 0.91 ***
98(94) 68 67 71 0.80 0.90 ***
31(28) 23 22 20 0.75 0.85 ***
N1: number of individuals for mtDNA analysis; N2: number of mtDNA haplotypes; N3: number of individuals for microsatellite analysis, in parentheses is the
number of individual data from previous work ; N4: number of individuals for MLG analysis excluding missing data; N5: number of individuals for population
genetic analysis after removing near-identical MLGs; G, number of MLGs; nc, not calculated because of small sample size; *: P < 0.05; **: P < 0.01; ***: P < 0.001;
NS: no significance.
diluted to an appropriate concentration and analyzed
on an ABI 3730 capillary automated sequencer using a
LIZ 500 labeled size standard. The allele sizes were read
using GeneMapper software (Version 4.0) and checked
Mitochondrial DNA analysis
Unique haplotypes for the mitochondrial DNA sequences
(concatenated nad1, nad4 and 16S-12S rRNA sequences;
hereafter combined mtDNAs) were identified using
Arlequin 3.11 . All unique haplotypes were aligned
in ClustalW . A phylogenetic tree was constructed using
three methods, namely neighbor-joining (NJ),
maximumlikelihood (ML) and maximum parsimony (MP), in MEGA
5.10 . A consensus tree was obtained after bootstrap
analysis using 104 bootstrap replicates. Schistosoma mansoni
(ID in GenBank: NC_002545.1) was used as an outgroup. A
median-joining network was generated to infer the
relationships among the haplotypes of S. japonicum using
Network . Nucleotide diversity () per population was
calculated in DnaSP Version 5 : comparisons between
regions (mainland China vs island region and lake vs
mountainous regions in mainland China) were made
using unpaired Students t test. Hierarchical analysis of
molecular variance (AMOVA) was applied to partition
the genetic variances into among regions (i.e. mountain
and lake regions), among populations within region and
within populations, for samples from mainland China, in
Multiple genotypes from a single snail are often very
similar to one another and possibly derived from a single
miracidium by mutations occurring during the proliferation
of cercariae inside the snail . We therefore retained
only a single individual from each cluster of near-identical
Multi Locus Genotypes (near-identical MLGs) which
formed tight single-sex clusters in principal-coordinates
analyses (PCoAs). After this, the PCoA was applied to
explore the relationships among remaining MLGs. For
each population, the genetic diversity was also examined
by calculating the expected heterozygosity (He) and
observed heterozygosity (Ho) in GenAIEx. In this and
following analyses, the individuals for which data were
missing were excluded, as were populations with N < 6.
Third, the inbreeding coefficient (FIS), which measures the
extent of nonrandom mating, was computed in Genepop
3 . Positive values of FIS indicate heterozygote
deficiency, whereas, negative FIS values suggest
heterozygote excess. Finally, deviations from Hardy-Weinberg
equilibrium (HWE) were tested in Genepop, with 104
Genetic population differentiation was estimated using
Wrights F-statistics (FST) in GenAIEx, and the
significance of the FST value was tested by 104 permutations.
AMOVA was applied to quantify intra- and
interpopulation and region variances in Arlequin. A Mantel
test was used to test whether there was a relationship
between pairwise geographical distance (km) and
pairwise genetic distance (FST, based on microsatellites) in
We used STRUCTURE 2.3.4  to cluster the
samples on the basis of their microsatellite genotypes. The
most likely number of subpopulations was estimated from
the same dataset by running the procedure with a value of
K from 1 to 13 using the admixture model . In this
process, 106 iterations were performed after a burn-in of
Mitochondrial DNA analysis
We successfully amplified and sequenced 42 S. japonicum
individuals for three complete mitochondrial fragments
(nad1, nad4 and 16S-12S rRNA). All the sequences
were submitted to GenBank under accession numbers
KP793743- KP793784 for nad1, KP793785-KP793826 for
nad4, KP793827-KP793868 for 16S rRNA and
KP793869KP793910 for 12S rRNA.
Among the 42 samples sequenced, 34 haplotypes were
identified (Table 1). No haplotype was found at more than
one geographical location. Phylogenetic trees, constructed
by different methods (i.e. NJ, ML, and MP), showed
identical or similar topologies with only a few differences
in bootstrap values (not shown). The Taiwanese sequences
formed a group very distant from the others. Lineages
from the mountainous region of China (Sichuan and
Yunnan) and the other island locations (Indonesia, the
Philippines and Japan) were each distinct, forming
monophletic clades (almost so in the case of Yunnan) supported
by relatively high bootstrap values (Figure 2). In contrast,
there was no obvious structure for samples from the lake
region in China, with samples from Anhui, Hunan and
Jiangxi intermingled across the tree (Figure 2). This
pattern was further supported by the median-joining network
(Additional file 1: Figure S1).
The nucleotide diversity () ranged from 0 to 0.005
(mean = 0.0016). Interestingly, the nucleotide diversities
of most (five of eight) populations from mainland China
were significantly higher than those from Chinese Taiwan
and each of the other island regions (mean for mainland
China 0.0022 vs 0.0005; N = 12, t = 2.52, P = 0.03).
However, we detected no significant differences in diversity
between populations from the mountainous area and
lake region of mainland China (N = 8, t = 1.80, P = 0.13).
We recognize that sample sizes are small and this might
influence the results. The four individuals from Anhui
Guichi shared the same haplotype. In a further seven
populations (Anhui Tonglin, Hunan Changde, Hunan Yueyang,
Sichuan Xichang, Yunnan Eryuan, Indonesia and Japan),
each individual had a unique haplotype (Table 1). The
AMOVA revealed that most of the genetic variation was at
the within-population level (62.53%), however, significant
Figure 2 Maximum-likelihood tree showing phylogenetic relationships of S. japonicum mitochondrial haplotypes (concatenated nad1 +
nad4 + 16S-12S rRNA). Abbreviations: AHGC: Guichi Country, Anhui Province; AHTL: Tongling Country, Anhui Province; HBSH: Shashi City, Hubei
Province; HNCD: Changde City, Hunan Province; HNYY: Yueyang City Hunan Province; JXDC: Duchang Country, Jiangxi Province; SCXC: Xichang City,
Sichuan Province; YNEY: Dali City, Eryuan Country, Yunnan Province; CTW: Chinese Taiwan; IN: Indonesia JP: Japan; PH: The Philippines.
genetic differentiation was found between the mountain
and lake regions (6.25%; P < 0.001).
At the 10 microsatellite loci, 317 unique MLGs were
detected from 353 individuals (individuals with missing data
were excluded: Table 1), and no MLG was shared among
populations. According to the PCoA, the MLGs of 262
worms (after exclusion of near-identical MLGs) could be
divided into three groups: individuals from the Philippines,
Indonesia and Chinese Taiwan were clustered according
to their geographical origins and all the worms from
mainland China and Japan were grouped together (Figure 3).
Identical results were obtained when the PCoA was run
using only the 42 individuals for which mitochondrial
sequence data were also obtained (not shown). Observed
heterozygosity (Ho) was consistently lower than the
expected heterozygosity (He), and most of the inbreeding
coefficient (FIS) values were positive, with the exception
of Indonesia and Japan (Table 1), indicating an excess
of homozygotes within subpopulations in comparison
to HWE. Indeed, all populations deviated significantly
from HWE (Table 1).
Although most of the genetic variation occurred at the
within-population level (74.91%) according to hierarchical
AMOVA, significant genetic differentiation was detected
among the populations. Pairwise FST values ranged from
0.01 to 0.51 (averaged over 10 loci; Table 2), suggesting
low to high population differentiation. Specifically, the
FST between the Taiwanese and Indonesian populations
was 0.51 (Table 2), indicating high population
differentiation. In contrast, relatively low differentiation was seen
among the populations from mainland China (FST ranged
from 0.01 to 0.05; mean = 0.03; Table 2). The Mantel test
for isolation by distance revealed a positive, but
nonsignificant, correlation between population genetic
differentiation (FST) based on the microsatellite data and
geographical distance (R2 = 0.44; P = 0.09; Figure 4).
Based on the highest value of K, the most likely
number (K) of clusters was eight (Figure 5A). Using K = 8 in
STRUCTURE, we found that samples from Indonesia,
the Philippines and Taiwan each formed a unique, distinct
cluster. On the other hand, lineages from mainland China
and Japan were difficult to separate (Figure 5B). Other
values of K were explored, without providing any
additional insights (not shown). This clustering was consistent
with the PCoA (Figure 3), i.e., both analyses recognized
distinct lineages from Indonesia, the Philippines and
Taiwan, but not within mainland China and Japan.
Populations from mountain regions (Sichuan and Yunnan)
were not distinguished from those in the lake regions.
To our knowledge, this report includes the widest
geographic coverage to date, surveying 12 S. japonicum
populations in four Asian countries to assess the degree of genetic
subdivision within the species.
The two classes of markers, mitochondrial DNA and
microsatellites, yielded results those were not completely
congruent. Based on the phylogenetic tree (constructed
using three mitochondrial DNA fragments), the Taiwanese
lineage obviously diverged from the others. Moreover, the
lineages from the mountain regions of mainland China,
Indonesia, Japan and the Philippines were distinct from
each other and from populations in the lake region.
However, no substructure was detected among the
lineages from the Chinese lake region. In agreement with
the phylogenetic analysis, the PCoA analysis, based on
Figure 3 Principal coordinates analysis (first two factors only) using 10 microsatellite loci based on co-dominant genotypic distances
of all S. japonicum individuals (after removal of near-identical genotypes).
Table 2 Pairwise genetic differentiation (FST) among populations based on 10 microsatellite loci
Abbreviations: AHGC: Guichi Country, Anhui Province; AHTL: Tongling Country, Anhui Province; HBSH: Shashi City, Hubei Province; HNCD: Changde City, Hunan
Province; HNYY: Yueyang City Hunan Province; JXDC: Duchang Country, Jiangxi Province; SCXC: Xichang City, Sichuan Province; YNEY: Dali City, Eryuan Country,
Yunnan Province; CTW: Chinese Taiwan; IN: Indonesia JP: Japan. All values are significant below the level of 0.05 (based on 104 permutations).
ten microsatellite loci, showed that individuals from
the Philippines, Indonesia and Chinese Taiwan formed
distinct clusters according to their geographical origins.
This pattern was further supported by a Bayesian approach
in STRUCTURE. The S. japonicum lineages from the
mountain and lake regions of mainland China and from
Japan, that were distinct according to the mitochondrial
DNA markers, could not be separated by the
Although some earlier studies failed to detect any
evidence of major genetic dissimilarities at the DNA level
between Chinese and Philippine S. japonicum , our study,
in agreement with the more recent studies using
microsatellites , has shown that schistosome populations in the
two countries are genetically distinct. Moreover, although
S. japonicum was first described from Taiwan in 1914
, there have been few studies investigating genetic
variability in this population . Here, we provide the
first report that the Taiwanese population is clearly
divergent from others.
Genetic divergence observed among regional Chinese
and Asian populations in this study may relate to the
distribution and morphs of Oncomelania hupensis, the
only intermediate host species complex involved in the
transmission of S. japonicum . In mainland China,
there exist two distinct morphological and allozyme forms
of O. hupensis: one with a smooth shell in the mountain
region and another with a ribbed shell in the lake region
[32,33]. A single phenotype of O. hupensis quadrasi occurs
in the Philippines, while O. h. nosophora is present in
Japan, O. h. lindoensis in the Indonesian islands, and O.h.
formosana and O. h. chiui are present in Chinese Taiwan
. Oncomelania hupensis from China is genetically
different from O. h. quadrasi in the Philippines ,
and the O. quadrasi populations in the Philippines have
a substructure associated with their geographic origin
Figure 4 Linear regression of genetic differentiation (FST) based on ten microsatellite loci versus geographical distance between
S. japonicum populations.
Figure 5 Assignment of specimens of S. japonicum to populations by the STRUCTURE program. (A) The peak of K represents the most
likely number of subpopulations; (B) Bayesian clustering results inferred by STRUCTURE with the most probable model (K = 8).
. These different phenotypic and genotypic morphs
of O. hupensis that exist in different regions might
contribute to the genetic divergences apparent among
populations of its parasite, S. japonicum .
In the present study, FST values ranged from 0.01 to 0.51,
indicating varied levels of pairwise population-genetic
differentiation. In fact, strong population genetic
differentiation has been detected between the mountain and
lake regions of China on a large geographical scale [3,10].
This could be explained by different ecological
environments and agricultural practices between the two habitat
types. Another explanation may relate to the
topographical isolation of the mountainous land from the lower
Yangtze River basin, resulting in restrictions to gene flow.
In agreement with a previous study which applied another
set of microsatellite loci , our Mantel test showed no
significant correlation between genetic and geographical
distance. However, identical multilocus genotypes were
not shared among the populations, which would be direct
evidence of gene flow.
Corresponding well with the previous studies indicating
high genetic diversity of S. japonicum, based on
microsatellites  and mitochondrial DNA , in this study high
genetic diversity was detected within most S. japonicum
populations, according to both mitochondrial DNA and
microsatellite markers. We also observed marked
deviations from HWE in most populations, in agreement with
findings of a previous study on S. japonicum  and
similar to the results of studies on S. mansoni .
This significant deviation could have resulted from the
effects of non-random mating, inbreeding and
population subdivision. Also, the deviation could also have
been caused by unintended non-random sampling, given
the way in which specimens for analysis were obtained.
Furthermore, only low numbers of individuals were
available for analysis from some populations.
In conclusion, high genetic diversity within and substantial
genetic differentiation among S. japonicum populations
were demonstrated using both mitochondrial DNA and
microsatellites. Future research should aim to generate
a comprehensive link between the population nucleotide
diversity/structure differences and corresponding
phenotypic variation, such as host preferences and differences in
pathology, as well as possible differences in the response
to anti-schistosome vaccines and immunodiagnostics.
Overall, the present findings provide fundamental
biological and evolutionary information on S. japonicum,
with significant implications for the control and
elimination of this parasite in Asia.
Additional file 1: Figure S1. Median-joining Network based on the
mitochondrial haplotypes (concatenated nad1 + nad4 + 16-12S) of S.
japonicum. Abbreviations: AHGC: Guichi Country, Anhui Province; AHTL:
Tongling Country, Anhui Province; HBSH: Shashi City, Hubei Province;
HNCD: Changde City, Hunan Province; HNYY: Yueyang City Hunan
Province; JXDC: Duchang Country, Jiangxi Province; SCXC: Xichang City,
Sichuan Province; YNEY: Dali City, Eryuan Country, Yunnan Province; CTW:
Chinese Taiwan; IN: Indonesia JP: Japan; PH: The Philippines. The sizes of
nodes are proportional to the frequencies of the haplotypes.
The authors declare that they have no competing interests.
MY and WH designed the study, HL and BX carried out the molecular work.
MY, ZY and HL contributed to data analyses. MY, DM, DB, ZF and WH wrote
the manuscript. All authors read and approved the final version.
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