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Characterization of Population Genetic Structure of red swamp crayfish, Procambarus clarkii, in China

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The red swamp crayfish (Procambarus clarkii) is one of the most economically important farmed aquatic species in China. However, it is also a famous invasive species in the world. This invasive species was dispersed most via human activities including intentional or unintentional carry in China. Thus, P. clarkiinaturally distributed in China provides us a desirable mode to investigate the genetic structure of an invasive species dispersed mainly by human-mediated factors. To reveal the impact of human-mediated dispersal on genetic structure of P. clarkii in China, a total of 22,043 genome-wide SNPs were obtained from approximately 7.4 billion raw reads using 2b-RAD technique in this study. An evident pattern of population genetic structure and the asymmetrical migrational rates between different regions were observed with 22 populations based on these SNPs. This study provide a better understanding of the population genetic structure and demographic history of P. clarkii populations in China, inferring that anthropogenic factors (aquaculture or by accident) and ecological factors (e.g., complicated topography and climatic environment), as well as its special biological traits could account for the current population structure pattern and dispersal history of P. clarkii.
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Characterization of Population
Genetic Structure of red swamp
craysh, Procambarus clarkii, in
China
Shaokui Yi
1,2, Yanhe Li1, Linlin Shi1, Long Zhang1, Qingbin Li1 & Jing Chen3
The red swamp craysh (Procambarus clarkii) is one of the most economically important farmed aquatic
species in China. However, it is also a famous invasive species in the world. This invasive species was
dispersed most via human activities including intentional or unintentional carry in China. Thus, P. clarkii
naturally distributed in China provides us a desirable mode to investigate the genetic structure of an
invasive species dispersed mainly by human-mediated factors. To reveal the impact of human-mediated
dispersal on genetic structure of P. clarkii in China, a total of 22,043 genome-wide SNPs were obtained
from approximately 7.4 billion raw reads using 2b-RAD technique in this study. An evident pattern of
population genetic structure and the asymmetrical migrational rates between dierent regions were
observed with 22 populations based on these SNPs. This study provide a better understanding of the
population genetic structure and demographic history of P. clarkii populations in China, inferring that
anthropogenic factors (aquaculture or by accident) and ecological factors (e.g., complicated topography
and climatic environment), as well as its special biological traits could account for the current population
structure pattern and dispersal history of P. clarkii.
e red swamp craysh, Procambarus clarkii (Girard 1985), native to north-eastern Mexico and south-central
United States, is one of the world’s most invasive species1,2, and its aggressive burrowing leads to the damages of
levee, dam and paddy eld3. In 1920s, P. c larki i invaded China from Japan and now widely distributed in almost
all types of freshwater habitats, e.g., swamps, sloughs, ditches and paddy elds, in China. Several traits of its life
history, e.g., polytrophism, rapid growth, high fecundity and disease resistance, make its invasion of the wild suc-
cessful4. Interestingly, it is widely favored and consumed in China, and is one of the most economically important
farmed aquatic species rather than a devastating invasive species. In the cities located in the middle and lower
reaches of the Yangtze River, annual consumption of P. clarkii reaches more than 600,000 metric tons during
the peak summer season (www.yyj.moa.gov.cn). us, recently, much attention has been paid to the cultivation
technique5, articial reproduction6, and antibacterial or antiviral mechanisms of P. cla rk ii7,8. e dispersal rate of
P. c la rk ii appeared to greatly exceed the estimated rates, and now it has invaded into most areas in China except
Tibet plateau. Study on the population genetic structure of this alien species would contribute to understanding
its biological invasions and establishing possible methodologies for its prevention and control9. Human-mediated
jump dispersal of P. clarkii including intentional or unintentional carry, combined with its natural expansion
inuenced the population structure and genetic diversity10,11. Especially, with P. clarkii consumption demand
increasing in recent years, the craysh was carried into many areas by commercial transportation. e wild cray-
sh populations were mixed with the craysh escaped from cultured population due to the natural disaster or
human negligence. erefore, the geneticstructure of P. c l ar k ii populationsin China is complicated. Information
on genetic structure and diversities of P. c l ar ki i populations, which are mainly dispersed by human-mediated fac-
tors, would be more helpful to nd a way to control its invasion and scientically utilize the germplasm resources.
Undoubtedly, P. c l ar k ii could provide a desirable mode for investigating the genetic structure of an invasive species
1College of sheries, Key Lab of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education,
Huazhong Agricultural University, Wuhan, 430070, P. R. China. 2Fish Genetics and Breeding Laboratory, The Ohio
State University South Centers, Piketon, 45661, United States of America. 3Institute of Fisheries, Anhui Academy of
Agricultural Sciences, Hefei, 230031, P. R. China. Correspondence and requests for materials should be addressed to
Y.L. (email: liyanhe@mail.hzau.edu.cn)
Received: 2 November 2017
Accepted: 23 March 2018
Published: xx xx xxxx
OPEN
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mainly dispersed by human-mediated factors. Previous studies6,12,13 proposed that the populations of P. cl arki i in
China exhibited a high level of genetic diversity aer successfully invading a new environment. Meanwhile, in
the past years, the human-mediated dispersal in aquaculture accelerated the invasion of P. c larkii in China and
might result in the diernet levels of gene ow among dierent populations11. However, the impacts of anthro-
pogenic factors on the population structure of P. c lar ki i need to be comprehensively assessed, and the population
genetic features of P. c larkii have changed during the past decades aer the invasion. erefore, the dynamics
of population genetics in P. clarkii should be evaluated with a powerful method. e population structure and
genetic diversity of this alien species help us revealing the historical dispersal pattern of P. cl ar ki i in China, and
also provide new insights into invasion prevention and utilization of genetic resource. Previously, population
genetic studies of P. cl ar ki i have generally used mitochondrial genes12, microsatellites14,15 and AFLP13, and that the
genomic data of P. clarkii is quite limited8,16. e growing accessibility to high-throughput sequencing technolo-
gies allows the production of massive data and the discovery of genome-wide resources at relatively modest and
decreasing costs. As a simple and exible technique for genome-wide genotyping, 2b-RAD method can provide
the rapid discovery of thousands of SNPs that are to some extent evenly distributed across the genome17,18. To
date, to our knowledge, there is no document about investigating the population structure and genetic dierenti-
ation of P. cla rk ii at genome level aer its successful invasion.
Recently, rapid adaptive evolution of invasive species has been a hot topic19,20. eoretical considerations
indicate that invading populations should be prime candidates for both adaptive and non-adaptive evolutionary
change21. It is clear that invasion can drive evolutionary changes in the phenotypic traits of invasive organisms
within periods of years to decades. To adapt the environmental conditions of its introduced area, invasive species
rapidly evolved adaptive clines in key morphological and life-history characteristics aer its invasion. Presently,
most researches in this area tended to concentrate on comparing genetic diversity between native and invasive
populations. Few studies have investigated the phenotypic divergence of the invasive animal species aer itsinva-
sion. Zhang et al.22 rstly reported that the morphological dierentiation of 12 dierent populations in P. cl ar ki i
and proposed the Chinese populations exhibited phenotypic variations (e.g., carapace, abdominal, body length)
compared to American populations. Based on the basis of that previous study, the investigation of morphological
characters between dierent populations of P. cl ar ki i could help us understanding the rapid evolution of pheno-
typic traits of P. cla rk ii aer its invasion.
In this study, amounts of genome-wide SNPs were identied in P. c larkii using 2b-RAD technique and then
used into clarifying the genetic structure of P. cl ar k ii in the main distribution areas in China. e genetic dynamics
and genetic structure impacted by human-mediated dispersal, and phenotypic divergence of P. c la rk ii in China,
would be presented in the study. We investigated thepopulation genetic signatures and conductedmorphological
analyses to test the hypotheses that (1) the P. c lar ki i populations impacted by anthropogenic factors show persis-
tent asymmetrical gene ow and exhibit an evident pattern of genetic structure; (2) rapid evolution or selection
aects the phenotypic divergence of dierent P. cl ar ki i populations. ese results would contribute to exhibiting
the genetic signatures of P. c lar ki i populations aer the successful invasion, providing management strategies for
the invasion of P. cla rk ii in China and utilization of germplasm resource for the hatchery breeding in aquaculture.
Results
Identication of SNP loci. Sequencing of the RAD libraries generated an average of 33.53 million reads
per individual, prior to any quality ltering. Aer quality ltering, on average, 26.54 million (79.22%) reads with
restriction site per individual were retained. Of the retained sequences, an average of 10.89 million (40.98%)
aligned to the P. cla rk ii genome survey sequences (not published) with the average coverage was 26.08 ± 6.29
reads per locus. e sequences were discarded due to alternative alignments and insucient depth of coverage
(Supplementary TableS1). A total of 20,691 loci with minimum 3X coverage were retained for SNP discovery, and
these RAD loci were distributed among 19,908 reference scaolds. Of these, 19,339 (93.47%) loci were monomor-
phic, 1352 (6.53%) loci showed two alleles per individual and were consequently eliminated from further analy-
ses, producing a total of 22,043 candidate SNP markers. Transition polymorphisms (Ti) outweighed transversions
(Tv), accounting for 56.14% of the SNP sites in our data set, with an observed Ti: Tv ratio of 1.28 (Supplementary
Fig.S1).
Population genetic dierentiation. Average observed and expected heterozygosity was 0.0047 and
0.0052, respectively. e nucleotide diversity (π) across all individuals ranged from 0.1335 to 0.1964 among
22 populations. e FST values between populations ranged from 0.0400 to 0.2521. Also, we calculated the FST
based on the SNP sites (Supplementary Fig.S2A). 4099 of 22,043 loci (18.60%) gave low or negative estimates
of FST, and 8695 of the loci (39.45%) had noticeably high FST values (>0.15), of which 2284 even exceeded 0.25.
Analysis of molecular variance (AMOVA) revealed that a signicant fraction of the total variance was due to
between-populations variance (12.69%, FST = 0.1269; P < 0.01) with evidence of signicant genetic dierentiation
(P < 0.05); most of the variance was due to variation within populations (87.31%). Notably, the pairwise compar-
ison of FST values from seven populations in the middle reaches of Yangtze River basin revealed the signicant
lower level of genetic dierentiation between these populations than others with the average of 0.085, indicating
the high level of genetic communication among the populations in the middle reaches of Yangtze River basin.
e Mantel test for matrix correlation between genetic similarity and geographical distances were not signicant
(r = 0.2946, P = 0.9780), indicating the pattern of isolation by distance for the P. clarkii didn’t exist in the main
distribution areas of China. Additionally, the mismatch distribution displayed a unimodal curve for P. c la rk ii
(Supplementary Fig.S2B) and t the expected distributions under sudden expansion model.
Population genetic structure. e Bayesian analysis of population structure in STRUCTURE and calcu-
lation of ΔK value from the STRUCTURE output showed the model value of parameter K was 13, thus indicating
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that the uppermost hierarchical level detected by STRUCTURE was 13 distinct genetic clusters (Fig.1). e clus-
tering of population structure revealed that each of the populations in the upper reaches of Yangtze River basin
had the specic ancestors. ree populations from the lower reaches (including WX, HZ and SH) grouped into
one cluster, and showed the similar population structure. Two populations from Huai River basin (SD and PZ)
exhibited the dierent ancestors with other populations. With the K equal to four, we found the obvious division
between the WX, HZ and SH populations, two populations from Huai River basin and the populations from the
upper reaches of the Yangtze River above Nanjing City, which was consistent with the population’s geographic
distribution. e WS population shared the ancestry sequences slightly with the WX, HZ and SH populations,
indicating high level of gene ow between these populations. e principal components analysis (PCA) also
recovered these groupings (Fig.2), with the rst and second eigenvectors separated the populations into three
subgroups, i.e., two populations of Huai River basin including PZ and SD, three populations of lower reaches of
Yangtze River including WX, SH and HZ, the other populations in the upper reaches of the Yangtze River above
Nanjing City. Here, we dened these three subgroups as three regions, including Huai River region, lower reaches
region and upper and middle reaches (UM) region, according to the population structure and geographic loca-
tion of these populations.
e neighbor-joining (NJ) tree of these craysh populations (Fig.3A) features two main clusters, with HZ,
SH and WX populations grouping together and the other populations forming the other cluster. Notably, the
individuals (e.g., CQ, YB and WS) from upper reaches irregularly clustered with the individuals from middle
reaches of Yangtze River basin. For instance, the YB and CQ populations were clustered with HK population. It
could be inferred that the populations from middle reaches invaded into the upper reaches of Yangtze River basin
with high level of human-mediated dispersal in the past few decades. e NJ tree of the 221 individuals (Fig.3B)
Figure 1. Population genetic structure of the 221 P. cl ark ii individuals. Analyses with STRUCTURE show the
clustering of individuals into 4 and 13 groups. e proportion of the individual’s genome from each ancestral
population is shown by the length of each colored segment.
Figure 2. e principal components plots of 221 individuals in Yangtze River basin based on the 1st an 2nd
eigenvectors. e dierent colors represent the dierent craysh populations. e dashed circles indicate the
individuals from the lower reaches of Yangtze River basin and Huai River basin, respectively.
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revealed that the individuals from same location grouped into one population-specic cluster except the individ-
uals in DJK population, which indicated the high level of genetic diversity in DJK population. e branch length
of the NJ tree exhibited the length of individual or population-specic branches account for the most proportions,
inferring that the major genetic variations of craysh derive from the among-individuals within population.
ese ndings also coincided with the results of analysis of molecular variance.
Gene ow of P. clarkii populations. e population structure and NJ tree revealed that these 22 popu-
lations grouped into three clusters, which coincided with the geographic distribution. Here, we constructed a
gene ow hypothesis that persistent asymmetrical gene ow occurred among these three regions. To investigate
the migration among these three regions, the population sizes and migration rates of each group were calculated
Figure 3. Neighbor-joining trees of the 22 craysh populations (A) and the 211 individuals (B). e dierent
colors in (B) indicate the individuals from dierent populations.
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using Migrate-n program. ree geographical regions were analyzed, and estimates of eective population size (θ)
were consistently low and ranged from 0.0487 for lower reaches region (SH, HZ and WX) to 0.0702 for the UM
region (Fig.4A). Estimates of migration rate between three regions were bi-directional and relatively low, which
ranged from 436.8 to 660.4. e highest M value was found for individuals from UM region to Huai River region,
while the lowest M value was for individuals from the lower reaches region to Huai River region. Signicant
asymmetrical migration rates between UM region and Huai River region were discovered by non-overlapping
95% condence intervals of each estimate. Skyline plots revealed that the population sizes of UM region and
lower reaches region increased prior to Huai River region, and the population sizes of UM and Huai River region
were higher than the lower reaches region (Fig.4B).
Phenotypic divergence of carapace width in P. clarkii. Since the level of genetic dierentiation is
relatively low between populations and is higher within populations. Investigation of morphological characters
between dierent populations of P. cl ar kii could contribute to understanding the rapid evolution of phenotypic
traits within populations of the craysh to some extent. A total of 378 female individuals from eight locations were
chosen for morphological investigations. Signicant positive relationships existed between body weight and the
other six traits (i.e., body length, carapace length, carapace width, abdominal segment length, abdominal segment
width, and rst abdominal segment length) in P. cl ar kii (P < 0.05). Path analysis showed that carapace length (LC),
carapace width (WC) and abdominal segment length (LA) had strong positive direct eects (Table1), indicating
these three characters, especially carapace length (LC) and carapace width (WC), are indicative of determining the
body weight of P. cl ar ki i . In total, 90.4% of the variation in weight could be explained by the variation of two inde-
pendent variables. e unexplained variation, 9.6% of the total, may be due to variation in the other components
under consideration. To investigate the phenotypic divergence of the individuals from dierent populations, we
compared the ve external characters among the populations, which were standardized by total length (LT). e
results showed that carapace widths of P. cl ar ki i were diered among these populations (Fig.5). ree popula-
tions (HS, YY and NJ) in the UM region exhibited a signicant lower carapace width/total length ratio than other
populations in lower reaches region and Huai River region (Wilcoxon rank sum test, P < 2.2 × 1016). e lowest
Figure 4. Estimates of the migration (M and θ) (A) and skyline plots among three geographic regions of
craysh (B). Θ represents the mutation-scaled population size, and the value M in (A) indicates the mutation-
scaled migration rate. Θ1, θ2, θ3 represents the population sizes of Huai River, lower reaches and UM regions of
Yangtze River basin, respectively. e map in (A) was generated by ArcGIS 10.2.
Variables Correlation
coecients Path
coecients
Indirect eect
LCWCLFA
LC0.948*0.758* 0.137 0.054
WC0.895*0.149*0.695 — 0.051
LA0.842*0.062*0.657 0.122 —
LT 0.916*0.064
LFA 0.805*0.006
WA0.802*0.016
Table 1. Path coecients for measured characters in P. cl arkii . Total length (LT), carapace length (LC),
abdominal segment length (LA), rst abdominal segment length (LFA), abdominal segment width (WA) and
carapace width (WC); Signicant at the 0.05 probability level.
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value of carapace width/total length ratio was observed in NJ population, where P. c larkii was rstly introduced
into China. ese populations were divided into three groups based on the geographic distribution. Further, the
phenotypic divergence of carapace width between the populations in the lower reaches (WX, SH and HZ) and
Huai River region (SD and PZ) was not signicant (P = 0.38).
Discussion
P. cl a rk ii , one of the most famous invasive species in China, dramatically became an important cultured species in
China. Since it invaded into China in 1920s, it has widely dispersed in Yangtze River basin and Huai River basin.
Barbaresi et al.3 proposed that the wide dispersal of P. clar ki i throughout the world is attributed to human activi-
ties. It is evident that aquaculture and aquatic product transportation promote the spread of this invasive species
in China. Also, the dispersal capabilities (behavioral exibility) of P. cl ar ki i contribute to the rapid and widespread
diusion. erefore, population genetic structure, genetic diversity and evolutionary history, and phenotypic
divergence of this alien species deserve a special mention in response to challenges in biodiversity conservation
and invasion control.
Characterization of population genetic structure. It is generally known that crustaceans were consid-
ered to be problematic for karyological and cytogenetic studies, as the majority of species have relatively high dip-
loid chromosome numbers (>150) and relatively small chromosomes23, and P. c l ar k ii is no exception24. erefore,
the genome assembly of P. c larkii becomes much more dicult even though the NGS technology rapidly devel-
oped in the past few years. As an ecient and exible method for genome-wide genotyping, 2b-RAD technique17
provides an excellent fractional representation of the targeted genome and has been widely applied in the pre-
vious studies2527. In this study, to reveal the characteristic of genetic structure of P. c la rk ii, a total of 22,043 SNP
sites were identied from the P. c larki i genome based on 2b-RAD technique, which were adequate for the study
of population genetics.
e high level of genetic diversity in P. clarki i populations was detected in this study, which is consistent with
the previous studies12 and could contribute to invasion success28. Meanwhile, the high level of variance within
populations rather than among populations was revealed in this study, which might help to the invasion success
of P. cl ar kii 11,12, and also it appears to be an typical genetic feature of invasive species aer its successful invasion,
which is also reported in other invasive species2931. Meanwhile, the high FST values revealed less genetic exchange
between most P. cl ar ki i populations, which may be related to its migrational ability in the wild and high fecun-
dity. In addition, the low rate of migration between natural populations, which is aected by the human activ-
ities (extensive shing of this economic species), to some extent reduced the level of genetic exchange between
the populations in China. Furthermore, other methods should be adopted to further conrm this conclusion
in the future studies, such as investigation with slow evolving sites or haplotype distribution. Additionally, no
pattern of isolation by distance (IBD) detected in the P. c la rk ii populations to some extent further veried that
human-mediated jump dispersal was the main dispersal pattern for P. c la rkii .
e populations from upper reaches of Yangtze River basin exhibited similar population structure with the
populations from middle reaches. Li et al.11 proposed that the P. c l ar kii was most likely originally introduced into
China from Japan, and then expanded its range primarily into the middle and lower reaches. Here, we infer that
the populations in upper reaches may originate from the populations in middle reaches of Yangtze River basin.
Notably, two populations (SD, PZ) from Huai River region and three populations (SH, WX, HZ) from lower
reaches region obviously separated from UM region, which coincided with the phenotypic divergence of carapace
width. ese three regions revealed asymmetrical migration and low migration rates. Meanwhile, the mismatch
Figure 5. e morphological divergence of carapace width in the eight populations. e symbols below the
diagonal in the top right corner represent the signicance level in ANOVA. N indicates P > 0.05; asterisk
indicates P < 0.05.
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distribution was adopted to investigate the demographic history of P. cl arkii , and the typical unimodal distribu-
tion indicated that populations had undergone demographic expansions. To estimating population expansion
through time, Bayesian skyline plots revealed that the population sizes of three regions were uctuant and expe-
rienced one or two period of rapid increase. Interestingly, the population size (θ) of UM region remained at high
levels in the longtime, which experienced two periods of rapid increase. ese ndings conrmed our hypothesis
that persistent asymmetrical gene ow occurred among these three regions, and UM region was the main distri-
bution area. e population sizes of lower reaches and Huai River regions were increasing, which were consistent
with the conclusion reported in Li et al.11,12. In summary, this successfully invasive species that was mainly inu-
enced by human-mediated dispersal revealed some characteristics of genetic landscape including asymmetrical
migration, population size rapid increasing, high genetic dierentiation within population, etc.
As the characteristics for an successfully invasive species mentioned above, the notion of fast evolving DNA
markers reaching saturation levels of genetic distance between populations and maximum genetic diversity
(MGD) theory32,33 might account for the phenomenon that the low genetic dierentiation among populations
and high dierentiation within population were detected in this study. Unfortunately, it was dicult to carry out
the related analysis in this species because it seemed impossible to identify the slow evolving sites based on the
limited SNPs information in this study. e bottleneck was that the reads outputted from 2b-RAD technique was
only 33 bp in length. A comprehensive conclusion could not be given for the results mentioned above due to the
limitations of the SNPs with limited information used in this study. e identication of more informative SNPs
in P. cl ar kii must be carried out using other better methods (e.g., genome resequencing, amplicon sequencing) in
the future studies. In addition, the craysh genomic information is still very limited although we had sequenced
the genomic sequences. e SNPs related with phenotypic divergence could not be identied even though the
morphological characters were investigated in the subsequent section. us we could only exhibit the trend of
divergence of morphological traits for P. cl ark ii as a whole.
Phenotypic divergence of P. clarkii after its rapid invasion in China. Generally, invasive species
show higher phenotypic plasticity than native species34, and high level of phenotypic plasticity should enable a
colonizing species to cope with, and become established under new environments. Many studies have focused
on the phenotypic dierences between populations in invasive plants3538 (e.g., Impatiens glandulifera, Bromus
tectorum, Agrostis capillaris, Eschscholzia californica), and fewer studies have proposed the phenotypic divergence
of invasive animals39,40. As a famous invasive species, P. clarkii provide a desirable opportunity to investigate
rapid adaptation of its phenotype. Given that the genome complexity of P. clarkii, phenotypic divergence is a
better breach for predicting future invasion scenarios and understanding how populations can evolve rapidly in
response to novel and changing environments. In the present study, the morphological dierentiation of carapace
width in P. cl ar ki i was observed aer invaded into China, and the lowest value of carapace width was observed
in NJ population. Since it was rstly introduced into Nanjing City and invaded other areas10, the hypothesis that
the carapace has been gradually widening for the rapid evolution of P. cl ar ki i aer its invasion was supported by
the comprehensive comparisons of morphological characters. erefore, it could be inferred that rapid evolution
or selection aects the phenotypic change of the dierent populations in P. c la rk ii . Path analysis revealed that
higher carapace width indicates the higher weight of individuals in P. cl ar kii, which are exclusively preferred in
aquaculture at present.
Management implications and scientic utilization of P. clarkii. P. clarkii plays two roles in China,
a famous invasive species and a popular aquaculture species. e middle reaches of Yangtze River dominates the
craysh industry of China in both aquaculture and wild capture sheries, where the industry contributes well in
excess of 56.4 billion RMB annually in 2016 (www.cnfm.gov.cn/). Although the burrowing activities of P. cl ar kii
can lead to damage to water courses and to crops, particularly rice, and its feeding can disrupt native ecosystems41,
Chinese farmers are adept at avoiding the ecological damage (i.e., building blocking net) and utilization of its bio-
logical characteristics (i.e., tolerating relatively low dissolved oxygen concentrations and high temperatures). For
example, the rice-sh culture system, a complex ecological aquaculture mode, is widely developed in southern
China42. P. cl ar kii is better adapted to growing in a rice eld, and the concurrent culture of rice and craysh makes
good use of land, resources, equipment, and infrastructure already being used for rice production. Also, the wild
capture of P. c larki i is still in a certain proportion in Chinese craysh industry presently, while the wild craysh’s
burrows result in dam damages and a huge loss of irrigation water, causing signicant economic loss43.
e high level of genetic diversity in P. c larkii populations and low level of genetic exchange between most
craysh populations were observed in our study. High level of genetic diversity promotes availability of natural
variations for use in breeding germplasm. e P. cl ar kii populations with high level of genetic variation might be
used as a source for selection of desirable germplasm and development of new varieties. In addition, the carapace
of P. cl ar kii has been widening since it was rstly introduced into China in 1920s. Path analysis revealed that the
characters in carapace play major role in body weight of P. clar ki i. It provides novel insights into future selective
breeding of P. clarkii , for improving the economic value of craysh culture. e results of the present study,
together with those of previous population genetic studies in P. clar ki i 11,12 investigated the comprehensive genetic
information of P. c l ar k ii populations in China, could provide a signicant guidance into utilization of the valuable
genetic resources and more ecient management of its invasion.
Overall, this study provided some suggestive clues for comprehensively revealing the impact of
human-mediated dispersal on the genetic structure and diversity of P. clarkii populations, and uncovered the
population genetic landscape of this famous aquatic animal as well as the phenotypic dierentiation between
dierent areas in China that could provide us some fresh ideas to conduct selective breeding program in future.
However, imperfectly, this study has two weaknesses that should be considered when interpreting these results.
First, we used only a small number of populations from the Huai River region to draw conclusions as a whole.
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A larger population size would be an important way to make more powerful conclusions about migration rate,
population structure, and genetic diversity. Also, the gene ow from unsampled populations in Huai River region
could be inuencing the poor estimates because Migrate-n assumes that all populations exchanging migrants
are sampled. Second, the sequencing method used in this study had few limitations that the SNPs identied
based on this method without high-quality reference genome were not informative due to the generation of short
reads (33 bp in length). However, such an ideal assumption cannot meet for most studies, yet this method has
been widely used to infer migration rates in natural populations. Given the above mentioned, our future work
will collect more samples from more areas especially the Huai River region if possible, and focus on evaluating
the evolving rates of genomic variants that contribute to understanding the genetic diversity and phylogeny of P.
clarkii and the potential connectivity between environmental conditions and genetic variations.
Methods
Samples collection. To explore the genetic dynamics and genetic structure of P. c l ar kii, a total of 22 natural
populations, which selected from the major cultivation areas and river basins where P. cl a rk ii distributed in China,
were collected (Fig.6; Supplementary TableS2). Ten specimens of each location were sampled, and the muscle
tissue was preserved in 95% ethanol and stored at 4 °C for later DNA extraction. Additionally, seven populations
from 22 populations mentioned above and one population from Huangshan, Anhui province were selected to
evaluate the phenotypic dierentiation (Supplementary TableS2; Supplementary Fig.S3).
SNP identication. Total DNA was extracted from muscle tissue using the ammonium acetate method44.
Extracted DNA was dissolved in DNAase-free water and concentration was determined using NanoDrop 2000
spectrophotometer (ermo Fisher Scientic, Waltham, USA). DNA integrity was assessed by 1.0% agarose gel
electrophoresis. e 2b-RAD libraries were constructed for each individual following the methods from Wang
et al.17 Dierent from original 2b-RAD technique, our procedure produced single-tag constructs using modied
adaptors and biotin-labeled primers, and then digested by the Bsa XI enzyme to generate distinct cohesive ends
and then ligated in a predened order to produce ve concatenated tags for Illumina paired-end sequencing.
Meanwhile, a dra genome of P. cl arkii was assembled with 102.73 Gb clean data, and the maximum contig was
83,480 bp in length and the contig N50 value of 1,138 bp (unpublished). is genome was adopted as a reference
which was used in the subsequent analysis. e restriction enzyme recognition sites of Bsa XI were detected on
the genome and the recognition sites number was 99,273 in the scaolds.
To assess the robustness of the method and subsequent data analyses, FH11 library was replicated (Technical
Replicates, TRs). e starting DNA was digested with the Bsa XI restriction enzyme and ligated to the two
library-specic adaptors. To reduce marker density, the adaptors with fully degenerate 5-NNC-3 overhangs were
chosen. For estimating Bsa XI sites coverage, simulant detections of the Bsa XI sites in P. cl ar ki i reference genome
were carried out. PE300 sequencing of 221 libraries was performed on Hiseq X10 platform (Illumina, Inc. USA).
Demultiplexed reads were returned by the sequencing facility in FastQ format and their quality was checked
by FastQC (www.bioinformatics.babraham.ac.uk/projects/fastqc/). e Perl scripts from Wang et al.17 were run
for quality ltering and adaptors trimming of the reads. Assembly of the PE reads and extraction the assembled
reads containing ve tags was performed on PEAR script. e processed ve-tag dataset from PEAR output was
divided into single-tag data sets using a Perl script, and then the single tags with restriction site were extracted.
ese tags of each individual were mapped to the reference genome using SOAP2 program (http://soap.genomics.
org.cn/) with the parameter M 4, v 2 and r 0 allowing four mismatches. e sequences that aligned with
only one location were regarded as unique tags and the expected potential markers. e aligned reads were per-
formed SNP calling using RADtyping45 with default Posterior probability is calculated for two possible genotypes
(homozygote and heterozygote) at a given locus using a maximum likelihood approach. To look for the rela-
tionship between sequencing data size and Bsa XI sites coverage, a correlation test was performed based on two
Figure 6. Geographic locations of 22 P. clarki i populations collected for 2b-RAD in China. e map was
generated using ArcGIS 10.2.
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Scientific RePoRtS | (2018) 8:5586 | DOI:10.1038/s41598-018-23986-z
repeated samples. To improve the accuracy of SNP genotyping, the loci that were present in all samples in at least
80% of the individuals from each sample, with at least three RAD tags per allele at each locus (3X coverage per
allele) were included. To avoid linkage bias for the SNP calling, only the loci containing 1~2 SNP and minor allele
frequency (MAF) >0.05 were retained in the nal analysis. e SNP dataset is publicly available at the website:
https://doi.org/10.6084/m9.gshare.5537584.v1.
Genetic diversity and population genetic analyses. Pairwise F-statistics (FST) per pair of populations
were calculated using Arlequin suite version 3.546 to identify genetically dierentiated localities. e potential
number of genetic clusters and the membership of each individual were estimated using STRUCTURE Ver.
2.3.447. e soware uses Markov chain Monte Carlo (MCMC) simulations to estimate those parameters, with
the number of clusters to be tested (K) specied by the user. e MCMC simulation was run for 300,000 repe-
titions, aer a burnin period of 100,000. For each value of K (number of potential ancestral populations), the
genetic ancestry of each individual was estimated based on the admixture model without any prior population
assignment; estimations were obtained from the 300,000 iterations that followed a burn-in period of 100,000
iterations. e correlation between genetic distance and geographic distance was assessed using IBDWS version
3.2148. Additionally, the distribution of genetic variation was analyzed by AMOVA analysis using the Arlequin
suite version 3.5. Principal components analysis (PCA) was performed using GCTA soware49, and the scatter
plot of the rst and second components was done using R (https://www.r-project.org/). We then used these aver-
age pairwise FST values to cluster populations by a neighbor-joining method implemented in the MEGA 6.0 pro-
gram50. Patterns of migration and past migration rates between populations were determined with MIGRATE-N
program following the procedure described by Beerli and Palczewski51. e tested connectivity model was chosen
based on the hydrography of the region and the previous genetic structure analysis. Full migration model with all
migration paths open (asymmetric gene ow allowed) was evaluated in the analysis.
Morphological dierentiation in P. clarkii. To investigate the divergence of morphological traits in P.
clarkii which evolved over a timescale of about 80 years in China, eight natural populations (see Supplementary
Fig.S3) were selected to evaluate the phenotypic patterns. More than 30 individuals of each population were
measured using an electronic digital caliper. Seven morphological characters, including Weight (W), total length
(LT), carapace length (LC), abdominal segment length (LA), rst abdominal segment length (LFA), abdominal seg-
ment width (WA) and carapace width (WC), were investigated. Simple correlation and stepwise multiple regression
analysis were performed using SPSS program (IBM, USA). e relative importance of direct and indirect eects
of measured traits on weight was determined by path analysis for the morphological data. In the path analysis,
weight was the dependent variable and the six characters (mentioned above) were considered as independent var-
iables. Given the variables of the individual size, total length was used to standardize the size eect in the statisti-
cal analysis. e statistics were executed in R (https://www.r-project.org/) using the package coin52. Signicance
was assumed if P < 0.05.
Data Availability. Raw sequence data were deposited into the NCBI Short Read Archive (SRA) with the
accession number SRP135662.
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Acknowledgements
This work was supported by the National Natural Science Foundation of China (No. 31501858) and the
Fundamental Research Funds for the Central Universities (No. 2662016QD009). We would like to thank
anonymous reviewers whose suggestions improved this work.
Author Contributions
Y. Li and S. Yi conceived the study; L. Shi, Q. Li and L. Zhang collected the samples; Y. Li and S. Yi analyzed the
NGS data; S. Yi and Y. Li wrote the manuscript; J. Chen revised the manuscript; and all authors approved the nal
manuscript.
Additional Information
Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-23986-z.
Competing Interests: e authors declare no competing interests.
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Supplementary resource (1)

... Measures of genetic diversity and inter-population variation can be used to make inferences about invasion history and to identify sources (e.g., Bryan et al. 2005;Cristescu 2015;Estoup et al. 2004). Accordingly, both microsatellite and mitochondrial data has been used to identify sources of RSC invasions globally (e.g., Oficialdegui et al. 2019), and to characterize dispersal dynamics within specific regions (Huang et al. 2017;Paulson and Martin, 2014;Yi et al. 2018). Such studies have documented how RSC populations are structured at global and regional scales; however, data are lacking for RSC in the Great Lakes region. ...
... Results largely corroborate findings from previous studies that used microsatellites and/or mitochondrial sequences to inference the sources of RSC invasions from regional to global scales. Regional studies within Nevada (Paulson and Martin, 2014), Italy (Barbaresi et al. 2003) and China (Li et al. 2012;Yi et al. 2018;Yue et al. 2010) found evidence of strong genetic structuring among sampling locales, which the authors suggested are likely indicative of strong founder effects and limited gene flow among local populations. Given that RSC have limited dispersal abilities (1-38 m per day; Gherardi et al. 2002), the above explanations are highly plausible. ...
... Paulson and Martin (2014), which focused on the efficacy of flumes to limit dispersal, point out that limited gene flow even within a single system can create strong structuring. Similarly, the high F ST estimates observed in Li et al. (2012) and Yi et al. (2018) may explain why high values for Ks (range in their study: 4-13) associated with STRU CTU RE analyses were supported despite historical accounts suggesting that a single introduction occurred in NanJiang, China in 1929. ...
Article
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Reducing propagule pressure is critical for preventing the establishment and limiting the spread of invasive species. To reduce the risks associated with propagule pressure, the relative impacts of potential vectors contributing to invasions must be understood. In 2017, the red swamp crayfish (Procambarus clarkii) invasion expanded to include two new invasion fronts in Michigan, USA. To determine potential sources and make inferences about expansion dynamics among introduced populations, we developed genomic resources using restriction-site associated DNA (RAD) sequencing to genotype 175 red swamp crayfish at 1321 single nucleotide polymorphisms. Samples included potential wild and industry sources and collections within each Michigan invasion front. Results based on unsupervised clustering and multivariate analysis indicated that multiple, genetically distinct sources founded the Michigan invasions, which may include sources from the biological supply trade. Results support the recommendation that prevention strategies implement a multifaceted approach targeting diverse user groups associated with the live trades (e.g., biological supply, live food markets, and pet trade). In addition, regulatory frameworks that minimize the presence of a live invasive species should be adopted to reduce the risk of incidental introductions, geographic and demographic expansion, and establishment.
... The red swamp crayfish (Procambarus clarkii), native to the United States and Mexico, was introduced from Japan to Nanjing, China, in the 1930s [1][2][3]. The red swamp crayfish is one of the most aggressive species in the world, with their dredging behavior causing damage to dikes, dams and rice paddies, but due to its high market value, including edible meat, high protein content, low fat content and high nutritional power, it is widely loved and consumed in China and is one of the most economically important aquatic species for aquaculture rather than a destructive invasive species [4]. Recently it has recently become a popular freshwater aquaculture species in China, especially in the middle and lower reaches of the Yangtze River [5]. ...
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The red swamp crayfish (Procambarus clarkii) is an important aquatic animal and has developed as a popular aquaculture species in China. In this study, a total of 72,839 SSR motifs were identified from transcriptional data, and 20 microsatellite markers of them were finally developed to assess the genetic diversities of seven wild populations from natural lakes and nine cultured populations from rice fields. Genetic diversity was slightly higher in the cultured populations than in the wild populations. The degree of genetic differentiation between cultured populations is slight, while a moderate to a large degree of genetic differentiation between wild populations and most of the variations occurred within individuals (79%). The analysis of cluster, principal coordinate analysis and STRUCTURE were similar, and they showed that isolation-by-distance pattern was not significant. The microsatellite markers developed in this study can not only be used for genetic monitoring of population but also provide important information for the management of breeding and cultured population in red swamp crayfish.
... Integrated aquaculture-agriculture technologies have shown great potential for food security and poverty alleviation due to their high synergistic effects [5]. Rice and crayfish are the two most popular food species in Asia countries, especially in China. ...
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In order to explore the breeding direction of morphological selection of Procambarus clarkii , the morphological characteristics of five P. clarkii basic populations from different regions in China were comprehensively analyzed by multivariate statistical analyses. The results showed that there were significant differences in most morphological parameters among populations and between sexes. In the discriminatory analysis, the most discriminant characteristics for distinguishing females among populations were body weight (BW), first abdominal segment width (FASW), third abdominal segment width (TASW) and third abdominal segment height (TASH), whereas for males, the characteristics were body weight (BW), carapace length (CL), carapace width (CW) and third abdominal segment width (TASW).The most significant variables of the differences between sexes were body weight (BW), third abdominal segment width (TASW) and double cheliped weight (DCW). This study would be beneficial to understanding the main morphological characteristics of P. clarkii , which could provide basic data of the collected germplasm resources and some reference for indicating the direction of P. clarkii morphology-based breeding. The germplasm resources with stronger abdomen, smaller carapace and smaller cheliped would be the selection targets, and all-female breeding would also be one of important breeding directions of for P. clarkii .
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Arlequin ver 3.0 is a software package integrating several basic and advanced methods for population genetics data analysis, like the computation of standard genetic diversity indices, the estimation of allele and haplotype frequencies, tests of departure from linkage equilibrium, departure from selective neutrality and demographic equilibrium, estimation or parameters from past population expansions, and thorough analyses of population subdivision under the AMOVA framework. Arlequin 3 introduces a completely new graphical interface written in C++, a more robust semantic analysis of input files, and two new methods: a Bayesian estimation of gametic phase from multi-locus genotypes, and an estimation of the parameters of an instantaneous spatial expansion from DNA sequence polymorphism. Arlequin can handle several data types like DNA sequences, microsatellite data, or standard multilocus genotypes. A Windows version of the software is freely available on http://cmpg.unibe.ch/software/arlequin3.
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The red swamp crayfish (Procambarus clarkii) is one of the most economically important farmed aquatic species in China. Compared with its relatively well-known antibacterial and antifungal mechanisms, the antiviral mechanism is still unclear. We used Illuminabased RNA sequencing and bioinformatic technology to obtain highquality sequence reads from the crayfish lymph organ. A total of 5933 differentially expressed genes (DEGs) were identified between normal and white spot syndrome virus-challenged samples. Of these, 4638 genes were differentially upregulated and 1295 differentially downregulated by more than two-fold. The DEGs were then mapped to different signaling pathways; the Janus kinase/signal transducers and activators of transcription, insulin, and Wnt signaling pathways were predicted to be involved in crayfish antiviral innate immunity. These results provide new insights into crayfish antiviral immunity mechanisms.