Nilaparvata lugens) are the most serious insect pests of rice, one of the world's most important staple crops. They reproduce year‐round in the tropical parts of their distribution, but cannot overwinter in the temperate areas where they occur, and invade seasonally from elsewhere. Decades of research have not revealed their source unambiguously.Brown planthoppers (We sequenced the genomes of brown planthopper populations from across temperate and tropical parts of their distribution and show that the Indochinese peninsula is the major source of migration into temperate China. The Philippines, once considered a key source, is not significant, with little evidence for their migration into China. We find support for immigration from the west of China contributing to these regional dynamics.The lack of connectivity between the Philippine population and the mainland Chinese populations explains the different evolution of Imidacloprid resistance in these populations. This study highlights the promise of whole‐genome sequence data to understand migration when gene flow is high—a situation that has been difficult to resolve using traditional genetic markers. This is due to the central role population connectivity plays in the evolution of traits such as insecticide resistance and biological traits that enhance the pest status of an insect, such as the ability to overcome crops bred to be resistant to insect damage  (Stål) (Hemiptera: Delphacidae)  and it is thought that they would not be able to migrate into central and eastern China because of mountainous barriers and unsuitable monsoon conditions  indicated high gene flow across the distribution of brown planthopper . Library preparation and sequencing was conducted by Total Genomics Solution . Sequencing was performed on Illumina HiSeq X Ten returning 150 bp paired end reads with an insert size of approximately 280 bp. Sequence data quality metrics were estimated with Fastqc Andrews,  and Fast2.2We mapped all reads to the brown planthopper reference genome, assembly GCA_000757685.1 of bioproject PRJNA177647 in GenBank (NCBI), which is approximately 1.2 Gbp  to quality Q30 using vcftools, then to a minimum depth of three  and a maximum depth of 30 (three times our highest coverages). Following this, we removed any marker that was missing more than 30% of the data , to remove the markers most affected by missing data. We then removed any locus with a minor allele count below three (e.g. one homozygote and one heterozygote). Applying hard filters for minor allele frequency can cause weak structure to be missed , and then, all markers with deviations from HWE in these populations were removed from the total data set (using a threshold of p = .05). This final data set contained 762,576 snps and is referred to as the snp genotype data. An alternative approach to calling snps and filtering them is to infer genotype likelihoods, with some analyses methods then being able to use the likelihoods rather than the raw genotypes themselves, preserving the uncertainty in the inferences. We also called genotype likelihoods with the program ANGSD  and COI (cytochrome oxidase subunit one) that was not supported by read mapping, so we removed this region  and used this edited sequence as the reference. We used a pipeline to call snps and prepared a consensus sequence for each individual. Briefly, after mapping, we removed the unmapped reads using Samtools  accessed using the R  .To assess population structure without predefining populations, we performed a principal component analysis (PCA) on the full 2.5We used assignment testing to infer the source of the immigrants into the nonoverwintering locations. Based on the PCA and admixture analyses, we designated the “core‐overwintering” populations (including Tengchong), which we assume to be the putative sources, into three groups: Philippines, Tengchong and Indochinese peninsular. Indochinese peninsular comprised Thailand and Vietnam. We generated linear discriminant functions based on these three groups, using DPAC  alleles removed.The number of population‐specific alleles in each population was calculated using a custom R script, which calculated allele frequency and then identified The genetic diversity summary statistics pi and theta were calculated from the genotype likelihood data for every 10 kb sliding window with 2.5 kb overlap across the genome in ANGSD . The highest pairwise FSTs were from comparisons of the Philippines to other populations  at close to 100% probability. When we plotted the linear discriminant scores for the nonoverwintering individuals and the three “core‐overwintering” groups, the nonoverwintering individuals were placed between the Indochinese peninsular group and Tengchong (but closer to Indochinese peninsular). This is likely a result of the admixture between Tengchong and the other mainland populations .4Our results show that a population genomics approach contributes considerably to our understanding of the migration dynamics of brown planthoppers. The Indochinese peninsular is indicated as a major source of brown planthoppers in their migration to the temperate regions of China (and most likely on to Japan). The Philippines population was genetically distinct from the other populations in all analyses, and little evidence of gene flow from the Philippines into temperate China was found. Tengchong (in western temperate China) also formed a separate group in the admixture analysis and DAPC, but the PCA indicated a mixed population Figures  and 4. Tsnp data, we were, however, able to make inferences into the more recent migratory dynamics of this species (as outlined above and below).Our mitogenome analysis is broadly in line with the results of Mun et al.  who founPhysiological comparisons of bugs from temperate, subtropical and tropical regions have led to suggestions that tropical populations of brown planthopper do not contribute to the migratory dynamics of this pest , as sugThis study highlights that it is still difficult to make precise estimates of migration using classic population genetic methods when gene flow is very high (high migration systems like brown planthopper). The number of individuals that could be included in this whole‐genome analysis was limited by cost. Increasing the number of individuals per population may have provided greater power in our assignment testing. The real promise of whole‐genome sequencing in this kind of study, however, likely lies in the potential to perform not only parentage analysis, but to leverage the information from linkage and recombination to infer deeper pedigree information and estimate migration across multiple generations. This approach would require many individuals to be sampled from each putative population and sequenced with sufficient depth, and is currently cost prohibitive, but will likely become feasible with further advances in sequencing technology.4.1Arsenophonus, has been associated with imidacloprid resistance  in that study is most likely the result of rare immigration of Philippines planthoppers into mainland China. This highlights the key role of migration in the evolution and spread of insecticide resistance (Denholm et al., The Philippines has been reported as a major source of the northward migration of brown planthoppers into temperate regions of China and Japan, based on trap catches, backwards telemetry and modelling (Otuka et al., vice versa, as seems to be the case for imidacloprid (Pang et al., Our results show that the Philippines is distinct, in population genetics terms, from the mainland populations and there is very little migration between them. Insecticide resistance traits that evolve in the Philippines are less likely to spread to the mainland and Our data also indicate that there may be a somewhat distinct population to the west of Tengchong, perhaps in India. Future studies should investigate populations further to the west of the insect's distribution, from Japan, and more sites in the South‐East Asian part of its distribution to further establish the migratory dynamics of this insect and the routes by which insecticide resistance traits travel.5In summary, we used whole genomes to make new inferences about brown planthopper migration. Chinese and Indochinese peninsular populations should be considered the same in terms of management and insecticide resistance. The Philippines population is distinct from these mainland populations, and this explains the prevalence of imidacloprid susceptibility there compared to elsewhere. Our results also indicate the potential for immigration from the west into the Chinese and Indochinese peninsular populations. This route was previously discounted but may present a means for new insecticide resistance traits to enter the Chinese and Indochinese peninsular populations. More generally, our results highlight the promise that population genomics holds for understanding the connectivity of pest insect populations characterized by high rates of gene flow.There are no competing interests to declare.J.P.H and A.M.M analysed the data and designed the figures. J.P.H and Y.M.W wrote the manuscript, with revisions and input from A.M.M, and G.H.W. and X.C prepared libraries for sequencing. C.X and Y.M.W collected the samples. Y.M.W designed the research.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.


Iron deficiency anaemia is a major health problem affecting approximately 1.2 billion people worldwide. Young children, women of reproductive age and pregnant women living in sub-Saharan Africa are the most vulnerable. It is estimated that iron deficiency accounts for half of anaemia cases. Apart from nutritional deficiency, infection, inflammation and genetic factors are the major drivers of anaemia. However, the role of genetic risk factors has not been thoroughly investigated. This is particularly relevant in African populations, as they carry high genetic diversity and have a high prevalence of anaemia. Multiple genetic variations in iron regulatory genes have been linked to impaired iron status. Here we conducted a literature review to identify genetic variants associated with iron imbalance among global populations. We compare their allele frequencies and risk scores and we investigated population-specific selection among populations of varying geographic origin using data from the Keneba Biobank representing individuals in rural Gambia and the 1000 Genomes Project. We identified a significant lack of data on the genetic determinants of iron status in sub-Saharan Africa. Most of the studies on genetic determinants of iron status have been conducted in Europeans. Also, we identified population differences in allele frequencies in candidate putative genetic risk factors. Given the disproportionately high genetic diversity in African populations coupled with their high prevalence of iron deficiency, there is need to investigate the genetic influences of low iron status in Sub-Saharan Africa. The resulting insights may inform the future implementation of iron intervention strategies. Pre-sch in 2017 ,4, parti in 2017 . This hi in 2017 –7.HFE) gene but are also seen in hepcidin ), transferrin receptor 2 (TFR2), solute carrier family 40 member 1 (SLC40A1), haemojuvelin (HJV) and transferrin (TF) genes [TMPRSS6 (transmembrane protease serine 6), have been associated with low iron status [TMPRSS6 genetic variants can lead to different degrees of iron deficiency and anaemia [TF gene, also important in iron transport to cells, have also been reported to affect iron status and lead to low iron status [DMT1), the duodenal apical iron transporter encoded by the SLC11A2 gene have been associated with an unusual syndrome characterized by microcytic anaemia and a paradoxical iron overload [Although iron supplementation can be effective in nutritional IDA, it is ineffective in non-nutritional IDA, particularly those caused by genetic factors . TherefoF) genes –11. ThesF) genes . Geneticn status –20 and an status –21. Indin status ,23. Alth anaemia –20. In an status –26. Furtoverload ,28.TF and HFE, which explain approximately 40% of variation in serum transferrin levels [TMPRSS6 associated with alterations of serum iron status, erythrocyte volume [TMPRSS6 with lowered haemoglobin concentration, and one SNP in TF with increased ferritin concentrations [A genome-wide association study (GWAS) investigating genetic determinants of relevant haematological traits and iron status have identified variants in n levels . Also, Ge volume , and haee volume . Africantrations . DiffereTMPRSS6, HAMP, TF, TFR2, SLC40A1 and HFE genes. We investigated the geographical distribution of studies and assessed the differences in allele frequency of these polymorphisms and their linkage disequilibrium patterns across global populations. We use genetic data from our Keneba Biobank in rural Gambia and from the 1000 Genomes Project. We also explored the possibility of natural selection acting on these genes and any resulting population-specific selection, as measured through large differences in allele frequencies between geographic regions. As part of this, we sought to summarize the geographical distribution of genetic determinants of iron status. The resulting insights may assist in designing future genetic association studies that are geared towards identifying population-specific genetic risk factors affecting iron status and, ultimately, guiding population-specific iron intervention strategies.Here, we conducted a review of the literature to identify genetic variants that have been associated with iron imbalances, with a special focus on SNPs in TMPRSS6, HAMP, TF, TFR2, SLC40A1 and HFE) or iron status measures. These include iron status biomarkers  alone or in combination with haematology traits . Animal studies, case reports, commentaries and articles not written in English were excluded. Rare variants reported in a single individual or family were discarded. Information on genomic and gene location, allele ancestry, minor allele variant and the predicted consequence of each SNP were obtained from the Ensembl dataset (release 98) [A literature search was conducted using the Human Genetic Epidemiology (HuGE) navigator, a database of published population-based human genetic epidemiology studies. This review was complemented using the PubMed site with search terms: “anaemia”, “iron”, “iron overload”, “iron deficiency anaemia”, “iron imbalance”, “hepcidin”, “genome-wide association study”, “GWAS”, “haematology traits”, and “haemochromatosis”. The search was conducted on articles published between 01 January 1999 to 31 October 2018. The assessment process included examining titles and abstracts of studies and excluding duplicates. Articles were included if they were: (1) original research papers conducted in humans; (2) tested for an association between at least one SNP in the genes commonly linked to dysregulated iron status (ease 98) .We obtained genotype data from the Keneba at MRCG at LSHTM   were calculated using the R package Genetics [We assessed the differences in allele frequencies for SNPs with genotype calls in both the Keneba Gambian and the pan-African populations in the 1000 Genomes Project. Linkage disequilibrium (LD) measures  , 1 (heterozygote) or 2 . Using this information, we determined the allele risk scores across populations for both low and high iron SNPs. For 23 SNPs it was not possible to identify the associated alleles (e.g. just a “A/T” label) or classify the direction of association (e.g. absence of regression coefficients). Also, for some SNPs (2111833)  we foundFST to assess differences in allele frequencies) were calculated from the genotype data for all iron-associated SNPs using a combination of the R packages Adegenet [FST values were calculated and range from 0 to 1, where a zero value implies that the two populations are interbreeding, and a value of one means that the two populations do not share any genetic diversity. Population Branch Statistic (PBS) values were calculated using the FST data from the comparison of three populations  according to methods described elsewhere [FST and PBS values, the results were compared with the empirical distribution of genome-wide SNPs reported by others using individuals from several geographical locations and including data from the HapMap and HGDP [The minor allele frequency (MAF), observed and expected heterozygosities and measures of population differentiation  [http://hgdp.uchicago.edu/cgi-bin/gbrowse/HGDP/) at the individual genes and surrounding regions.Statistical differences between MAFs were analysed using the two-proportion Z-Test in R. The integrated Haplotype Score (iHS) ,47 statigo.edu/)  and HGDPThe Keneba Biobank Project received ethical approval from the MRCG at LSHTM Scientific Coordinating Committee and the MRCG at LSHTM/ Gambia Government Joints Ethics Committee (SCC1185). Written informed consent was obtained from each participant.TMPRSS6, HAMP, TF, TFR2, SLC40A1 and HFE)  . More thand HFE) . Nine SNTMPRSS6 gene region (n = 23), where the majority were associated with IRIDA, iron deficiency or indicators of low iron status  were also associated with biomarkers indicating elevated iron status (The highest number of SNPs were identified in the n status . The mosn status .TF gene had the second highest number of SNPs related to either low or high iron status (n = 18). The most common of these (rs3811647) was reported by ten studies  [TFR2 (rs7385804) , both of which were found to be associated with increases in haemoglobin and alterations serum in ferritin concentrations [HFE gene, we found four SNPs that have been associated with alterations in haemoglobin and/or an increase in the genetic risk of hereditary haemochromatosis [HFE variant is rs1800562 (C282Y) [The  studies . This vaomatosis ,51,55–570421768) ,55,58–60trations ,58,61–63omatosis ,62,64–74 (C282Y) ,29,71,72TF, TMPRSS6, HFE and SLC40A1 genes, were available in the Keneba Biobank, because not all the SNPs were on the Exome chip that was used for genotyping this population. When we compared the allele frequencies of the SNPs with data from The Gambians in the Keneba Biobank with the pan-African populations in the 1000 Genomes project, we observed minimal differences  and the 1000 Genome project  . The 100ferences .SLC40A1, and in several SNPs in TMPRSS6 (including rs855791 and rs855788). The intronic variant rs1439816 in the SLC40A1 gene has a MAF of ~20% in the non-African populations but reaches >73% frequency in Africa (TMPRSS6 rs855791) is the most reported SNP associated with iron deficiency and has a MAF of ~50% across all non-African populations, but in Africa it only reaches 10% (7% in the MRCG Keneba Biobank population)  . The intulations .TMPRSS6 rs78174698 and TF rs1799899; associated with increased serum ferritin: SLC40A1 rs11568350; associated with haemochromatosis: HFE rs1800562). The TMPRSS6 rs78174698 (P555S) MAF is low overall (<2%) across most populations, except in South Asia where the minor allele is >10%. The minor allele for rs1799899 (G277S) is rare in Africa and East Asia (<0.2%), and only reaches >4% MAF in European, American and South Asian populations. For SLC40A1 rs11568350 (Q248H), the minor allele reaches 5% in Africans, including in both The Gambian populations in the two datasets analysed. In the other global populations, the ancestral allele is almost fixed. The variant A allele of rs1800562 (C282Y) has the highest frequency in European populations . The frequency of this variant is extremely low in Africans (0.2% in the 1000 Genomes project) and it was not detected (MAF = 0) in the Keneba Biobank population.From the selected SNPs, several in African (n = 10 SNPs) and East Asian (n = 11 SNPs) populations have fixed ancestral alleles or low MAF (<5%) . These STF gene still showed a pattern of high LD in the African populations.We also investigated the population-specific linkage disequilibrium (LD) patterns between SNPs in the candidate genes. There were blocks of high LD in the non-African population, and the overall levels of LD were lower in the African populations , S1 Fig,TMPRSS6  and TF   .The South and East Asian populations had the highest number of low iron risk alleles, whereas, Africans had the lowest and were significantly different from the other populations .-16)  .HAMP, TMPRSS6, TF, SLC40A1, TRF2 and close to HFE) with their risk alleles indicated . These three SNPs were associated with haemochromatosis. Since haemochromatosis is predominantly common in those of European descent and rare in other populations, we analysed these SNPs separately. The European populations have the highest number of high iron risk alleles, significantly different from the other populations (P < 0.00850) (Twelve SNPs were clearly associated with high iron or related biomarker (SNPs in ndicated . Three o0.00850) .TMPRSS6 rs5756506, TF rs1799852 and SLC40A1 rs11568350 (Q248H) were available for the Gambians in the Keneba Biobank. When we compare the frequencies of the high iron risk alleles at these three SNPs across populations (Data for two of the SNPs (rs1799945 and rs1800562) were available for the Keneba Biobank population, but the frequency of risk alleles was low . Therefore, we could not compare the frequencies of risk alleles of these SNPs between the Keneba Biobank population and the 1000 Genomes project populations. Furthermore, we compared the frequencies of the high iron risk alleles of the remaining nine SNPs associated with elevated iron status in other genes. The African population in the 1000 Genomes Project had a significantly lower number of high iron risk alleles than the other populations (P <0.0001) . The disulations , Gambianulations .FST) across the 5 populations to assess population divergence for all iron-associated SNPs. The overall FST across the populations was 0.076. The pairwise FST between the continental groups shows that African versus non-African populations had the greatest allele frequency differentiation  and highest allele frequency differences were rs1439816 in SLC40A1 and rs855791, rs855788 and rs5756506 in TMPRSS6  [We then investigated the individual SNPs driving the differentiation between African and other populations . The var TMPRSS6 . The ave > 0.28) .FST-based test involving the comparison of three populations, to investigate if the differentiation between populations could be driven by positive selection [FST results. In particular, the highest PBS values were present in: SLC40A1  and TMPRSS6   values, an election . We used = 0.07) . These vHAMP, TMPRSS6, TF and TRF2 (iHS<1). However, values of iHS scores close to 2 were found for the regions containing SLC40A1 (e.g. rs1439816: iHS = 1.8 (East Asian-Hapmap ASN), iHS = 2 (European HapMap CEU) and HFE (e.g. rs198846: iHS = 1.8 ASN), suggesting a high frequency of longer haplotypes with the ancestral allele. Other studies have suggested that the HEF locus could be under positive selection in both European and Asian populations [Finally, we investigated if any signals of recent positive selection could be detected in these genes by using the Integrated Haplotype Score (iHS) values from the Haplotter and HGDP selection browsers. The iHS statistic is based on the LD surrounding a positively selected allele compared with the LD around the alternative variant in the same position . A positulations .In this study we identified a significant lack of data on the genetic influences of iron status in African populations. This finding highlights a critical gap since African populations have high genetic diversity, and information from other populations may not be transferable to Africans ,77. AfriWe used genotype data of populations from the Keneba Biobank at MRCG at LSHTM, The Gambia  and the SLC40A1 and TMPRSS6 genes. SLC40A1 encodes ferroportin, a transmembrane transport protein which is the only known mammalian iron exporter [SLC40A1 Q248H variant (rs11568350) is rare globally except in populations of African ancestry populations, where it reaches frequencies of ~5% [SLC40A1 Q248H is associated with modest protection against anaemia and iron deficiency in African children [Substantial differences in minor allele frequencies were observed when comparing the African versus non-African populations. The major differences occur in SNPs in exporter . The SLCs of ~5% . The Q24s of ~5% ,81. Alsochildren ,82.TMPRSS6 gene which encodes for Matriptase-2, a type II transmembrane serine protease that negatively regulates hepcidin synthesis [TMPRSS6 had allele frequencies that are significantly different between African and non-African populations. These variants include rs855791, which has a low MAF (<10%) in African populations and reaches more than 35% in other populations. TMPRSS6 rs855791 is associated with iron deficiency anaemia and IRIDA, with elevated hepcidin, reduced iron and reduced haemoglobin indices [We found significant differences in allelic frequencies for variants in the ynthesis ,83. Impaynthesis ,85, whicynthesis . Several indices ,84,86,87 indices ,89 ThereSLC40A1 and three SNPS in TMPRSS6, which have the highest FST and PBS values in Africa.To understand if the differences in the observed allele frequencies could lead to differences in over- or under-representation of risk alleles leading to iron imbalances, we explored the frequencies of the combined risk alleles across the genes. We found that African populations, including the Gambian population from the Keneba Biobank, had a significantly lower number of alleles associated with the risk of anaemia or low iron. Similarly, we observed a lower number of risk alleles associated with high iron, or iron overload in Africans. This observation is likely because most of the studies were conducted in non-African populations. However, it is also possible that these differences are due to natural selection processes to balance the environmental risk factors to which African populations are exposed. For example, malnutrition and infections  can lead to anaemia or limit iron overload which can increase susceptibility to certain infections . It is possible that the allele frequency differences between populations we described have occurred through founder effects as humans migrated out of Africa rather than through selective pressure. Possible signals of selection have only been observed for one SNP in Our study has limitations. These include the potential for bias in the SNPs selection from the literature as there is an overrepresentation of studies related to genetics of iron imbalances in European and Asian populations. Also, it was difficult to ascertain the risk allele for several variants either because they were not described by the original study and/or the different studies used different genotyping platforms. In addition, although some risk alleles have been confirmed in more than one ethnic group (46% of the SNPs), for other SNPs it is possible that the alleles have different effects across populations and this could affect the risk allele analysis. Overall, our study highlights a major gap in genetic studies in Africa and the need to perform genetic studies in African populations.TMPRSS6 rs4820268 is in strong LD with TMPRSS6 rs855791 in Europeans [We also observed a lower linkage disequilibrium between SNPs in African populations. For example, the uropeans , but we In conclusion, this study identified a substantial disparity in allele frequencies of genetic variants associated with iron, between Africans and other populations. We also, identified the scarcity of data on the genetic influences of iron status in Africa. Given the high burden of iron deficiency in sub-Saharan Africa, particularly in child-bearing women and children, comprehensive mapping of the genetic influences on iron status may help lay the foundation for future studies and assist in developing future iron intervention strategies.S1 FigLD plot showing D prime values in SNPs associated with iron imbalances in (A) African populations, (B) European populations and (C) Gambian population in the Keneba Biobank.(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.


Rhamdia quelen, a Neotropical fish with hybridization between highly divergent mitochondrial DNA (mtDNA) lineages, represents an interesting evolutionary model. Previous studies suggested that there might be demographic differences between coastal lagoons and riverine environments, as well as divergent populations that could be reproductively isolated. Here, we investigated the genetic diversity pattern of this taxon in the Southern Neotropical Basin system that includes the La Plata Basin, Patos-Merin lagoon basin and the coastal lagoons draining to the SW Atlantic Ocean, through a population genomics approach using 2b-RAD-sequencing-derived single nucleotide polymorphisms (SNPs). The genomic scan identified selection footprints associated with divergence and suggested local adaptation environmental drivers. Two major genomic clusters latitudinally distributed in the Northern and Southern basins were identified, along with consistent signatures of divergent selection between them. Population structure based on the whole set of loci and on the presumptive neutral vs. adaptive loci showed deep genomic divergence between the two major clusters. Annotation of the most consistent SNPs under divergent selection revealed some interesting candidate genes for further functional studies. Moreover, signals of adaptation to a coastal lagoon environment mediated by purifying selection were found. These new insights provide a better understanding of the complex evolutionary history of R. quelen in the southernmost basin of the Neotropical region. Rhamdia quelen   is distributed from the northeast end of the Andes mountain range to the central part of Argentina [R. quelen has been proposed as a priority species for conservation in its southernmost distribution because of its cultural and economic value [Rhamdia genus, more than 100 species were synonymized, 46 of which were synonymized with R. quelen [R. quelen [cytb) and cytochrome c oxidase subunit I (COI); however, analyses at different geographical scales based on both markers have not been performed to date. Five R. quelen cytb mtDNA lineages  have been identified in the basin system encompassing the La Plata Basin, Patos-Merin lagoon Basin, as well as rivers and lagoons draining to the SW Atlantic Ocean  [Rq4 and Rq6) in several basins of this region  [R. quelen from southwestern Atlantic coastal lagoon environments appeared geographically isolated and genetically differentiated and showed small effective population sizes associated with putative bottleneck events. In such an evolutionary context, it was hypothesized that genomic footprints of selection might be associated with local adaptation to different environments and could therefore be retrieved using genomic screening across populations [The high Neotropical diversity of freshwater fishes is mostly explained by the hydrogeological history, paleontological evolution and climatic change of that region . Over thrgentina ,10,11. Mic value . Due to . quelen . Previou. quelen ,15, whil. quelen . Those sP-PM-AO) . Microsa basins) . In addi basins) . Homing ulations .Restriction site-associated DNA sequencing (RAD-seq) is a high-throughput technique that enables the simultaneous identification and genotyping of thousands of SNPs (Single Nucleotide Polymorphisms), indels and microsatellite markers in non-model organisms ,19,20. GR. quelen in the LP-PM-AO basin system using 17,559 RAD-seq derived SNPs, at an average density of 60 kb assuming a genome size of 1 Gb [R. quelen using a consistent statistical genomic scanning approach. A further purpose was to integrate previous phylogenetic and population data of the species, particularly those focused on the mitochondrial cytb lineages using the COI gene to extend and redefine mtDNA lineage distribution in the LP-PM-AO basin system of the Neotropical region.The purpose of this study was to characterize the genetic structure patterns of  of 1 Gb . The stuAll sampling protocols were approved by the CNEA  from Uruguay.R. quelen individuals were analyzed, respectively, for cytb and COI mtDNA markers . The voucher specimens were stored in ethanol 95% either at Sección Genética Evolutiva, Colección de Zoología de Vertebrados de Facultad de Ciencias, or at the Museo de Historia Natural of Uruguay. Total genomic DNA was extracted from liver or muscle tissue using proteinase K digestion, followed by sodium chloride extraction and ethanol precipitation . The DNcytb were amplified using fish universal DNA primers Gludg-L and CB3-H [R. quelen individuals and other Rhamdia species were analyzed for the cytb dataset, comprising 22 new samples collected for this study  and 45 ng of template DNA.In order to integrate the data from both mtDNA markers reported in previous studies ,14,15,37 GenBank . The COI HC02198 . The PCRcytb and COI datasets were selected in jModelTest v.2.1.17 [Imparfinis mirini Haseman, 1911 and Pimelodella chagresi   were used as outgroups in the COI phylogenetic tree. The nucleotide substitution models that best fit the v.2.1.17 , based ov.2.1.17 ), the cov.2.1.17 ) and thev.2.1.17 ). A heurv.2.1.17 . SequencAlfI (Thermo Fisher), an IIb type restriction enzyme (RE) that cleaves DNA on both sides of the restriction site. As a result of this digestion, the genome is fragmented in thousands of fragments of 36 bp in length to obtain a fraction of the genome. The 74 individual 2bRAD-seq libraries were quantified using Qubit 2.0 fluorometer  and equimolarly pooled. The pool of libraries was sequenced on a NextSeq 500 Illumina sequencer using a 50 bp single-end chemistry at the FISABIO Bioinformatic Service . Library preparation followed the 2b-RAD protocol  with sliAlfI recognition site in the correct position 12GCA(N)6TGC(N)12) were removed; (3) reads with uncalled nucleotides or more than eight nucleotides with average Phred quality score below 30  were excluded using the process_radtags module in STACKS v.1.46 [IS [IS ≥ 0.50 and ≤ −0.50 were discarded to minimize the presence of null alleles in the former and of paralog sequences in the latter [Raw demultiplexed data were subjected to three filtering steps: (1) reads were trimmed to 36 nucleotides length and shorter reads were removed; (2) reads without S v.1.46 . In the S v.1.46 . SimilarS v.1.46 ) and BowS v.1.46 , which wS v.1.46 ). SNP ge1.46 [IS  (loci wie latter ). AdditiFor the 2b-RAD v2.1 pipeline, we applied the same parameter criteria as in STACKS for analogous parameters. Thus, a minimum coverage of 10 reads was required (-c 10), two mismatches were allowed among global references  and two mismatches were allowed to align reads with reference . Reference was built by selecting 300,000 reads of each sample  to produce a combined dataset with more than 10 million reads. To determine genotypes from alignments, an interval for minor allele frequency (0.1–0.2) was established (consistent with the mean coverage observed in loci built by STACKS) to define if a locus is homozygous , unknown (10–20%) or heterozygous (> 20%). SNPs were filtered using an MAF threshold of 0.05 and based on the total percentage of individuals genotyped (70%) and on the percentage of individuals genotyped per populations (60%) (in at least seven populations). For both loci-building pipelines, SNPs at extreme RAD-tag positions (positions 1 and 36) were not used, following criteria proposed by Wang et al. [IS values (FIS ≤ −0.50 or FIS ≥ 0. 50) in at least three locality samples. Finally, a consistently integrated catalog was constructed with the SNPs shared by both STACKS and 2b-RAD v2.1 loci-building pipelines. For this purpose, RAD-tag sequences with selected SNPs from both pipelines were merged and clustered with cd-hit-est  to identify exclusive and shared RAD-tags from both catalogs. Coincident SNP positions within each RAD-tag pair were confirmed with both catalogs using ad hoc spreadsheets. The final catalog of SNPs was obtained considering a unique SNP per RAD-tag, after eliminating those loci that showed low or high FE-value ˂ e−4, but not matching against Teleostei were filtered out. All RAD-tags bearing outlier SNPs of 36 bp were annotated using the GenBank database and Blastn search of BLAST . SNPs iST-based methods is recommended [p-value ˂ 0.01 in Arlequin analysis were selected as putative outliers. Different population groupings, according to previous data [Rq2, Rq4 and Rq6; The Total dataset comprised 17,559 SNPs. Among these, the Outlier dataset comprised those SNPs identified by both BAYESCAN v.2.1  and Arleommended  and thisommended . The Bayous data  and the Ictalurus punctatus  genome [I. punctatus in Ensembl opened opportunities for further comparative mapping purposes in this study (see below), although limitations for annotations may not be ruled out since R. quelen and I. punctatus belong to different Siluriformes families. An E-value ˂ e−4 was considered as the significance threshold for homologies in both searches. Hypothetical chromosomes that would include the annotated genes were analyzed through a BLASTn search against the I. punctatus genome. Gene mining around outlier loci was performed using Ensembl within the I. punctatus genome. Gene mining was performed only when two or more loci were anchored in the same chromosome region within a range of < 500 Kb. The biological process of the gene lists identified by BLAST and gene mining was performed with BLAST2GO [All SNP-bearing RAD-tags were annotated using the GenBank database and BLASTn search. Additionally, these RAD-tags were annotated using the ) genome  by means) genome . AvailabBLAST2GO . IS. Na, FIS, and HE were calculated in Arlequin [For each locality, the mean number of alleles per locus (Na) and the expected heterozygosity were calculated. The magnitude and sign of departure from Hardy-Weinberg equilibrium (HWE) were analyzed using FArlequin . ST estimates with Arlequin v.3.5, for Total, Neutral and Outlier loci. Finally, Isolation by distance (IBD) was analyzed using Mantel test based on FST and geographical distance matrixes. IBD was performed in Arlequin and based on Neutral loci because they might explain the geographic isolation in absence of gene flow [Population Structure analyses were performed using the Total, Neutral and Outlier SNP datasets. Analysis of clusters and graphical representation of genetic structure were performed by Discriminant Analysis of Principal Components (DAPC). Scatter plots were performed using the R package ADEGENET . For Totene flow , withoutA total of 19 bioclimatic variables  were retrieved from WORLDCLIM database  for the Population history was inferred using TREEMIX software . This soR. quelen mtDNA lineages, a total of 22 new partial sequences of cytb were obtained in this study  [cytb dataset. The cytb topology obtained by PhyML was similar to that by Ríos et al. [Rq2, Rq4 and Rq6  in this study to anchor both types of markers , as well as several sequences, which collapsed in a basal polytomy. Sequences belonging to each mtDNA lineage are detailed in Partial sequences of  GenBank . COI aliplotypes . Accordis et al. , togetheA total of 341,495,357 reads were retrieved from the sequencing platform, representing, on average, 4,614,802 reads per specimen . After applying sequence quality and RE site position filters, 223,148,950 reads were retained. IS values in at least three locality samples were discarded, and the catalog was reduced to 17,575 loci. Finally, a total of 16 loci were discarded because RAD-tags were not homologous to Teleostei sequences, thus the Total dataset was composed of 17,559 loci , a consistent Outlier dataset was identified considering the two highly differentiated clusters and by comparing the Northern and Southern basins of the sampled area. In this analysis, a total of 174 and 524 outlier loci were found, respectively, by BayeScan and Arlequin. A final set of 73 loci detected by both methods was retained as the most confident Outlier dataset between the two major genomic clusters of I. punctatus chromosomes  across all populations. Na ranged from 1.20 (6-AO-BL) to 1.60 (4-NR-RB) (E ranged from 0.249 (5-LP-SL) to 0.328 (6-AO-BL). No significant signals of HWE departure were detected, and FIS values were close to zero across all populations except for 4-NR-RB and 9-ML-QC, which showed high and significant positive FIS values  and Merin Lagoon basin (9-ML-QC) might be explained by a Wahlund effect. When we split these samples by their genomic cluster assignment  and 0.286 (H4-NR-RB) , while HS values . The amoDAPC results based on the Total, Neutral and Outlier loci showed rather similar patterns a–c. The Structure analyses using the Total, Neutral and Outlier SNP datasets showed the best clustering outcomes for K = 2 a–c, wellStructure histograms for K = 2 based on the Total, Neutral and Outlier data a–c were Data analyses of the Total and Neutral datasets with fineRADstructure  showed aST values were similar between Total and Neutral datasets , the Outlier dataset did not evidence genetic differentiation among coastal lagoons  than when using the Total and Neutral datasets . Mantel test was not significant; hence, it did not support the IBD hypothesis (p = 0.016).Pairwise genetic differentiation results were significant for most comparisons tested . Pairwisdatasets . Total, p ˂ 0.01; Selection analyses of Total, Neutral and Outlier datasets resulted concordant, and the “maximum temperature of the warmest month” was the most significant variable explaining genetic differences (p ˂ 0.005). The topology including these four migration events  that coexist in the Uruguay River , Negro River and Merin Lagoon basins (Rq4 and Rq6), the study of genomic differentiation becomes crucial to understand R. quelen population genetics history. Here, we provide evidence of a population structure characterized by two major clusters differentiated by latitude as well as by its demographic history. We identified and annotated several loci putatively under selection. Finally, we unraveled the underlying substructure of populations.This is the first study where the genetic diversity and structure pattern of R. quelen using cytb and COI mitochondrial markers arrived at important evolutionary conclusions in the Neotropical region, but a comparative analysis between mtDNA lineages reported separately for both markers was lacking in the literature [cytb mtDNA lineages that coexist in some basins and that would have diverged in allopatry 5.94–4.55 million years ago (MYA) [Rq2 and Rq4+Rq6 would belong to different clades, A and C, respectively. Clade A (Rq2) would have diverged in the Amazon Basin and would have spread to LP-PM-AO. Clade C (Rq4 and Rq6) would have diverged within LP-PM-AO, which is consistent with marine transgressions in this region [Rq2 individuals here analyzed showed the same COI mtDNA lineage reported for specimens from Miranda River [Rq2 in the Amazon Basin. The interchange of biota between the Amazon and La Plata rivers  was previously proposed by Carvalho and Albert [Serrasalmus and Pygocentrus genera  [Jubiaba acanthogaster  (Characidae family) [COI results showed that the Rq4 mtDNA lineage not only inhabits the lower Uruguay River, Merin Lagoon and Negro River basins, but it is also present in the upper Uruguay and Parana River basins. Rq6, the most abundant cytb mtDNA lineage in the Southern region of LP-PM-AO [COI mtDNA lineage as the specimens from the La Pampa Plain [Rq6 COI mtDNA lineage. The wide distribution of Rq6 could be explained by the exchange among basins as a consequence of low topographic relief, as proposed by Albert and Reis [Rhamdia branneri  from the Iguaçu Basin [Rq6 COI lineage. R. branneri is considered endemic of the Iguaçu River in the far northeast of LP-PM-AO [Rq6 and warn about the need to review the taxonomic status of R. branneri. Moreover, R. branneri could hybridize with R. quelen, not only in hatcheries, as proposed by Scaranto et al. [Previous studies in terature ,6,9,10. go (MYA) , Rq2 ands region . Rq2 indda River    as well  family) , supportLP-PM-AO , was clupa Plain , which ipa Plain  as well and Reis . AlternaLP-PM-AO . These ro et al. .ST values among 1-UR-CR, 2-UR-AR and 3-UR-QR could be due to their common origin in the Uruguay River basin. Total and Neutral pairwise FST analysis recovered significant differentiation among coastal lagoons as previously reported based on microsatellite loci  and lack of structuring among coastal lagoons (FST = 0.000). Bayesian Structure analyses of the three datasets were similar and revealed the presence of two clusters. Like DAPC, the distribution of these clusters follows a North-South pattern. A North Cluster was detected in the Northern basins (Uruguay River basin (UR): 1-UR-CR), 2-UR-AR and 3-UR-QR river basins); the second one, the South Cluster, inhabits the Southern basins (La Plata Basin (LP): 5-LP-SL coastal lagoon; and coastal lagoons draining to Atlantic Ocean (AO): 6-AO-BL; 7-AO-RL and 8-AO-CL). The two highly divergent clusters were detected in the Negro River (4-NR-RB) and in the Merin Lagoon (9-ML-QC) basins. Latitudinal differentiation associated with water temperature has also been reported in the Chinese Sea Bass ) by RAD-seq analysis on populations of the continental marginal seas along the Chinese coast [Salmo salar Linnaeus, 1758) also showed clinal SNP variation associated with latitude in North Atlantic Ocean populations [Genomic differentiation was higher among the Northern samples than among the Southern ones, while central samples (4-NR-RB and 9-ML-QC) were genetically closer to the South samples . These rs et al. : 0.125–0se coast . Furtherse coast . Genomiculations . ST analysis based on a strict set of 73 candidate loci under selection recovered much higher differentiation values among the Northern and Southern locality samples  than those derived from the Neutral (0.336) and Total (0.527) datasets. This could indicate that a strong divergent selection is acting over these genomic regions where outlier loci are located, and consequently, differentiation significantly increases along the genome background. According to Fowler et al. [Gadus morhua Linnaeus, 1758) parallel adaptive evolution associated with temperature clines. Conversely, the Outlier dataset unraveled genetic similarity among the four coastal lagoons studied with common SNP variants fixed at each locus. Because these coastal lagoons would have originated recently , which is involved in osmoregulation and in a possible adaptation through ion homeostasis in plasma in a changing external salinity environment [scn8a is fixed for the same allele in all coastal lagoons might be associated with adaptation in lacustrine populations , a hypothesis that should be further investigated at a functional level. Evidence of adaptation to different salinities has been documented for several fish species through genes involved mainly in osmoregulation [According to the analyses of environmental variables, the higher latitudinal differentiation associated with outliers could be explained by temperature, specifically, the maximum temperature of the warmest month. Both the North and South clusters coexist in central basins, i.e., 4-NR-RB and 9-ML-QC, which is in accordance with absence of isolation by distance and could be the result of secondary contact. The pairwise Fr et al. , temperar et al.  identifiears ago ), selectears ago . Strong ears ago  in theseironment ,87. The gulation ,88,89. Rq4 and Rq6) evidenced by Ríos et al. [Heliconius spp. butterflies [Helianthus spp. sunflowers [Populus spp. poplar trees [Gasterosteus spp. three-spines stickleback [I. punctatus genome  [G. morhua [R. quelen reference genome does not allow analyzing the genomic architecture of the divergence process in this species. Another interesting outlier locus 33517_8 in our study was anchored to sperm acrosome membrane-associated protein 6 gene (spaca6), which is involved in mediating sperm fusion through binding to an egg membrane receptor [R. quelen lack acrosomal structures [The pattern of genetic structure observed revealed strongly differentiated North and South clusters that would represent major lineages of the species. The past migration events evidenced by TREEMIX between locality samples from the North and South clusters would point to a scenario of divergence with gene flow/introgression. These migration events between genomic clusters might be associated with ancestral mtDNA lineage hybridization , or limckleback ,95; Baltckleback ). Some o. morhua ) or with. morhua . Howeverreceptor . This geructures ,102; thuructures , who sugR. quelen follows a geographical pattern and is composed of different genetically divergent populations. In this study, a population substructure pattern was disclosed based on the Total and Neutral datasets. Specifically, the Total and Neutral datasets evidenced a complex substructure . More recent population differentiation within each genomic cluster may have been the result of geographic isolation and genetic drift. The presence of fish assigned to either the North or South genomic clusters in the Negro River and Merin Lagoon basins, suggests the absence of recent introgression, although caution should be exercised due to the limited sample size. Ríos et al. [R. quelen in the LP-MP-AO basin system. Genomic differentiation associated with homing behavior cannot be ruled out, although this behavior has never been described for the Rhamdia genus.Based on 10 microsatellite loci, Ríos et al.  found thtructure  with mortructure e, K = 5 s et al. , based oR. quelen (North and South clusters) from the LP-PM-AO basin system that may be driven by divergent selection. This latitudinal structure pattern could be explained by temperature gradients, which may affect adaptive variation at specific genomic regions. The two major genomic clusters detected show restricted gene flow and represent valuable adaptive variability for the species. In particular, the coastal lagoons should be considered for conservation and management purposes, given the potential population adaptation to the lacustrine environment. Novel insights into the phylogeographic composition of mtDNA lineages within LP-PM-AO were found, and Rq2, Rq4 and Rq6 appeared to be more widely distributed within that basin system than previously suggested. Population genomic analysis has identified high latitudinal differentiation between two clusters of


Neolucanus swinhoei is a widespread species of stag beetle distributed across the subtropical Taiwan Island. The biological features of weak flying capability, two-week adult lifespan, and strong dependence on forests are the key factors determining its genetic differentiation. We investigated the influence of historical climatic oscillations in Pleistocene on the distribution of N. swinhoei. The complex population genetic structure and dispersal routes of eastern populations are affected by the geographic isolation of the north/south-oriented Central Mountain Range (CMR) during periodical Pleistocene glaciations. Four possible shelters were inferred for the distribution of N. swinhoei in each geographic region during the periodical glaciations. The coexistence of widely distributed ancestral haplotypes and the locally derived ones are deduced to be the possible explanation of long-distance dispersal events for N. swinhoei. In the presence of the CMR, the northern and southern low mountains are proposed as alternative routes for N. swinhoei. The present study proposes dispersal routes from eastern to western Taiwan, in contrast to the more common scenarios of western-to-eastern dispersal found in other studies.Neolucanus swinhoei complex was driven by its biological features and habitat requirements as well as hindrance by the CMR during periodical Pleistocene glaciations. The results revealed a tendency of geographical differentiation and major and sub- lineage divergences before and after the Riss glaciation, followed by stable population growth during Würm glaciation. At least four refugia were inferred for N. swinhoei during the Riss–Würm glaciations. The ancestral haplotype retention in the cytochrome oxidase subunit I (COI) gene and compensated substitution in 16S rRNA gene is a possible evolutionary scenario resulting in the inconsistent evolution pattern between COI and 16S rRNA gene coupled with the long-distance dispersal of N. swinhoei. Although the CMR did hinder the dispersal of N. swinhoei, its ancestors may have dispersed to eastern Taiwan through the northern and southern low mountains of the CMR before the Riss glaciation. Our finding suggests that the population growth in the Würm glaciation led a dispersal back to western Taiwan, which is contrast to the more common dispersal scenario from western Taiwan to eastern populations proposed in other studies.The present study demonstrates that the complicated genetic structure of the hilly lineage of the  Historical climatic fluctuations during the Pliocene‒Pleistocene period have been considered a significant factor affecting species diversification and population dynamics of extant organisms ,2,3. In Loxoblemmus appendicularis shifted to eastern Taiwan through a southern route [Kurixalus eiffingeri via a northern route [The Island of Taiwan, situated majorly in a subtropical region, emerged above sea level approximately 5–6 Mya due to collisions between the Philippine Sea plate and the Eurasian plate ,14. The rn route  and the rn route .Neolucanus swinhoei complex (NSC), a group of diurnal stag beetles endemic to Taiwan, are characterized by their complicated phylogeographical history due to the influence of the CMR and periodical Pleistocene glaciations [The ciations . Two majciations . Calibraciations . Anotherciations  reportedN. swinhoei, including weak flight capability, a 2-week adult lifespan, and an altitudinal distribution limited to <1500 m, may shape its differentiation pattern under CMR hindrance [N. swinhoei may be closely related with the recurrent climatic oscillations and CMR hindrance during the Pleistocene.Determining the population genetic structure and historical dispersal of organisms driven by periodical Pleistocene glaciation and geographical barriers in the Island of Taiwan has always been a challenge ,21,24. Tindrance . The strindrance ,25. TherWe propose a hypothesis involving refugia isolation during the Riss glaciation and secondary contact triggered by the Würm glaciation, namely LGM, for the hilly lineage of NSC, which has been demonstrated in plants and other insects ,26,27,28N. swinhoei, repeated isolation in and expansion from refugia driven by the 100,000-year Pleistocene glacial cycle may have caused the evolution of a complicated population genetic structure. The coexistence of widespread haplotypes and geographically specific ones in each population provides evidence for refugia isolations and ancestral synapomorphy during glacial cycles. In the present study, the mitochondrial genes of cytochrome oxidase subunit I (COI) and 16S rRNA gene were used to investigate the differentiation history of the hilly lineage of NSC without the use of nuclear amplicons because of the genetic admixture proposed by Tsai et al. [In N. swinhoei from the Department of Plant Medicine, National Pingtung University of Science and Technology, Pingtung County, Taiwan, and 4 dried specimens from the National Museum of Natural Science, Taichung City, Taiwan. In addition, the COI and 16S rRNA gene sequences of 88 specimens published by Tsai et al. [Neolucanus nitidus from China and Vietnam and individuals from the other three NSC lineages were used as outgroups  and following the protocol of Tsai et al. . The priN. nitidus and specimens from the other three NSC lineages mentioned in Tsai et al. [8 generations with sampling every 1 × 104 generations. The effective sample size (ESS) for the posterior distribution of estimated parameter values were analyzed in Tracer v. 1.5 [The timing of diversification for the hilly lineage of NSC was estimated in BEAST v. 1.6.1  using a i et al.  were usei et al. . The suii et al. ; (2) 1.1i et al. . The Marr v. 1.5  until thhttp://www.google.com/intl/zh-TW/earth/index.html (accessed on 1 October 2016)). Analysis of molecular variance (AMOVA) was performed in Arlequin 3.1  to estimStatistical parsimony network analysis was conducted in TCS v. 1.21 on the basis of a 95% connection limitation , and the8 generations with sampling every 1 × 104 generations. Historical population expansion was inferred from COI + 16S rRNA haplotypes using a mismatch distribution analysis with 1000 bootstrap replications in Arlequin 3.1 [The coalescent-based Extended Bayesian Skyline Plot (EBSP) based on COI + 16S rRNA genes was implemented to reconstruct historical population demographics for the hilly lineage using BEAST v. 1.6.1 . The fitquin 3.1 . Then, tquin 3.1 . eN μ, where eN is the effective population size and μ is the mutation rate) and migration rate M  were estimated using FST-calculation for start values and a starting tree by UPGMA for parameters. The migration rate (M) was estimated at a mutation rate of 2.69 × 10−8 per generation and 2.3 × 10−8 per generation for COI + 16S rRNA genes and the COI gene, respectively [To evaluate the extent of dispersal routes among populations in the hilly lineage of NSC, the coalescent based maximum likelihood method implemented in MIGRATE v. 3.6.4 was employed ,39 usingectively . The parN. swinhoei. The sequence lengths for COI and 16S rRNA genes were 686/669 bp and 550 bp, respectively. The average base compositions of G, A, T, and C were 16.5%, 30.8%, 30.9%, and 21.8% in the COI gene and 20.9%, 29.2%, 40.0%, and 9.9% in 16S rRNA gene, respectively. A total of 83 new sequences amplified in the present study have been deposited in GenBank under LC590052–LC590134 for the COI gene and LC590135–LC590217 for 16S rRNA gene.Both COI and 16S rRNA genes were successfully acquired for 171 specimens of Based on the origin time of approximately 0.2 Mya, phylogenetic inference using COI + 16S rRNA genes revealed four lineages in the hilly lineage of NSC, with several minor sublineages correlated with the geographical distribution of populations . LineageMolecular dating based on COI + 16S rRNA genes  showed that major lineage divergence occurred 0.18–0.28 Mya in the middle Pleistocene, and the subsequently formed sublineages diversified around the time of the LGM, which was 0.01–0.11 Mya . PhylogeThe substitution rates applied for insects, namely COI: 1.15%/lineage/Myr, 16S rRNA gene: 1%/lineage/Myr, revealed that the origin time of each lineage was approximately 0.3 Mya . Four maN. swinhoei differs in eight substitution steps from the outgroups  in populations A, B, C, D, F, and I, whereas significant negative values were found only in population A in 16S rRNA gene. Moreover, Fu’s Fs values from the COI gene revealed significant negative values (p < 0.05) in populations A, B, C, D, E, F, and I; in 16S rRNA gene, significant negative values were found in populations A, B, C, D, F, H, and I (Nucleotide diversities (π) of all populations ranged from 0.0009 to 0.0029 for the COI gene and from 0.0019 to 0.0061 for 16S rRNA gene . NucleotH, and I . FST < 0.05) was detected for most populations in the COI gene, the indices from 16S rRNA gene showed greater differentiation (p < 0.05), except population C. Populations from western Taiwan, namely populations B, C, D, and E, revealed moderate to high genetic differentiation from the eastern populations G and H. Moreover, slightly positive relationships between nucleotide divergence and relative geographical distances were observed in both genes ulations . The posulations . Studies and NSC ,53. A poormosana ,53,54. Sterflies ,56. N. swinhoei might have promoted frequent gene flow between adjacent populations, namely, between southern populations D and I and between northern populations A and F , crabs (Candidiopotamon rathbunae/Geothelphusa), common cricket (Loxoblemmus appendicularis), damselfly (Euphaea formosa), swallowtail butterfly (Troides aeacus formosanus), and the southeastern lineage of NSC [Euphaea formosa, on the basis of genetic structure analysis [N. swinhoei may have had stable growth and dispersed back to the western region through lower mountains in northern and southern Taiwan (The altitudinal distribution of l routes . The 16Sulations ; thereafe of NSC ,57,58,59n Taiwan . Owing to the weak dispersal capability and two-week adult lifespan of NSC, the long-distance dispersal events from the eastern populations to western Taiwan might have been induced by the retention of the ancestral haplotypes in both 16S rRNA and COI genes; the intermediated haplotypes in 16S rRNA gene with geographical association could also account for the long-distance dispersal events . For insThe present study revealed the peculiar genetic structure of the hilly lineage of NSC and investigated possible phylogeographical scenarios on the subtropical Taiwan Island. The inconsistent evolutionary patterns between COI and 16S rRNA gene were likely the result of functional constraint, biological features of 2-week adult life span and weak flying capability, and secondary contact after stable demographic growth during the LGM. Together, these factors resulted in ancestral haplotype maintenance and new mutations originating in different genealogical periods and geographical populations. Four possible refugia were identified in northern, northwestern, southern, and eastern Taiwan. The southern and northern dispersal routes to eastern Taiwan are congruent with those identified in previous studies, although the extent of refugia and dispersal duration may reveal a different differentiation history. The westward dispersion from eastern populations due to population expansion during the LGM is the first reported case in the phylogeographical studies in Taiwan.


Lindera aggregata (Lauraceae), a dominant species in South-East China was selected to address this issue. Fifteen low-copy nuclear genes (LCGs) and four chloroplast DNA (cpDNA) fragments were used to detect its evolutionary history. In LCGs, STRUCTURE and dated Bayesian phylogeny analyses detect distinct subtropical-tropical divergence since late Pleistocene. Approximate Bayesian calculation (ABC) further supports the distinct subtropical-tropical divergence, and close related Taiwan and South China populations are diverged at the last interglacial. Isolation by distance, isolation by environment and isolation by resistance analyses suggest the current climatic difference rather than geographical distance contributes to the genetic differentiation. Principle component analysis shows populations of tropical cluster occur in warmer area with higher precipitation. Ancestral area reconstruction based on Bayesian phylogeny indicates that ancestral L. aggregata populations are distributed in tropical region. In cpDNA, although unique haplotypes are found in tropical region, distinct subtropical-tropical divergence is absent. In conclusion, distinct late Pleistocene subtropical-tropical divergence of L. aggregata is triggered by climate. It is likely that L. aggregata is originated in Southwest-South China and experienced hierarchical dispersal from south to north. The South China Sea land bridge has dual role in connecting or isolating Taiwan and mainland populations since the last glaciation.In East Asia, genetic divergence is usually considered to be correlated to different floristic regions, however, subtropical-tropical divergence is largely ignored, compared to widely explored temperate-subtropical divergence.  Floristic region, defined as the assemblage of all plants in a given region and time, is formed through tempo-spatial evolution and distribution of plants under historical and contemporary geo-climate conditions2. Different floristic regions are usually relate to different geologic structures, geomorphic units or climatic zones2. Different floristic regions in temperate, subtropical and tropical climates from north to south in East Asia have uneven biodiversity3 and likely distinct evolutionary histories4.East Asia harbours a great biodiversity in Northern Hemisphere because of extreme geo-diversity with conjunction of historical climate and sea level changesAcer mono (Sapindaceae), the northern temperate populations were formed through range expansion recently while the southern subtropical populations were long-term persisted, and intermediate area in North China was occupied with admixed populations6. Similar genetic pattern was revealed in Juglans ssp. (Juglandaceae) and contribution of temperate-subtropical environmental difference to genetic divergence was further emphasized7, as different climate may has resulted in local adaptation after long-term independent evolution since late Miocene. Sharper divergence without genetic mixture was found in Lindera obtusiloba (Lauraceae)8 and Euptelea (Eupteleaceae)9. These species are more arid-sensitive and the inland Taihang-Qingling migration route in North China is not available9. Since no suitable habitat in the East China Sea (ECS) shelf was predicted during the last glacial maximum (LGM) using ecological niche modeling (ENM)9, the ECS acts as geographical barrier for genetic interchanges in L. obtusiloba (Lauraceae)8, Euptelea (Eupteleaceae)9 and some other species10. So, contemporarily climatic and topographical condition are both responsible for the temperate-subtropical genetic divergence.Genetic divergence corresponding to temperate-subtropical differentiation has been widely explored. The temperate-subtropical divergence was firstly found in 2, while subtropical-tropical differentiation related genetic divergence was poorly understood. Although numerous phylogeograhic studies were conducted in subtropical region, limited species stretches its distribution to the tropical region11. Shared chloroplast genetic components without clear subtropical-tropical divergence were widely found15. In Tetrastigma hemsleyanum (Vitaceae), Wang et al.12 found early divergence in chloroplast genome (late Pliocene) while recently genetic mixture in nuclear genome between subtropical and tropical populations. The failure to uncover subtropical-tropical divergence may due to two reasons. The first reason may relate to the real absence of subtropical-tropical divergence as the only differences are weak climatic difference and topographic barrier2, and the boundary of subtropical-tropical climatic zones16 is further south compared to that of the two floristic regions2. The second reason relates to methodological issues. Chloroplast genome with low mutation rate is prone to trace early demographic history before the Pleistocene, while recent divergence and admixture since the Pleistocene should turn to nuclear genome17, which is very limited in previous studies12. So, whether genetic divergence occurs between subtropical and tropical regions remains to be tested.Different floristic division also occurs in subtropical (Sino-Japanese Floristic Region) and tropical  regionsLindera aggregata (Lauraceae) is a dominant shrub or small tree of evergreen broadleaved subtropical forests that is widely distributed in subtropical region but it can stretch to the tropical region as well , there are two accepted infraspecific taxa, L. aggregata var. aggregata, and L. aggregata var. playfairii which is only distributed in South China. The difference between the two varieties lies in type of pubescence, and size of leaf and flower. Our previous phylogeographic work using four chloroplast DNA (cpDNA) fragments, rpl16, psbA–trnH, trnL–trnF and trnS–trnG, 15 self-developed low-copy nuclear genes (LCGs) and ENM uncovered distinct south-north divergence. The northern populations experienced extensive post-glacial range expansion that contrasts with its generalized long-term population stability in the southernmost range, and noteworthy post-glacial gene flow into long-term refugial populations was also found20.ell Fig. a18. It iL. aggregata20 suggests that it is possible to detect potential subtropical-tropical divergence. However, the sampled populations were biased in subtropical region in our previous work20. Populations in tropical region and contact zone between south-north clusters are limited. To resolve this problem, more populations in these two regions were sampled  was conducted to detect if there is any subtropical-tropical divergence. Ancestral areas were reconstructed to infer distributions of ancestral populations. Isolation by distance (IBD)21, isolation by environment (IBE)22 and isolation by resistance (IBR)23 were calculated to infer driving force for genetic differentiation.South-north divergence accompanied with environmental difference in ed Table  and the L. aggregata, (2) if yes, whether geographical or climatic condition contributes to the genetic differentiation, and (3) the demographic history of L. aggregata.The specific aims of the present study are to detect (1) whether subtropical-tropical divergence exists in 20. In the present study, six additional haplotypes are obtained through six additional substitution sites  and haplotypes from populations in South China (H11 in NAN and H1 in BLSH) form a monophyletic lineage  was widely distributed in our previous workes Table . The hapes Table . All thees Table . Althoug20, eighteen sampled populations were divided into three distinct clusters with one located in south, the second in the north-eastern part and the third in the north-western part using LCGs. In the present study, K = 5 is the most possible number of genetic clusters using 29 populations based on LnP(D) and ΔK  and 112 ka , respectively. Other parameter estimations are shown in Table In DIYABC, scenario 3  which supports the subtropical-tropical divergence receives the highest support (PP = 0.67) compared to scenario 1  and 2   and they experience vicariance between tropical and South China region after dispersal . Based on geographic, environmental and genetic distance, Mantel test and partial Mantel test show significant IBE , or  when accounting for geographic distance, while no significant IBD , or  when accounting for environmental distance  while not IBD . Mantel test further shows significant IBR .The environmental and geographic distance show weak correlation  of the total variation. Figure 20, more detailed genetic structure is revealed and distinct subtropical–tropical divergence is detected. Although one widespread haplotype (H3), more diverse haplotypes in southern region and no distinct structure are consistently found using chloroplast markers20, various analyses including genetic differentiation distribution, genetic structure, Bayesian phylogeny, ABC modeling of nuclear markers, which have much higher mutation rate24, successfully detect the recent subtropical–tropical divergence that can be only traced back to late Pleistocene (109.7/154 ka).With additional 11 sampled populations, especially in the tropical and genetic admixture regionL. aggregata. The PCA analysis shows that tropical populations are tend to occur in warmer areas with higher precipitations compared to subtropical populations, similar to our previous study20. It should be noticed that a significant IBE does not necessarily imply the occurrence of adaptation to local environments26. Short evolutionary history (since late Pleistocene) occurred in small scale compared to the late Miocene origin Juglans spp. that are wildly distributed in East Asia7. Further, the two varieties are very hard to discriminate as the difference lies only in type of pubescence, and size of leaf and flower, and intermediate types are also found during our field work. Thus, whether adaptive evolution exists between subtropical and tropical populations needs further investigations. The significance of IBR may largely due to climatic difference because the IBR implicitly conflates IBD and IBE22, and the resistance distance was calculated based on the ENM at present using 10 low correlated bioclimatic variables20.The IBD and IBE analyses indicate climatic difference rather than geographical distance is responsible for the genetic differentiation of 28 would act as dispersal corridor in L. aggregata as introgressions from three subtropical clusters are found29. The distinct clustering of subtropical red and green or even yellow cluster may be caused by demographic expansions. Waters et al.30 suggest the genetic partitioning of re-colonizing genotypes could potentially produced by a combination of occasional northward long distance dispersal (LDD) and high-density blocking. In L. aggregata, occasional LDD dispersed by birds31 combined with high-density hindering would cause genetic partitioning in subtropical region32 although low-level of genetic differentiations are found (Table 34 and South China Sea (SCS)35 should act as geographical barriers between Taiwan and the mainland populations even though no significant IBD is found. High pairwised genetic differentiations between Taiwan and other clusters combined with low genetic diversity  in LCGs and two unique chloroplast haplotypes indicate Taiwan population is long-term isolated36. Bayesian phylogeny of LCGs further implies that it is likely a relict population. Thus, the Taiwan Strait and SCS have successfully impeded both pollen and seed dispersal between Taiwan and the mainland. While in Qiongzhou Strait, shared nuclear and chloroplast genetic component between Hainan island and the mainland  between Taiwan and South China populations with BEAST (90.7 ka). The time falls into the last interglacial when sea level raised37. The sea level fluctuations of SCS due to glacial-interglacial alternations can both provide dispersal corridor or barrier39. The L. aggregata populations likely showed continuous distribution between South China and Taiwan in early stage, while geographic isolation afterwards has resulted in genetic distinctness. Previous study on Quercus championii (Fagaceae) also suggests the Pleistocene SCS land bridge contributed to flora of Taiwan island by dispersal from ancestral Southwest China-Southeast Asia35. Some terrestrial vertebrates also display similar patterns40.In Taiwan, its cpDNA haplotypes are closely related to that from South China although limited variations are found  and the remaining seven populations in Paleotropical Floristic Region 2  from 1 to 10 with 100,000 Markov chain Monte Carlo (MCMC) steps of burn-in, followed by 1,000,000 steps. LnP(D) and ΔK were applied to determine the most likely number of clusters. Pairwised genetic differentiation (FST) and genetic diversity as measured by π and AR of LCGs among potential clusters were calculated in SPADS 1.045.In cpDNA, DnaSP 5.10.0146 in BEAST 2.447 with all partitions unlinked. The substitution models for all loci were the same as Ye et al.20. A strict clock model and Yule process with a piecewise linear and constant population size model were applied. The length of the MCMC algorithm was set to 2 × 109 steps with sampling every 2 × 104 steps, and the first 20% was discarded as burn-in. The estimated substitution rate of 3.4 × 10−9 with 95% HPD of 1.8 × 10−9–5.8 × 10–9 site−1 year−1 was applied to estimate divergence time20.In LCGs, the Bayesian phylogeny of 29 populations (population WGSH was excluded due to sequencing problems) was inferred using *BEAST48. All individuals were subdivided into TW, southern cluster, and northern cluster  in subtropical region according to STRUCTURE and Bayesian phylogeny analyses (see “As the lineage including the Taiwan population (TW) and populations in southern cluster  does not receive significant support see “”, the suses see “”. Three ses see “.FST between pairs of clusters. The simulation was repeated 1 × 106 times for each scenario. To compare the posterior probability of three scenarios, the 3 × 104 (1%) simulated data sets closest to the observed data set were selected for the logistic regression and 300 for the direct approach. After choosing the best scenario, we estimated parameter posterior distributions taking 1 × 104 (1%) simulated data sets closest to the observed data set for the local linear regression, after applying a logit transformation to the parameter values. Two independent runs were performed in all simulations.Each simulation was summarized by the following summary statistics: number of segregating sites, mean and variance of pairwise differences, private segregating sites, and mean and variance of number of rarest nucleotide at segregating sites with cluster, mean of segregating sites, mean of pairwise differences, and L. aggregata, the Bayesian binary MCMC (BBM) analysis implemented in RASP 3.249 was performed using 80% post burn-in trees retained from the BEAST analysis of LCGs. Four geographic regions representing the current distribution were defined according to the floristic division of China2: A, Central China; C, South China; B, East China, and D, Tropical China. The number of maximum areas at each node was set to five. We applied 20 MCMC chains with the JC + G model running for 1 × 106 generations, and sampled the posterior distribution every 100 generations with the first 10% treated as burn-in.In order to reconstruct the geographical diversification of FST) of LCGs was calculated in SPADS and FST/(1 − FST) was used as genetic distance. The geographic distance was transformed by natural logarithm. Climate variables of the 29 sampled populations of 19 climatic variables, which was downloaded from WorldClim 1.451 at a 2.5-arcmin resolution, were extracted using Spatial Analyst Tools in ArcGis 10.2 . Pairwised Euclidian distances calculated (‘dist’ function in R 3.2.350) using the matrix of all 19 retrieved climatic variables was adopted to represent environmental distances. A PCA (‘princomp’ function) was performed in R50 on the 19 retrieved climatic variables to detect tropical-subtropical environmental differences.To infer the contributions of current geography and environment to genetic differentiation, IBD, IBE and IBR was analyzed. Pairwised genetic differentiation (52) using 126 occurrences of L. aggregata20. The ENM resulted in a map of environmental suitability values ranging from 1 to 0 with ratio of potential distribution from high to low. Resistance values were the reverse of the suitability scores (1 − suitability), because higher suitability is expected to have lower resistance53. Least-cost path distances are calculated by finding the minimum total cumulative resistance between two populations53 using SDM Toolbox in ArcGis 10.2. ‘partial.mantel.test’ function in R50 was used to perform Mantel tests between genetic distance and geographic distance, environmental distance or resistance-based spatial distance, and partial mantel tests between genetic distance and geographic distance  while accounting for environmental distance (or geographic distance) using 1 × 104 permutations. MMRR54 that provides a straightforward method for estimating linear regressions among distance matrices was further used to qualify IBD and IBE. The effect of environmental distance and geographic distance, as explanatory variables, on genetic distance, as the response variable, was analyzed using the ‘MMRR’ function in R50 with 1 × 104 permutations.The resistance-based spatial distance was calculated based on ENM at present. The predicted distribution was modeled by 10 low correlated (r < 0.8) bioclimatic variables  at a 2.5-arcmin resolution through maximum-entropy modelling technique (Maxent 3.3.3Supplementary Information.


Corbicula fluminea s.l. is an edible freshwater bivalve of economic value in Asia. The species has been particularly well studied in the invaded range. However, there is a lack of knowledge in its native range where it supports an increasing commercial harvest pressure. Among Asiatic countries, China accounts for 70% of known commercial harvest and aquaculture production. We aim to characterize here wild C. fluminea s.l populations exposed to commercial harvest pressure in Poyang Lake Basin. We found higher biomass, density and genetic diversity in lake populations compared to peripheral populations . Given that lake habitats support more intense harvest pressure than peripheral habitats, we suggest that demographic and genetic differentiation among subpopulations may be influenced in some degree by different harvest pressure. In this regard, additional demographic and/or genetic changes related to increasing harvest pressure may place population at a higher risk of extirpation. Altogether, these results are especially relevant for maintaining populations at or above viable levels and must be considered in order to ensure the sustainability of the resource.The freshwater clam  The comThe main changes associated to unnatural selection are related to demographic and genetic characteristics on wild populations. For example, harvesting large individuals may cause a reduction of biomass, and biases populations towards younger ages and smaller sizes at maturation . In the Corbicula fluminea s.l., is an edible freshwater bivalve of economic value in Asia  populations in the native distribution range. First, we aimed to test whether lake and peripheral  populations show different biomass density parameters. Second, we aimed to test whether (1) genetic diversity will be related to drifting organisms from lake to peripheral populations or (2) genetic diversity will be related to migration from peripheral populations to the main lake. Finally, we discuss wether observed demographic and genetic parameters could be related with different harvest pressure between lake and peripheral habitats, and how these results could be of utility in the invaded range.The goal of this study was to provide one first demographic and genetic characterization of http://www.fao.org) of the United Nations food balance sheets (2016). To the best of our knowledge this service constitutes the best available source of information for landings’ estimation. Data were aggregated by region  and country , fishing area , animal group  and species .First, commercial harvest data (in tons per year) for the period 1950 to 2017 were obtained from the Food and Agriculture Organization  across six sites . Observations from multiple visits were used to estimate the number of harvest boats in each zone. Corbicula fluminea individuals were directly purchased from local fishers between 2018 and 2019. Fishers were identified by their on-site location and obtained clams were bagged, labelled, returned to the laboratory and preserved in 95% ethanol. Following, samples were counted and measured to the nearest 0.1 mm along the longest posterior-anterior axis using Vernier calipers.Second, we aimed to characterize 2) was used for sampling in Poyang Lake and the Yangtze River. Second, a quantitative Surber sampler for benthic macroinvertebrates  was used in mid-size rivers. All samples were collected in triplicate at each sampling site. Retrieved C. fluminea samples were transported to the laboratory at the University of Nanchang in a low temperature incubator. Specimens were counted and weighed with an electronic balance (HANGPING FA1204B) to the nearest 0.1 g.In addition, we established a total 84 sampling points covering 28 sampling sections and eight sampling areas, which included (1) lake habitats representative of high harvest pressure, and (2) peripheral habitats representative of low harvest pressure . SamplesEnvironmental data for the 28 sampling sections were referenced from C. fluminea among habitats were tested using Analysis of Variance (ANOVA) on ln (x) transformed data. No significant differences were found in density or biomass between sampling time points . Hence, the four sampling time points were pooled together as one sample in order to enrich the data used. In addition, the intercepts of biomass-density relationships were compared between habitats by a unifactorial Analysis of covariance (ANCOVA). The ANCOVA tested for the effect of a predictor  beyond the effects of one known covariate (density).Data were tested for homogeneity of variance using Levene’s Test and Kolmogorov–Smirnov’s Test for normality. Differences on the size of harvested C. fluminea (n = 216) were collected during 2018 and 2019 from the main area of Poyang Lake , the connected river channel of Poyang Lake , Ganjiang River , Fuhe River ; Xinjiang River ; and Raohe River  in Poyang Lake Basin. Tissues of C. fluminea samples were preserved in 95% ethanol until DNA extraction. The TINAamp Marine Animals DNA Kit was used to extract the genomic DNA. DNA was evaluated by spectroscopic methods  and agarose gel electrophoresis.Samples of C. fluminea. The PCR reaction was performed in a 25-µL volume containing 12.5 µL 2x Taq PCR MasterMix (TianGen), 9.5 µL ddH2O, 1.0 µL forward primer, 1.0 µL reverse primer and 1 µL genomic DNA. PCR amplifications were performed with an initial denaturation of 94 °C for 2 min, followed by 35 cycles of 94 °C for 1 min, 50 °C for 1 min and 72 °C for 1 min; a final extension temperature of 72 °C for 7 min. Gel electrophoresis was used to confirm successful amplification. An EZ-10 Spin Column PCR Product Purification Kit  was used to purify the PCR products. An ABI 3730XL DNA Analyzer  was used to sequence the purified DNA.We used the cytochrome c oxidase subunit-I (COI) primer LCO22me2 (5′-GGTCAACAAAYCATAARGATATTGG-3′) in combination with a reverse primer HCO700DY2 (5′-TCAGGGTGACCAAAAAAYCA-3′) to screen for reliable amplification using the 216 samples of H), haplotype diversity (H d) and nucleotide diversity (π) for C. fluminea population from mtDNA COI sequences. MRBAYES v.3.2.2  was used to test the statistical significance of genetic divergence within and among populations and habitats  based on ARLEQUIN 3.5  of the RDA gradient, and the eigenvalues of the first 2 axes were used to measure their importance (X + 1) transformed to improve their normality before data analysis. CANOCO v 4.5  occurring in China.C. fluminea was 19.05 ± 4.95 mm (n = 411). However, most of the captures  were ≥ 15 mm. There was not a significant effect of habitat on the size of harvested C. fluminea .The total observation number of harvest boats was distributed between 120 and 160 in the Poyang Lake Basin, including lake and peripheral habitats. Lake habitats concentrate more harvest pressure  than peripheral habitats; i.e., half of the harvest boats distributed across the four sites in peripheral habitats. In general, across the six sampling sites within Poyang Lake Basin, the mean harvestable size ± SD for C. fluminea density and biomass was higher in lake habitats than in peripheral habitats  = 30.753, p = 0.001) indicated different regression slopes. After accounting for the density effect, the individual of C. fluminea from peripheral habitats was heavier and larger than the individual from lake habitats (habitats . A signihabitats .MN233672-MN233683) were found among 216 sequenced C. fluminea individuals around Poyang Lake Basin  contained 82 individuals and was shared among individuals from PY, GJ, CR, FH, XJ and RH. The analysis of haplotype network showed similar geographic structure with a general lack of geographic resolution as the phylogenetic analysis . Among lake population, the genetic variance was 1%, but not significant . Based on the AMOVA results, a total genetic variance of 0.002% was found between lake and peripheral populations, and the differentiation was not significant between them .There was a lack of significance for the mismatch distribution and neutrality tests based on mitochondrial COI sequences (p < 0.01), which indicated that C. fluminea population did not experience recently demographic expansion , 25.13% of the total genetic variance was found among populations, and the differentiation was significant among populations . These harvest data are necessary to verify our initial assumptions that a fishery such as that described here is phenotypically non-random. In this regard, we hypothesized that harvest can induce demographic and genetic changes in wild C. fluminea s.l. populations. Partially supporting this assumption is the observation that the stock of C. fluminea s.l. is in decline compared with 20 years ago (Maximum density: 156 ind. m−2 in 1997–1999 to 30 ind. m−2 in 2016–2017), which reflects the increasing fishing pressure  have dramatically affected Poyang Lake biodiversity . Environ species .C. fluminea were correlated with the dissolved oxygen, water velocity, turbidity and water temperature. In addition, these environmental factors have been also related with survival and growth of C. fluminea (C. fluminea populations (C. fluminea survival in water of 0 °C decreased from 100% to 17.5% with increasing exposure from 4 to 9 weeks. In addition, low river flow and low dissolved oxygen have been related with high mortalities in several macrozoobenthic species, such as C. fluminea (Anthropogenic alterations such as dam construction, water pollution and sand mining at Poyang Lake Basin are important factors responsible of environmental changes . It has fluminea . For exafluminea . Unusualulations . Increasulations . Müller fluminea .C. fluminea s.l. has become one of the most successful invasive species all over the world (In addition to commercial harvest, harvest management has been also proposed as an effective strategy for the containment, and long-term control of invasive species . Among fhe world . The spehe world . On thishe world . NeverthC. fluminea s.l. supports an important fishing industry. In the particular case of the Poyang Lake Basin, the number of harvest boats has been estimated on the basis of personal unstructured observations which do not give us perfect answers, but it gives an estimate to characterize this particular fishery and how lake and peripheral habitats differ in demographic and genetic characteristics. In this regard, lake populations exposed to higher harvest pressure showed demographic and genetic differentiation. Hence, it is likely to think that harvest management may affect population parameters in some degree. These results are especially relevant for those species of economic value because sustainable management should maintain populations at or above viable levels. In addition, our results have shown that harvest management can reduce population numbers. It should be noted, however, that harvest management as control strategy can be a high risk undertaking because it could incentivize deliberate long-distance introductions increasing the genetic diversity and, in consequence, enhancing its invasive potential.Present results have important implications in the native and invaded range. First, in the native range, 10.7717/peerj.9657/supp-1File S1DNA sequence data and the number of individuals and weight of Corbicula fluminea among the different habitats, sampling sites and seasons in the Poyang Lake Basin. These DNA sequence data, the number of individuals and weight were used for statistical analysis.Click here for additional data file.10.7717/peerj.9657/supp-2File S2Click here for additional data file.