Pectinophora gossypiella (Saunders) infestation on Bt cotton is a major concern to cotton production in India. The genetic diversity and phylogeographic structure of the insect in light of PBW resistance needs to be revisited. The objective of this study was to identify different haplotypes of pink bollworm and their distribution in India. To achieve this we studied the population structure in 44 cotton growing districts of India. The partial mitochondrial COI sequence analyses of 214 pink bollworm populations collected from 44 geographical locations representing 9 cotton growing states of India were analysed. Genetic diversity analysis exhibited presence of 27 haplotypes, among them Pg_H1 and Pg_H2 were the most common and were present in 143 and 32 populations, respectively. Distributions of pairwise differences obtained with partial COI gene data from the overall Indian populations are unimodal, suggesting population expansion in India. Significant neutrality test on the basis of Tajima’ D and Fu’s Fs presented a star-shaped haplotype network together with multiple haplotypes. The unimodal mismatch distribution, rejection of neutrality test with significant negative values supported the theory of demographic expansion in cotton pink bollworm populations in India. Genetic data not only provides us with a perspective of population genetics, but also that the two populations of pink bollworm, those occurring early in the season are genetically close to the late season populations with respect to their partial CO1 region. Resistance to Cry toxins does not seem to have had an impact on this region of the mt DNA in populations of pink bollworm.Pink bollworm,  In India, cotton is mainly cultivated in three distinct agro-ecological zones 4. Cotton is cultivated on an area of 12.43 million ha with average productivity of 505.46 kg/ha in India during 2017–185. Several biotic and abiotic stresses are constraints in cotton production including the bollworm complex and sucking pests6. Among the bollworm complex, pink bollworm (PBW), is one of the major damaging insect pests of cotton with an extensive range across India leading to severe loss to cotton production8.Cotton is one of world’s most essential fiber crops having global significance, cultivated in tropical and subtropical regions of more than 70 countries9. The PBW is assuming a major pest status even in some regions of northern India where there are ginning and oil extraction units which are procuring cotton seeds from central and south Indian cotton states where PBW has demonstrated resistance to Cry toxins in the field. So there is possibility of resistant pink bollworm infesting Bt cotton in North India through movement of seed. The evolution of resistance and pest adaptation to Bt crops containing Cry1Ac and Cry2Ab has been observed recently12. The development of resistance is due to multiple factors such as absence of refuge13 or supply of fraudulent refuge13, mono cropping, cultivation of long duration hybrids, extended cropping season14.Pink bollworm has become apparent as a threat to cotton cultivation in south and central cotton growing zones of India where the pest has developed resistance to Cry1Ac and Cry2Ab expressing cotton also developing resistance to insecticides and infesting late season cottonG. hirsutum cotton in India. BGII was expected to be effective against the pink bollworm especially after resistance to the single gene Cry1Ac was reported as heavy field infestations of PBW in Bollgard (BG), that was confined to Gujarat state in 200915. Despite reports in 2015 of plausible breakdown of BGII resistance the contribution of stakeholders of the technology was grossly inadequate to ensure its sustainability16.Bollgard-II (BGII) cotton expressing two proteins, Cry1Ac and Cry2Ab occupies approximately more than 90% of the area cultivating Bt-cotton expressing Cry1Ac (BG) and ‘Cry1Ac + Cry2Ab’ (BGII) was assesed in 10 major cotton-cultivating states of India during 2010–2017. However the PBW larval incidence during this period on Bt-cotton was found less in north cotton cultivating zone of India, where as in central and south India, PBW larval recovery from BGII cotton bolls was high in the range of 28.85–72.49% during 2014–20178. PBW infestation causes locule damage of 37.5% and 13.58% on non-Bt and Bt cotton respectively, at about 160 days of planting17. Presently PBW is assuming a major pest status even in northern India where it had minor pest status earlier. Recently the pink bollworm strain having 300-fold resistance to Cry1Ac, 2.6-fold cross-resistance to Cry2Ab identified and analyzed with novel cadherin allele (r16) builds its life cycle on transgenic Bt cotton containing Cry1Ac18.Pink bollworm (PBW) adaptation to transgenic 20. Mitochondrial DNA (COI gene) is maternally inherited, well conserved and evolves in a nearly neutral fashion so it reflects the divergence times, which makes it a robust marker for determining genetic relationships and geographical studies25. Further the genetic constitution of a pest population is very vital in determining its capacity to tolerate adverse climatic conditions and adoption to new conditions26. Population genetic structure and genetic diversity defines the level of adaptation of a population to environmental change and susceptibility to selection pressure28. Gene flow through dispersion and migration which is responsible for determining genetic variation leads to evolution of local populations29. Even in some of the lepidopteran species, the genetic diversity and genetic structure are reported to be related to their migration capacity as well as number of generations31.Mitochondrial DNA is widely used in taxonomy and systematics to explore the phylogenetic relationships of insectsBt cotton by PBW populations34. Liu et al.35 studied the population genetic structure of Chinese PBW using mitochondrial COII and Nad4 primers, and found extremely low genetic variability among all populations examined from nine provinces of China. Sequence variation in the Nad4 region differentiated the Chinese populations from the Pakistani and American populations. Haplotypes and differentiation in PBW populations of China was identified using piggyBac-like elements33. Sridhar et al.34, based on the analysis of pink bollworm population from 19 districts in 2011 and 2012 found that pink bollworm population in India exhibited low level of genetic diversity, and based on haplotype diversity results opined that the populations might be experiencing population expansion but could not provide the evidence through neutrality tests owing to the small population size.Studies on population structure and genetic diversity of PBW has been explored more in Asiatic countries such as India, Pakistan and China owing to the development of resistance to Bt resistant field collected Indian populations of P. gossypiella from three distinct cotton growing eco-zones of India spanning nine cotton growing states and forty four major cotton growing districts using mitochondrial cytochrome oxidase I (COI) gene. The study also aimed to understand the genetic diversity of populations of pink bollworm infesting early, collected from damaged rosette flowers and late season populations from infested bolls to understand the diversity of the two temporally separated populations of the cotton crop to attribute plausible reason for early outbreak of PBW.Present investigation was designed to analyze the genetic diversity and distribution and to validate the theory of population expansion in different P. gossypiella were collected from green bolls on cotton growing in different geographic locations of 44 districts of 9 states of India during 2017 and 2018  −2 µl, 10x taq buffer-2.5 µl, MgCl2(25 mM)−2 µl, dNTP(10 mM) −0.5 µl, forward primer (10 µM)-0.5 µl, reverse primer (10 µM)-0.5 µl, TaqDNA polymerase (3U)-0.4 µl and distilled water-16.6 µl. The PCR reaction was carried out in thermal cycler (Applied Bio systems) with the conditions such as initial denaturation at 94 °C for 5 min followed by 35 cycles of 94 °C for 45 s, primer annealing at 52 °C for 1 min and extension at 72 °C for 1 min with final extension at 72 °C for 5 min. The amplified products were resolved on 1.2% agarose gel, stained with ethidium bromide (10 mg/ml) and visualized in a gel documentation system (Biorad). Gel Extraction kit (QiagenVR) was used for extraction and purification of desired sized sample from agarose gel. After purification of samples from gel, samples were sequenced (both strands) through service of agrigenome Lab, Cochin, India using COI forward and reverse primer on automated ABI PRISM 3100 genetic analyzer (Applied Biosystems).Partial sequence of mitochondrial CO1 gene (850 bp) was amplified with universal primer COIF 5′TTGATTTTTTGGTCATCCAGAAGT3′ and COIR 5′TCCAATGCACTAATCTGCCATA TTA 3′ reported by Simonato ver. 7.0.9 software38. Descriptive statistics number of haplotypes (H), haplotype diversity (Hd), variance and standard deviation of haplotype diversity were calculated using DnaSP ver 5.10.01 software39. To depict the evolutionary and geographical relationships among haplotypes, a median-joining (MJ) haplotype network was constructed with Propart ver. 1.7 software  method was performed using MEGA rch Fig. . The rel43 were also performed using DnaSP ver. 5.10.01 software for detecting the range of population expansions. The genetic differentiation (FST) between different state populations in range referring to the criterion by Wright44 defined genetic differentiation as low for FST < 0.05, moderate for 0.05 < FST < 0.15, high for 0.15 < FST < 0.25 and very high for FST > 0.2546. The goodness of fit of population expansion analysis was done with Raggedness indices model according to Harpending47.Fu and Li’s D_test and F_test and Tajima’s D tests of neutrality index and genetic differentiation will be useful for demographic history information, with demographic expansion related to negative values and subdivided populations at equilibrium leading to positive valuesver. 7.0.9 and used for further analysis. The trimmed sequences were deposited in NCBI Gene Bank and accession numbers  were obtained.To determine the genetic diversity and distribution, 686 bp trimmed nucleotide sequences of 214 mt-COI region represented by South (52), Central (129) & Northern (33) zones of three cotton-growing zones of India were used for analysis. The homology search of sequences using NCBI BLAST matched with the mt-COI sequences of pink bollworm in database, and the sequence similarity varied from 98–100% to that of available sequences of pink bollworm populations. The nucleotide sequences were aligned using MEGA The study identified a total of 2712.61%) unique haplotypes in PBW populations of the three cotton growing zones of India Fig. . Among t2.61% uniP. gossypiella the descriptive statistics Haplotype (gene) diversity (Hd), Variance of Haplotype diversity and Standard Deviation of Haplotype diversity were calculated with DnaSP ver. 5.10.01 software at 0.531, 0.00149 and 0.039, respectively, which suggest that entire population exhibited low level of genetic diversity. In this study zone-wise clustering revealed that central zone recorded low level of Hd (0.499) as compared to south (0.560) and north (0.595). Similarly, a state wise analysis revealed low levels of genetic diversity in Rajasthan (0.356) and Maharashtra (0.446) populations  showed a slightly bimodal curve in north region while multimodal pattern in Haryana and Punjab region mismatch distributions may indicate strong population subdivision which confers a stable population size Fig. . The ragThe pairwise FST values ranged from −0.12018 to 0.10469. Of 28 comparisons, 9 showed moderate and the remaining 19 showed low genetic differentiation. The values of pairwise genetic distance between Gujarat population and other populations range from (−0.0359 to 0.10469) indicating low to moderate genetic differentiation.The low values (−0.01141 to 0.07145) were observed in Maharashtra population as compared to other populations. Comparison of Punjab populations with other populations revealed that genetic differences ranged from −0.00327 to 0.05875. Low to moderate values were observed in comparisons of the Haryana populations as compared to other populations (0.05875 to 0.10469)  haplotypes in 79 individuals distributed in 19 populations of Indian subcontinent. The eight haplotypes were shared by at least two populations other 19 haplotypes were unique and did not share the same ancestral haplotype. The haplotype (Pg_H1) was most distributed as it was found in 143 individuals, and the second most predominant haplotype (Pg_H2) was shared with 32 individuals as confirmed with the star-like haplotype network shared by populations across different zones  in 79 adult individual moths during 2011–2012 populations of pink bollworm before full blown resistance to both Cry1Ac and Cry2Ab in India. Similarly extremely low level of population genetic variation was observed in the two mitochondrial regions (COII and Nad4) among the nine Chinese Pink Bollworm populations, with only one to four mitochondrial haplotypes which were attributed to invasion bottlenecks, which had subsequently strengthened by its non-migratory biology and the mosaic pattern of agricultural activities32.Accurate assimilation and understanding of the genetic diversity of an insect pest is found essential to mitigate and improve its monitoring that further facilitates implementation of need based independent strategies. In this study zone-wise clustering revealed that the central zone recorded low level of Hd as compared to south and north zones. Low mitochondrial DNA variations are reported in taxa that might have undergone severe bottlenecks or founder effectsBt cotton might have influenced the evolutionary status of PBW as it is having narrow host range and limited dispersal ability53. The non-migratory behavior might also have influenced the genetic variation as dispersal patterns influence genetic variation in Lepidoptera species due to limited gene flow among populations55. To control broader range of pests Bt crops expressing new genes and multiple genes can be developed also in some countries like United States and Australia the non Bt cotton refuge strategy in which 25% of the area planted to cotton with a single Bt toxin protein as this area can be reduced by using two Bt proteins58. The high-dose refuge management strategy in which mating between susceptible and resistant individuals has been key to postpone counteract evolution of Bt resistance59.Commercialization of 61. Overall, the haplotype network showed a star-shaped cartography as characteristic of population expansion after a bottleneck, wherein newer mutations form groups of lower-frequency haplotypes budding from a central haplotype  obtained with COI gene data. Similar trend was observed in early and late season populations of PBW. Genetic data thus obtain not only provides us with an perceptive of population genetics, but also that the two populations of pink bollworm, those occurring early in the season is genetically close to the late season populations with respect to their CO1 region.We investigated the population genetic diversity and structure of 214 populations and haplotype distribution of Supplementary Information.


Procambarus virginalis) represents a very recently evolved parthenogenetic freshwater crayfish species that has invaded diverse habitats in Europe and in Madagascar. However, population genetic analyses have been hindered by the homogeneous genetic structure of the population and the lack of suitable tools for data analysis. We have used whole-genome sequencing to characterize reference specimens from various known wild populations. In parallel, we established a whole-genome sequencing data analysis pipeline for the population genetic analysis of nearly monoclonal genomes. Our results provide evidence for systematic genetic differences between geographically separated populations and illustrate the emerging differentiation of the marbled crayfish genome. We also used mark-recapture population size estimation in combination with genetic data to model the growth pattern of marbled crayfish populations. Our findings uncover evolutionary dynamics in the marbled crayfish genome over a very short evolutionary timescale and identify the rapid growth of marbled crayfish populations as an important factor for ecological monitoring.The marbled crayfish ( Olena Maiakovska et al. provide whole-genome sequencing of the parthenogenetic and invasive marbled crayfish and develop a computational framework for data analysis of monoclonal genomes. These data and methodology allow the authors to demonstrate genetic separation between two populations and provide the first size estimate for a marbled crayfish colony, which they used to model population growth patterns. Arthropod examples include parthenogenetic lineages of silkworms (Bombyx mori), water fleas (Daphnia pulex), weevils (Otiorhynchus scaber), and stick insects (Timema spp.)5. Complete genome sequences from parthenogenetic animals have presented unique features, and it has been suggested that parthenogenetic genomes evolve toward effective haploidy, as predicted by the Meselson effect7. However, these processes require parthenogenesis over millions of years. Also, a comparative analysis of 24 parthenogenetic genomes revealed a substantial degree of diversity in their features, which reflects the considerable biological differences among the corresponding species8.Parthenogenetic reproduction, i.e., the development of embryos from unfertilized eggs, has been described in many groups of animalsProcambarus virginalis) are obligate parthenogenetic freshwater crayfish that were first described in the German aquarium trade10. The species is a recent descendant of the slough crayfish , which is an abundant, sexually reproducing freshwater crayfish species in peninsular Florida and southern Georgia11. Marbled crayfish may have originated from P. fallax through an evolutionary very recent macromutation, which resulted in instant reproductive isolation12. All known animals can likely be traced back to an initial population that was founded in 199510. As such, the evolutionary age of the species appears to be ~25 years.Marbled crayfish  of animals from various aquarium lineages and from wild populations suggested that the marbled crayfish meta-population represents a single, genetically homogeneous clone18. Genetic variants are generated by the ongoing accumulation of natural mutations18, but their numbers are greatly limited by the extremely young evolutionary age of the species.A de novo draft assembly of the marbled crayfish genome with a genome size of approximately 3.5 Gbp and >21,000 genes has been published recently19. In parallel, the animals have increasingly colonized wild habitats. It is generally assumed that these populations were founded by anthropogenic releases and thus represent offspring of a single animal. This is exemplified by the situation in Madagascar, where the animals were first detected around 200621, and have now populated an area of 100,000 km2 that encompasses many diverse habitats22. Isolated wild populations have also been described in Germany and in several other European countries29, but their genomic variation has not been analyzed yet.Due to their overall robustness and aesthetic appeal, marbled crayfish have become popular and widely distributed aquarium pets31. Our study addresses these issues and provides information about the emerging genetic differentiation within the marbled crayfish population.The clonality of marbled crayfish genomes presents unique challenges and requirements for the analysis of genomic variation. Marbled crayfish populations are groups of individuals that coexist spatially and temporally, but appear to miss classical attributes of panmictic populations, like random mating of individuals and active recombination. Hence, the population genetic structure of clonal marbled crayfish cannot be characterized by allele frequency dynamics, as the underlying processes are typical for sexually reproducing populationsFor a systematic genetic comparison of marbled crayfish populations in Europe, we surveyed known populations. This revealed that some reports had described transient appearances of single animals or groups of animals (probably from recent anthropogenic releases), while others described wild populations that have now stably persisted over several years. We restricted our study to stable wild populations and confirmed the taxonomic identity of the animals by genetic authentication of multiple specimens from 15 populations and from nine different European countries  patterns (see “Methods” for details), using the genome sequence of a specimen from the oldest known aquarium lineage of marbled crayfish (founded in 1995) as a reference. The numbers of SNVs ranged from 5722 to 6007. This is an order of magnitude below the frequency of SNVs in clonal daphniidsage Fig. . Paralle15 and suggest that genetic variation in marbled crayfish is driven by natural mutation.Further analysis showed that the proportion of unique, partially shared, and fixed polymorphisms were similar for all representative individuals Fig. . In addiTo characterize the genomic variation of a single population, we analyzed WGS datasets from five animals from the type locality . After processing the dataset through the previously established pipeline (Supplementary Table S18 and a newly generated dataset from Madagascar (Supplementary Table SN = 10) and Malagasy (N = 5) samples  that was identified in our analyses. The results showed that the majority (74%) were located in intergenic regions and only 4% were located in coding regions . Subsequently, we applied three different growth models  to simulate the population growth, based on a range of physiological parameters for marbled crayfish reproduction (see “Methods” for details). We then calculated the probabilistic total genetic variability for the populations with different simulated growth models considering a well-established arthropod reference mutation rate37. Further analysis showed that the genetic variability with a simulated exponential growth intersects with the variability of the current Lake Reilingen population after 3.7 (±0.3) years  of the European and Malagasy populations, respectively40. Key examples are provided by bdelloid rotifers and darwinulid ostracods, which represent ancient generalist asexual organisms with wider geographical distribution and broader ecological tolerance than their sexual parent species. Clonal variation has been demonstrated in darwinulid ostracods and has been attributed to mutation accumulation over long evolutionary timeframes42. The analysis of marbled crayfish genomes will facilitate our understanding of the mechanisms of asexual genome diversification and adaptation. This could conceivably include the preservation of high heterozygosity levels, the selection of specific genetic variants, and changes in genome regulatory mechanisms44.The lack of sexual reproduction and genetic recombination limits genetic variation and is often considered to constrain phenotypic diversity and environmental adaptation. However, the general-purpose genotype model suggests that asexuality can be evolutionarily successful through the selection of generalist lineages with large phenotypic plasticity that can occupy a variety of ecological niches45 might facilitate the detection of additional populations in the future and thus uncover the full extent of the marbled crayfish spread. Of note, our study also revealed a considerable number of animals in a relatively small habitat. The rapid growth of marbled crayfish populations likely represents a key factor in the emerging economic development of the animals in Madagascar46. Importantly, this rapid growth also suggests that marbled crayfish populations should be monitored to determine their impact on freshwater ecosystems.In conclusion, our study describes 15 stable populations of marbled crayfish from various European countries. It is very likely that a large number of additional populations has not been identified yet. This can be explained by the recent emergence of the animals, their nocturnal activity and the lack of awareness about their existence. eDNA-based methods22. Genomic DNA was isolated and purified from abdominal muscular tissue using a Tissue Ruptor (Qiagen), followed by proteinase K digestion and isopropanol precipitation. The quality of isolated genomic DNA was assessed via agarose gel electrophoresis and/or via TapeStation (Agilent). PCR genotyping was performed using the mitochondrial cytochrome B and the nuclear Dnmt1 markers18. Whole-genome sequencing was performed on an Illumina HiSeq platform (DKFZ Genomics and Proteomics Core Facility).Animals were collected from various European wild populations  with default parameters48. The resulting alignments were processed and filtered for duplicates using samtools (version 1.3)49. Also, alignments on short contigs (≤10 kb) were discarded to avoid loss of time in processing fragmented sequences. Sequencing statistics for each individual are summarized in Supplementary Table STrimmed reads were aligned to the marbled crayfish genome (assembly version 0.4)50 and SnpSift51. In addition, SNV association with genetic features was analyzed using SnpSift51. Filtering for indels was performed by custom filtering pipelines. The comparative analysis included all P. virginalis WGS datasets, and a WGS dataset from the oldest known aquarium lineage sample (Heidelberg) as a reference18. Only sites that were covered in the reference were included in the analysis. To streamline the analysis in the triploid background, heterozygous sites in the reference were eliminated, which focused the analysis of polymorphic sites on non-heterozygous loci. Polymorphic sites were identified by the presence of a single-nucleotide substitution relative to the reference sequence. Other types of substitutions such as insertions or deletions were also discarded from the analysis.The Bayesian genetic variant detector Freebayes was used for variant calling with parameters set for triploidy, minimum mapping quality of 30, and minimum base quality of 20, to exclude low-quality reads and bases from the analysis. Filtering was based on a minimum read depth of 20, a maximum read depth of 200, and a minimum Phred-scaled quality score of 30 for each sample, using gatk-4.1.3.052. Finally, an unrooted phylogenetic tree was generated using the Phytools package53 and default parameters.The re-coded matrix was used for the calculation of distances between pairs of individuals followed by neighbor-joining tree estimation with default parameters using the Ape packageP. virginalis mitochondrial genome reference sequence (GenBank KT074364.1). After filtering for quality parameters and duplicate removal, the alignment files were used for the generation of consensus sequences of mitochondrial genomes. The genetic divergence was calculated by multiple sequence alignment in Clustal Omega with default parameters. The phylogenetic tree was constructed based on maximum likelihood estimation using PhyML54 with default parameters.For the mitochondrial tree, WGS reads were trimmed and mapped onto the The initial SNV dataset was used for the selection of heterozygous positions in the reference genome sequence. Sites were filtered for a Phred-scaled quality score >30 and a sequencing coverage >5×. This resulted in 250,453 heterozygous positions for VAF analysis. VAF per site was calculated as the ratio of the alternative allele read count to the total number of reads.55. The heatmap was generated based on the permutated matrix using the Pheatmap R package with default clustering parameters and a row aggregation parameter of kmeans_k = 200.After variant calling, each genotype of the analyzed sample was permutated with integers from 0 (0/0/0) to 3 (1/1/1) according to the alternative allele dosage. A PCA for each set of markers was run using the R package PCAMETHODSThe population size estimate was performed at Lake Reilingen  in July 2018. A bathymetric map of Lake Reilingen was established using a Smart Sonar PRO + (Deeper) equipped with a GIS system and sonar. A total of 18 traps (mesh width: 1 cm) were deployed at various water depths , of the surface covered by each trap (arbitrarily fixed SE = 10%), and the ratios of the considered populations relative to all measured populations .We evaluated the standard error (SE) by taking into account the uncertainties of the Schnabel estimate, of parameter −9 per basepair and per year37 were used. Growth models included classical exponential growth, logistic growth, and growth based on a strong Allee effect. The classical exponential growth model describes a population size at any time point t byN(t0) = 1 for a clonal population with a growth rate of (exp(g) = 200 ± 100) per year. The logistic population growth model is described byNmax = 192,000 (SE = 34.98%) for the current population. The growth with strong Allee effect described by56 of −3.1.To simulate marbled crayfish population growth kinetics, the observed population estimates, a growth rate of 200 (±100) offspring per animal and per year, and a mutation rate of 3.6 ×1037 of the 3,511,656,756 bp marbled crayfish genome assembly18 were multiplied by population size estimates.For calculating the genetic variability of the Lake Reilingen population, the identified SNVs and the estimated mutation rateP. virginalis specimens from 15 independent geographic locations. For one population (Lake Reilingen), five independent specimens were analyzed. Variant calling was performed on SNVs with a read coverage >20×. All statistical analyses and visualizations were performed using R (v3.5.0).This study is based on the analysis of WGS datasets of 19 Further information on research design is available in the Peer Review FileSupplementary InformationReporting Summary


Chilo suppressalis , is an important agricultural pest that has caused serious economic losses in the major rice-producing areas of China. To effectively control this pest, we investigated the genetic diversity, genetic differentiation and genetic structure of 16 overwintering populations in the typical bivoltine areas of northern China based on 12 nuclear microsatellite loci. Moderate levels of genetic diversity and genetic differentiation among the studied populations were detected. Neighbour-joining dendrograms, Bayesian clustering and principal coordinate analysis (PCoA) consistently divided these populations into three genetic clades: western, eastern and northern/central. Isolation by distance (IBD) and spatial autocorrelation analyses demonstrated no correlation between genetic distance and geographic distance. Bottleneck analysis illustrated that RSB populations had not undergone severe bottleneck effects in these regions. Accordingly, our results provide new insights into the genetic relationships of overwintering RSB populations and thus contribute to developing effective management strategies for this pest.The rice stem borer (RSB),  Chilo suppressalis , is a devastating rice pest in Asia, the Middle East, Europe and Oceania [The rice stem borer (RSB),  Oceania . It atta Oceania ,3. In Ch Oceania . It has  Oceania ,6.RSB is a polyphagous pest with high fecundity and low dispersal capabilities ,6. ThereCOI, COII, 16S, ND1 and Cytb) and microsatellite loci [COI gene sequences [Population genetics is a useful tool to illuminate the population genetic structure and to predict insect migration routes . The genite loci ,19,21. Iite loci . In addiequences .To our knowledge, RSB has relatively limited dispersal capacity, and adults disperse less than 1–3 km . DiscontIn this study, we investigated RSB population genetics in the typical bivoltine areas of northern China. Pure overwintering populations were collected in April 2019 through dissected the rice stubble. Twelve microsatellite loci were employed to investigate the genetic diversity, genetic differentiation and genetic structure of these RSB populations. The purpose of this study was to understand their population genetics and to provide a useful theoretical basis for developing effective control strategies for this pest.By dissecting rice stems, a total of 367 RSB individuals were collected from 16 overwintering populations in Liaoning Province, Northeast China, from March to April 2019, and the numbers of individual samples per locality ranged from 8 to 30, averaging 22.6 (± 7.79) samples per locality . Each saIn this study, genomic DNA was extracted from individual samples using a Qiagen DNEasy Blood & Tissue Kit . Twelve microsatellite loci developed specifically for RSB were used ,26. AmonFST with and without using the excluding null alleles (ENA) correction conducted in the FreeNA programme [Na), Shannon's information index (I), effective number of alleles (Ne), expected heterozygosity (He), unbiased expected heterozygosity (uHe) and observed heterozygosity (HO) were estimated using GenAlEx 6.5 software [FIS), fixation index (FST) and allelic richness (AR) were calculated with FSTAT 2.9.3.2 [PIC) analysis was conducted by CERVUS 2.0 [The probabilities of the presence of null alleles, large allele dropouts and scoring errors were detected using Micro-Checker 2.2.3 . After Mrogramme . Hardy-Wrogramme . Correctrogramme . The measoftware . The inb 2.9.3.2 . PolymorRVUS 2.0 .F-statistic (FST) in Arlequin 3.0, and the significance of the FST values was computed with 10,000 permutations [FST values and geneflow (Nm) values [Population genetic differentiation was estimated using Wright’s utations . The genutations . Heat ma) values .K) from one to sixteen [K for 500,000 iterations after a burn-in period of 50,000. The most likely number of clusters was determined based on the ΔK approach, which was implemented in Structure Harvester [K in CLUMPP 1.1 [FST/(1-FST) and ln (geographic distance) in GenAlEx 6.5 [Five methods were used to infer the population genetic structure of RSB. First, an unrooted phylogenetic tree was constructed based on the neighbour-joining (NJ) method using POPTREE 2 . Second, sixteen ,40. The arvester ,41. The UMPP 1.1 . In addiUMPP 1.1 . Fourth,UMPP 1.1 . Fifth, AlEx 6.5 . FinallyAlEx 6.5  and PeakAlEx 6.5 . Moran'sAlEx 6.5  were estAlEx 6.5 .Population genetic bottlenecks were investigated using Bottleneck 1.2.02  based ona = 0.05). In addition, the average FST values obtained with and without applying the ENA correction were 0.063 and 0.061, respectively, and these FST did not differ significantly . Thus, the presence of null alleles did not influence the FST estimation (Na) ranged from 2.750 in Cs117 to 17.500 in Cs381, with an average of 6.646. The effective number of alleles (Ne) ranged from 1.401–29.155 (average = 5.693). The Cs381 locus had the highest numbers of alleles and effective alleles. The observed heterozygosities ranged from 0.199 to 0.718, whereas the expected heterozygosities ranged from 0.287–0.967. The PIC values ranged from 0.888 in Cs248 to 0.380 in Cs133 and had a mean value of 0.616. The basic summary statistics of genetic variation among different RSB populations in the typical bivoltine areas of northern China are presented in Ho = 0.468) was similar to the mean expected heterozygosity (He = 0.595) in all populations. There were different levels of genetic variation among these populations. The unbiased expected heterozygosity (uHe) ranged from 0.543 in Beizhen (BZ) to 0.680 in Qingyuan (QY). The inbreeding coefficients (FIS) of the 12 microsatellite loci in all the RSB populations were very low (FIS = 0.198), indicating an excess of heterozygotes, which reflected a balance in the sex ratio of these populations. The Liaoyang (LY) population had the highest level of genetic diversity, while the Fushun (FS) population had the lowest level of genetic diversity. The observed Na values ranged from 4.333 in FS to 8.500 in LY and had a mean value of 6.646. The Ne values across the microsatellite loci ranged from 2.959 in Xinglong (XL) to 4.414 in Liaoyang (LY).In the present study, a total of 367 individuals from 16 populations in Liaoning Province, Northeast China, were genotyped using 12 microsatellite loci. Low null allele frequencies per locus were obtained, ranging from zero to 0.206 . No locutimation . The genFST = 0.111). The pairwise FST values for genetic differentiation varied from 0.004 to 0.193 based on the nuclear data, and only 19 of the 136 pairwise comparisons were no significant genetic differentiation  and Zhangwu (ZW), LH (Linghai), and Donggang (DG) and those Liaoyang (LY) and Haicheng (HC), Zhangwu (ZW) and Huanren (HR). Overall, a low level of gene flow  was detected among the studied populations. Meanwhile, a high level of gene flow was found between some populations, such as Qingyuan (QY) and Zhuanghe (ZH)    .The unweighted NJ tree based on the microsatellite data of the 16 RSB populations defined three major groups . One groK at K = 2 , Shenyang (SY), Fushun (FS) and Zhuanghe (ZH) in the second cluster and the remaining ten population grouped in the third group. This result was similar to that of the above NJ tree based on the same data.The Bayesian clustering model-based method allowed the degrees of genetic structure and admixture to be assessed among populations. An initial STRUCTURE analysis indicated a maximum Δat K = 2 , which cK values . In addis   . There wThe results of bottleneck analysis based on twelve microsatellite loci in the typical bivoltine areas of northern China are summarized in The results of GENECLASS analysis revealed that the assignment rate of individuals to their original location was 89.10% (40/367). The remaining 40 individuals (10.90%) were putative migrants and were evenly distributed in the typical bivoltine areas of northern China. In addition, a total of 13 potentially first-generation migrants were detected in the studied populations, and only four first-generation migrants were found in the Donggang (DG) population, which indicated that the low level of gene flow and non-directional spread occurred among most RSB populations in the typical bivoltine areas of northern China .He = 0.648) was found. This is in agreement with the results of Meng et al. [He in these 18 Chinese populations ranged from 0.591 to 0.725 [He in South China ranged from 0.625 to 0.833 [FIS values of four RSB populations, Tieling (TL), Linghai (LH), Xinglong (XL) and Zhuanghe (ZH), were slightly less than zero, indicating an excess of heterozygotes . A large number of RSB could migrate from southern to northern China annually, and this genetic exchange could lead to high heterozygosity in the RSB populations, enhancing the adaptability of these populations towards changing environmental conditions and thus leading to the high level of genetic diversity observed in the populations in these areas.The study of intraspecific variation can not only uncover new putative species but also allow inference of evolutionary origin and population history . In thisg et al.  and Liu g et al. . The valto 0.725 , and theto 0.833 . Moreoveozygotes . NotablyCytb gene and found significant genetic differentiation among the populations [COI gene sequences [Nm values indicate less than 2 migrants per generation, which suggests that the observed differentiation levels were the result of migration–drift equilibrium. If Nm > 4, then the local populations belong to one panmictic population [Nm = 2.002) were found among most of the populations, which attributed to the habitat barriers or environmental isolation in these regions [We found a moderate level of genetic differentiation among these overwintering RSB populations in the typical bivoltine areas of northern China based on microsatellite data, which was consistent with previously reported findings. For example, another group found a moderate to high level of genetic differentiation and no genetic structure in seven Chinese RSB populations by using microsatellite markers  [ulations . The limequences . The difpulation . In our  regions .Understanding the genetic structure of species helps to provide insights into the evolutionary and ecological processes . In thisRSB is one of the most destructive rice pests in China. In the past few decades, chemical control has remained the most efficient primary means of controlling this pest. Unfortunately, it has developed resistance to some kinds of insecticides, and insecticide resistance loci may show selection footprints . TherefoOur microsatellite results illustrated a moderate level of genetic diversity and a moderate level of genetic differentiation among the RSB populations in the typical bivoltine areas of northern China. We also noted a distinct population genetic structure, which divided these populations into three clades: western, eastern and northern/centra. IBD and spatial autocorrelation analyses demonstrated no correlation between genetic and geographic distance, while bottleneck effect analysis indicated that RSB had not undergone a severe bottleneck in these regions. The results of GENECLASS analysis led us to speculate that low level of gene flow and non-directional spread occurred among most RSB populations located in the typical bivoltine areas of northern China. Our results provide new insights into the fine-scale genetic structure of overwintering RSB in the typical bivoltine areas of northern China and contribute to understanding the dispersal pattern of this species in China.S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S3 Table(DOC)Click here for additional data file.S4 Table(DOC)Click here for additional data file.S5 Table(DOC)Click here for additional data file.S6 Table(DOC)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.


Spodoptera exigua, is a significant agricultural pest of numerous crops and has caused serious economic losses in China. To effectively control this pest, we analyzed its genetic variation, population genetic structure and demographic history. We used mitochondrial DNA (mtDNA) fragments of the cytochrome oxidase subunit I (COI) and eight nuclear microsatellite loci to investigate genetic diversity and population genetic structure of S. exigua populations at 14 sampling sites in western China. Both mtDNA and microsatellite data indicated low levels of genetic diversity among all populations. A moderate genetic differentiation among some S. exigua populations was detected. Neighbor-joining dendrograms, STRUCTURE, and principal coordinate analysis (PCoA) revealed two genetically distinct groups: the KEL group and the remaining population group. Isolation by distance (IBD) results showed a weak significant correlation between geographic distance and genetic differentiation. Haplotype networks, neutrality testing, and mismatch distribution analysis indicated that the beet armyworm experienced a recent rapid expansion without a recent genetic bottleneck in western China. Thus, the results of this population genetic study can help with the development of strategies for managing this highly migratory pest.The beet armyworm,  Spodoptera exigua (Lepidoptera: Noctuidae), is a significant polyphagous pest on vegetables, maize, cotton, soybeans, and ornamental plants [S. exigua larvae feed on host leaves, decreasing crop yields and leading to the death of plants. Originating from South Asia, this species is already widely distributed in the tropical and temperate regions of Europe, Africa, North America, and Asia [S. exigua was first recorded in Beijing in the 1890s. It has a wide distribution in the main crop-producing areas of China and has caused severe economic losses in recent years. For example, S. exigua has spread to some provinces in North and East China and has infested a total area of over 2.7 million hectares [The beet armyworm, l plants ,2. Generand Asia . In Chinhectares . This pehectares ,6.Spodoptera exigua was a polyphagous insect and has high fecundity and long-distance flight capabilities [S. exigua rapidly developed resistance to many of them, such as chlorinated hydrocarbons and carbamates [bilities ,7. In gebilities . Chemicarbamates ,10. Thercytochrome oxidase subunit I (COI) gene is suitable for reconstructing species phylogenies [S. exigua on large spatial scales is lacking, and only few studies being conducted to date [The genetic variation and population genetic structure of a species can be affected by many factors, such as climate change, ecological factors, natural barriers, migration behavior, and human activities –14. Nowalogenies ,17. Due logenies ,19. Thes to date –22. Due S. exigua in western China. Both the mitochondrial DNA (mtDNA) COI gene and microsatellite loci were used to infer the genetic variation, genetic differentiation, population genetic structure, and demographic history of 14 S. exigua populations in western China. We discussed the management strategies for this species. This study aims to recognize the population genetics of this moth and provide a useful theoretical basis for developing effective management strategies for this pest.In the present study, we investigated the population genetics of S. exigua in the 14 locations were collected by using sex pheromone traps from June to October from 2012 to 2018, including 11 populations collected in western China, 2 populations collected in Multan, Pakistan, and 1 population collected in Hanoi, Vietnam. Details of the locations of populations and the numbers of samples are provided in S. exigua samples were preserved in 95% ethanol at −20°C and deposited in the Plant Protection College, Shenyang Agricultural University, Shenyang, China. Genomic DNA was extracted from individual samples using Qiagen’s DNEasy Blood & Tissue kit  according to the manufacturer’s protocols.All individuals of COI mitochondrial genes was amplified by PCR using the primers LCO1490 (5′-GGTCAACAAATCATAAAGATATTGG-3′) and HCO 2198 (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′) [®buffer , 0.5 μM of each primer, 1 μL of DNA template, and 2.5 mM dNTP mixture. Conditions for PCR amplification were as follows: 94°C for 5 min; 35 cycles at 94°C for 30 s, 52°C for 30 s, and 72°C for 45 s; and a final extension at 72°C for 5 min. Amplification products were purified and then sequenced by Sangon Biotech (Shanghai) Co., Ltd. Chromatograms, including sense and antisense, were edited and assembled using DNASTAR 5.0  to obtain a single consensus sequence. Microsatellite alleles were analyzed using GeneMapper 4.0 software (Applied Biosystems).A fragment of the ATCA-3′) . Each PC®buffer , 0.5 μL of DNA template, and 0.4 μM of each primer, which were labeled with fluorochromes HEX or FAM . Conditions for PCR amplification were as follows: 94°C for 4 min; 30 cycles at 94°C for 30 s, 58°C for 30 s, and 72°C for 30 s; and a final extension at 72°C for 5 min. Following amplification, the products were visualized at Sangon Biotech Co., Ltd.  using an ABI 3730XL automated sequencer .In this study, individuals were genotyped using eight microsatellite loci, and technical details were provided by Kim et al. . These mCOI sequences were aligned manually using the CLUSTAL X 1.81 program [π) and nucleotide diversity (h) were estimated using DnaSP 4.0 software [COI haplotypes was conducted in NETWORK 2.0 [FST) statistics between populations using Arlequin 3.0 [FST and haplotype frequencies were completed using Arlequin 3.0. Isolation by distance (IBD) was determined by testing the correlation between geographical distance and pairwise FST/1–FST using the Mantel test with Arlequin 3.0 [S. exigua using two neutrality tests, Tajima’s D [FS [The mtDNA  program  and were program . For eacsoftware . The medWORK 2.0 . We calcquin 3.0 . Analysiquin 3.0 . Demograjima’s D  and Fu’s’s D [FS , across ’s D [FS .Na), the effective number of alleles (Ne), Shannon's information index (I), observed heterozygosity (HO), expected heterozygosity (He), and unbiased expected heterozygosity (uHe) were determined using GenAlEx 6.41 [AR), the fixation index (FST), and the inbreeding coefficient (FIS) were calculated with FSTAT 2.9.3.2 [PIC) was calculated in CERVUS 2.0 [The software Micro-Checker 2.2.3  was usedlEx 6.41 . Allelic 2.9.3.2 . PolymorRVUS 2.0 . The levRVUS 2.0 .K), measures of change in the likelihood function (ΔK) and FST (ΔFST) were calculated using the package CORRSIEVE [S. exigua using a partial Bayesian method [We used POPTREE 2 to construct an unrooted phylogenetic tree by the neighbor-joining method with 1,000 bootstrap replicates . The sofORRSIEVE , and theORRSIEVE . The sofORRSIEVE  was usedORRSIEVE . PCoA, bORRSIEVE . The sigORRSIEVE . We usedORRSIEVE . We perfORRSIEVE . Asymmetn method .FST, which was not used, and the excluding null alleles (ENAs) per the locus correction method, was 0.184 and 0.171, respectively, and these FST estimations did not differ significantly (P = 0.462). Therefore, the presence of null alleles did not influence FST estimation (S. exigua in western China was found (Na) across microsatellite loci ranged from 4.250 in Xilengrad (NMXM) to 8.875 in Kunming (KM). The effective number of alleles (Ne) ranged from 2.908 in Haikou (DLH) to 4.166 in Guiyang (GY). Shannon's information index (I) across loci ranged from 1.130 to 1.545. The inbreeding coefficients (FIS) of the eight microsatellite loci in all 14 populations of S. exigua were nearly zero (FIS= 0.131). Kunming (KM) population had the highest genetic diversity, whereas the DLH population had the lowest genetic diversity.In this study, 312 individuals were collected from 14 populations in western China and genotyped with eight microsatellite loci. The null allele frequency per locus was mostly less than 0.1 . The Hartimation . Modern as found . Overallas found . The estCOI gene sequence was sequenced for 291 S. exigua individuals (deposited in GenBank under accession nos. KY069907–KY069921). Of the 576 total characters, 566 were conserved sites and 10 were variable sites. We observed 10 haplotypes in 291 individuals. The average number of haplotypes in each population was 2.286 ± 0.914 and ranged from 1 to 4, and the Korla (KEL) population had the highest number of haplotypes. Hap 2 was the most common haplotype, present in all populations, and was shared by 247 individuals. Hap 4 was the second most frequent haplotype, being shared by 20 individuals from two populations. Genetic diversity indices haplotype diversity (h) and nucleotide diversity (π) are shown in h and π were 0.273 and 0.00069, respectively. The highest genetic diversity  was detected in Taiyuan (GSTY). In contrast, the genetic diversity value of Xilengrad (NMXM) and Hanoi (HN) was zero because no variable sites were found . Although a significant difference existed between most populations including Korla (KEL) and Lanzhou (GSTY), we found no significant differentiation among the other remaining populations.A global ficantly . Overallof 0.109 . We idenS. exigua populations defined two major clades  values increased slowly starting from K = 2, likely representing the most appropriate number of major clusters. The maximum ΔK was reached at K = 2. This result was consistent with the hypothesis that these populations could be divided into two groups: the Korla (KEL) group and the remaining group of 13 populations. Further structuring indicated the divergence of Delingha (DLH), Multan (BM), and Faisalabad (BF) from the other populations at K = 3. This result was consistent with the NJ phylogenetic tree analyses.Based on microsatellite data, we used a clustering algorithm in STRUCTURE 2.3.3 to infer the relationships between the 14 rn China . The meaS. exigua populations , and the remaining genetic variation  could be explained by the variation among populations  was explained by variation within populations. Consistent with the results of the NJ tree, PCoA and STRUCTURE, the AMOVA analyses were also supported one genetic group.The global AMOVA indicated that greater genetic variance was partitioned within individuals  (Based on the mtDNA jima’s D  and Fu’s’s D [FS  statisti –7.987) . The wid –7.987) . The uni –7.987) .S. exigua had expanded demographically without experiencing a severe population bottleneck in western China  under three models , which is consistent with a normal L-shaped distribution of allelic frequency. Therefore, these results indicated that rn China .P value of 0.05 based on the partial Bayesian method. Our results showed 130 migrant individuals from all of the 312 individuals, and the majority of putative migrant genotypes (110/135) were restricted to three populations of southwestern China, including Kunming (KM), Guiyang (GY) and Chengdu (SC). It also provides a highly directional from southwest China to other western regions populations. In addition, we also observed 11 individuals as potentially first-generation migrants (F0), of which 5 individuals migrated from three southwestern populations to almost all the other populations of S. exigua  were definitively classified at a . exigua .S. exigua was found to have low haplotype diversity and low nucleotide diversity based on the mtDNA COI gene , and a moderate expected heterozygosity (He) of 0.605 based on the microsatellite loci, demonstrating a low level of genetic diversity in S. exigua in western China. This result is consistent with previous studies [Danaus plexippus) [S. exigua is a significant agriculture pest, but little information has been published about its dispersal ability [Plutella xylostella, where sampling locations throughout temperate and subtropical Chinese regions were based on the climate division in China [In the present study,  studies ,20,21. Sexippus) . In gene ability . Overallin China .Locusta migratoria [Paeonia rockii [S. exigua, resulting in lineage differentiation. For example, Mt. Tianshan might have acted as a substantial geographical barrier, producing significant population differentiation between KEL and the remaining populations. Similarly, genetic structuring on a local scale has also been reported in the migratory moth Helicoverpa armigera in Australia, where it is thought that drought conditions impacted its migratory capacity and resulting genetic structure [S. exigua populations in western China, and this result is consistent with those reported in previous studies [Knowledge of the population genetic structure can provide insight into the evolutionary and ecological processes of species. In the present study, we found no distinct distribution pattern of this pest based on mtDNA haplotypes phylogenetic tree. In line with that, the mtDNA haplotypes network analysis showed that the dominant haplotype (Hap2) was evenly distributed in the western regions of China, indicated that gene flow occurred among the populations of this species in recent history and blurred the original relationship between different populations. These genetic features are evidence of migration, as demonstrated in other migratory species ,50–52. Pgratoria  and Paeoa rockii . We spectructure . We foun studies ,21,22. I studies .S. exigua is a polyphagous species that has been described as feeding on more than 300 plants, which implies its considerable adaptive potential. This pest originates from South Asia. It is currently widely distributed in most of the main crop-producing areas in China. We collected five individuals of S. exigua using sex pheromone traps for the first time in Yichun, Heilongjiang, in 2012. Therefore, this species has not accumulated additional genetic variation due to its short history of expansion in Northern China. In the present study, we found a high level of genetic diversity in the Kunming (KM) population. This high level of diversity can probably be explained by S. exigua not experiencing serious founder effects, genetic bottlenecks, or strong selection  in this region. Resistance to insecticides in insects is an example of evolutionary adaptation to environmental changes [S. exigua. S. exigua has developed resistance to some insecticides. For example, this species has a long history of exposure to carbamate pesticides and has developed a high level of resistance to methomyl in California, USA, since 1989 [S. exigua populations are essential. Understanding the dispersal ability, genetic structure, and population demography of this pest is critical for determining both theoretical aspects of its evolution and the effective implementation of pest forecasting systems. In the future, we will explore population genetic differentiation and population genetic structure based on the genomic level of S. exigua, and deeply reveal the evolution relationship, reconstruct population history, it will help us to gain insight into the adaptation to climates and ecology factors at the genomic level. These works will be carried out before a reasonable control strategy is developed for managing S. exigua. changes . Since tcytochrome oxidase subunit I (COI) and eight nuclear microsatellite loci to investigate genetic diversity and population genetic structure of S. exigua populations at 14 sampling sites in Western China. Low levels of genetic diversity and a moderate genetic differentiation among S. exigua populations were detected. Population genetic structure analysis revealed two genetically distinct groups: the KEL group and the remaining population group. Isolation by distance (IBD) results showed no significant correlation between geographic distance and genetic differentiation. Population demographic history analysis indicated that the beet armyworm experienced a recent rapid expansion without a recent genetic bottleneck in western China.We used mitochondrial DNA (mtDNA) fragments of the S1 Figx-axis represents the numbers of pairwise differences; the y-axis represents the relative frequency. The expected distribution under a model of population expansion is given as a continuous line, and the observed distribution is given as a dashed line.The (TIF)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(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.S8 Table(DOCX)Click here for additional data file.


Aedes aegypti populations’ susceptibility status, collected in 2016 from six different municipalities of Rio de Janeiro state (RJ), to temephos, pyriproxyfen, malathion, and deltamethrin. We collected eggs of Ae. aegypti in Campos dos Goytacazes (Cgy), Itaperuna (Ipn), Iguaba Grande (Igg), Itaboraí (Ibr), Mangaratiba (Mgr), and Vassouras (Vsr). We followed the World Health Organization (WHO) guidelines and investigated the degree of susceptibility/resistance of mosquitoes to these insecticides. We used the Rockefeller strain as a susceptible positive control. We genotyped the V1016I and F1534C knockdown resistance (kdr) alleles using qPCR TaqMan SNP genotyping assay. Besides, with the use of Ae. aegypti SNP-chip, we performed genomic population analyses by genotyping more than 15,000 biallelic SNPs in mosquitoes from each population. We added previous data from populations from other countries to evaluate the ancestry of RJ populations.Vector control largely relies on neurotoxic chemicals, and insecticide resistance (IR) directly threatens their effectiveness. In some cases, specific alleles cause IR, and knowledge of the genetic diversity and gene flow among mosquito populations is crucial to track their arrival, rise, and spread. Here we evaluated Ae. aegypti populations were susceptible to pyriproxyfen and malathion and highly resistant to deltamethrin. The resistance ratios for temephos was below 3,0 in Cgy, Ibr, and Igg populations, representing the lowest rates since IR monitoring started in this Brazilian region. We found the kdr alleles in high frequencies in all populations, partially justifying the observed resistance to pyrethroid. Population genetics analysis showed that Ae. aegypti revealed potential higher migration among some RJ localities and low genetic structure for most of them. Future population genetic studies, together with IR data in Ae aegypti on a broader scale, can help us predict the gene flow within and among the Brazilian States, allowing us to track the dynamics of arrival and changes in the frequency of IR alleles, and providing critical information to improving vector control program.All RJ  Aedes aegypti regarding their IR status towards insecticides previously or currently in use, their frequency of IR alleles and resistance ratio, their genetic ancestry and structure, and the gene flow. We observed that Ae. aegypti populations from RJ are becoming susceptible to the larvicide organophosphate temephos. The resistance ratios of temephos are lower among populations where gene flow might be occurring. All RJ populations are highly resistant to deltamethrin, partially explained by the high frequency of kdr alleles. Besides, all populations are susceptible to pyriproxyfen’s insect growth regulator and the organophosphate malathion. Population genomic analyses using more than 15,000 SNPs identified three potential genetic clusters within RJ populations, with higher gene flow to and out of the touristic region called Região dos Lagos. IR data, together with knowledge of the genetic structure and gene flow among different mosquito populations, can help with vector control program decisions.Monitoring of insecticide resistance (IR) is mandatory for the effectiveness of vector control programs. We investigate six populations of  In 2016, Brazil’s number of dengue cases was near 1.5 million, representing 69% of the dengue cases in South, Central, and North America combined. Within the Southeast region of Brazil, Rio de Janeiro State (RJ) is a crucial hub for the import and export of goods and a tourist attraction and port of entry. In 2016, RJ registered high dengue, chikungunya, and Zika, with 443.7, 108.1, and 414.2 cases per 100,000 inhabitants, respectively  while Cgy is the least one [slope value = 4.65]The concentration required to kill 50% of the susceptible strain (Rock) in 24h exposition, the LCWe evaluated the populations’ susceptibility status against the insect growth regulator (IGR) pyriproxyfen in a qualitative bioassay with the diagnostic dose of 0.18 mg/L. The experiment took 7 to 10 days, and none of the individuals died in the control treatment. At these time points, mortality in the treatments was between 82 and 99% . Rock exA qualitative dose-diagnostic assay with the OP malathion (20 μg/mL) using the CDC bottle guidelines showed all populations are ’susceptible’ to this OP. Rock and all natural populations achieved 100% mortality during 1h of exposure . WHO-tubkdr alleles in Ae. aegypti populations from RJ since they were resistant to deltamethrin. We genotyped 266 samples for two NaV SNPs: 1016  and 1534 (Phe or Cyskdr)  was found under very low frequencies, from absent in Cgy up to 9% in Ipn. The kdr NaVR1  varied between 3% (Cgy) to 34% (Mgr), while NaVR2 (1016Ilekdr + 1534Cyskdr) was the most frequent haplotype, with 58% in Vsr and 96% in Cgy. The sum of the "resistant" genotypes  was above 80% in all RJ populations, ranging from 82% (Ipn) to 100% (Cgy)  . The NaV0% (Cgy) .Aedes aegypti populations’ genetic ancestry. We used 14,095 SNP loci, which passed our filtering thresholds, to infer the genetic ancestry. The best number of clusters was four for the LEA analysis. Similar to what is described by [First, we used samples from other countries described previously  S1 Tabl to evaluribed by  populatiSecond, we analyzed the populations from Rio de Janeiro separately. We used 15,321 SNP loci that passed our filtering thresholds to infer their genetic ancestry. The best number of clusters based on the lowest entropy was three. We observed that Ibr, Vsr, and Cgy have the lowest admixture proportions Figs  and S2. The PCA analysis generated similar results when comparing populations from Rio de Janeiro and other countries . We alsoAedes mascarensis as an outgroup following [Finally, we constructed a phylogeny with our populations and used ollowing . After aFst values for all populations using 15,321 SNP loci. The overall values are relatively low, but we could observe that mosquitoes from Vsr and Ibr showed the highest genetic differentiation (Fst = 0.06) (Fst = 0.02). Since a single Fst estimate may not reveal genetic differentiation in specific genomic regions, we used sliding window analysis to see if we could find such areas. We were able to find sites with higher genetic differentiation even though the global Fst values were low  . The popwere low . We coulWe sought to obtain an overall migration trend among RJ localities by detecting first or second-generation mosquito migrants with BA3-SNPs. We found the highest migration estimates from Igg to Cgy Figs  and S1. Ae. aegypti is usually low. The Brazilian government strengthened vector control measures at the beginning of May with a program named MIRA 360°. Based on WHO protocols of monitoring and targeted actions, the plan used the additional employment of ultrasonic pulses for killing mosquito larvae [Ae. aegypti in August, or because of the intensified vector control measures and self-protection [Ae. aegypti in RJ. This includes monitoring insecticide efficacy [Ae. aegypti within RJ.In Brazil, the 2015 Zika outbreak led to health officials believing that the 2016 Summer Olympic and Paralympic games events, held in Rio de Janeiro, could cause a more significant higher risk of infection of Brazilians, tourists, and athletes . Based oo larvae . Accordiotection . So far,otection . Adding efficacy , and knoAe. aegypti originated from Africa and spread to the Americas 400 to 500 years ago [Aedes aegypti aegypti (Aaa) that breeds in human-made habitats and is present throughout the world [Ae. aegypti in the Americas was via Brazil or northern Argentina [Ae. aegypti in Brazil in the 1950s, which was successful until mosquitoes likely from Venezuela and the Caribbean, re-invaded the country [Ae. aegypti from RJ is genetically differentiated from other localities and population structure within RJ.We used different molecular and population genetic tools and bioassays to characterize populations of mosquitoes collected in cities representing most RJ regions. ears ago . The anthe world  and widergentina . Only Arrgentina ,67. One  country , and conAe. aegypti [Ae. aegypti within RJ populations. Although the chip presents more than 50,000 polymorphic sites, we know it cannot capture all the species’ genetic diversity. After stringent filtering, we were left with approximately 15,000 high-quality SNPs. The previous knowledge about genetic diversity Ae. aegypti from RJ was limited to populations from the city capital and based upon isoenzymes and microsatellite markers. The level of genetic diversity within the capital limits using isoenzymes was high [Taking advantage of a recently developed high-density SNP-chip for  aegypti , we evalwas high , while mwas high . These cwas high . Compariwas high . Such obAe. aegypti in Brazil are originated from re-invasion events, likely from Venezuela, after the eradication efforts in the 1950s [Ae. aegypti populations from this country show admixture with African populations differently from other South American and Caribbean populations [Using about 15,000 biallelic SNP loci, we observed significant genetic differentiation within RJ populations. Population-based analysis, such as PCA and DAPC, indicates three distinct genetic clusters . Howeverhe 1950s . Howeverulations ,68. Althulations . As a peulations .kdr resistant alleles at high frequencies. The highest resistance levels to temephos come from Ae. aegypti populations in Brazil and other Latin American countries, such as French Guiana and the Caribbean [Ae. aegypti populations came from collections in urban localities around the state capital in 1999, where RR50 varied from 3.0 to 7.6 [50 above 10 in Rio de Janeiro City (13.2 in 2002), São Gonçalo (15.5 in 2001), and Niterói (13.5 in 2001) [Regarding insecticide resistance analyses, RJ populations were resistant to the PY deltamethrin and the OP temephos while susceptible to the IGR pyriproxyfen and the OP malathion. We found the aribbean . The usearibbean . Due to aribbean . First t0 to 7.6 . In the in 2001) . In 2006in 2001) ,74.95 dropped from 16.3 to 9.8 between 2009 and 2012 in the Ae. aegypti population from Duque de Caxias in RJ [90 of 7.8 in 2001 [90 of 2.6 in 2016). Ipn registered the highest RRs in RJ populations: RR95 equal to 16.8 in 2003, increased to 25.6 in 2011, and decreased to 7.3 in 2016 [A decrease in the resistance levels to temephos occurred in populations from different Brazilian regions due to the substitution of this OP by IGR compounds. For instance, the temephos RRas in RJ . Among t in 2001 , represe in 2016 ,76. WithAe. aegypti in Brazil. Despite resistance to neurotoxic insecticides in natural populations across the State, the selected mechanisms did not confer any cross-resistance to the IGR in Ae. aegypti populations from RJ. In our study, all localities were susceptible to pyriproxyfen. As the use of IGRs is recent for mosquito control, few studies reported resistance to these compounds. In one of the few examples, an Ae. aegypti population from Malaysia showed resistance to the juvenile hormone analog methoprene (RR of 12.7) but low RR (1.4) to pyriproxyfen [Ae. aegypti from Brazil detected resistance to pyriproxyfen (dose-diagnostic assay) in localities from Bahia and Ceará States, in the Northeast of the country [The IGR pyriproxyfen, a juvenile hormone analog, started being used in 2013 against proxyfen , suggest country .Ae. aegypti populations since the beginning of the 2000s. For instance in 2003, low mortality to cypermethrin was recorded in Cgy (73.3%) and Ipn (54.8%) [90 equal to 29.7 to deltamethrin later in 2011 [kdr alleles. Accordingly, in the Ipn population, 48.1% of genotyped mosquitoes presented a kdr resistant genotype  in 2011 [Ae. aegypti populations [Culex quinquefasciatus mosquitoes, which end up reaching Ae. aegypti populations. The application is made by fogging or ULV equipment, with pyrethroid based compounds, especially because of their comparatively less irritating smell. These treatments occur several times per week or even daily in some regions during the summer. Therefore, the fact that pyrethroids are no longer deployed by governmental campaigns to control mosquito populations, we still have selection pressure coming from other forms of use of pyrethroids.There is evidence of adulticide pyrethroids resistance in RJ  (54.8%) . Due to  (54.8%) , the MoH (54.8%) , which i in 2011 . Althoug in 2011 , we obse in 2011  and incrulations ,83. BesiAe. aegypti were utterly susceptible to malathion, even though its use in RJ officially started nine years ago. The Brazilian national insecticide resistance program tested a population from our study, Cgy, and reported it as susceptible in 2018 [In our study, all populations of  in 2018 . Other p in 2018 .kdr mutations were near fixation in all RJ populations . F. Chikungunya cases in the cities of the Rio de Janeiro State. G. Percentual of Chikungunya cases of each city proportional to total cases in the State. H. Incidence of Chikungunya cases (%). I. Zika cases registered in the cities of the Rio de Janeiro State. J Percentage of Zika cases registered in the cities of the Rio de Janeiro State. K. Incidence of Zika cases (%).(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Tablep-values and confidence intervals (95%).We used 100 bootstraps to calculate (PDF)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.


Acanthochitona cf. rubrolineata , which geographically corresponds to hydrographic discontinuity among the coastal regions of Korea, Japan, and China. The assumed limited dispersal ability of  A. cf.  rubrolineata, coupled with northeasterly flowing, trifurcate warm currents, might have contributed to the genetic differentiation among the three groups. Meanwhile, a high level of within-group genetic homogeneity was detected, indicating extensive coastal currents might facilitate gene flow among the populations within each group. Bayesian skyline plots demonstrated significant population expansion after the Last Glacial Period (110-25 thousand years ago) for all studied populations except the Japan group. Together these results suggest that the present-day phylogeographic patterns of A. cf. rubrolineata are strongly affected by the interplay of historical and/or contemporary oceanography and species-specific life-history features.Chitons are a group of marine mollusks (class Polyplacophora) characterized by having eight articulating shell plates on their dorsal body surface. They represent suitable materials for studying the spatiotemporal processes that underlie population differentiation and speciation in ocean environments. Here we performed population genetic analyses on the northwestern Pacific chiton olineata  using tw The northwestern Pacific (NWP) is a vast region comprising the coastlines of China, Korea, Japan, and Russia. It features a unique tectonic and hydrological setting and provides an ideal system to test some interesting biogeographic questions that underlie marine species’ evolution . During 2, it is characterized by an extensive continental shelf with an average depth of 55 m (The Yellow Sea (YS), including the Bohai Gulf, is a marginal sea of the NWP located between the west coast of Korea and the east coast of China . With a  of 55 m . Past cl of 55 m . Marine  of 55 m . When th of 55 m . These p of 55 m .Present-day oceanographic features also shape phylogeographic patterns of marine species . Two oceAcanthochitona is a genus of the family Acanthochitonidae (Polyplacophora) and is distributed worldwide. It is one of the most taxonomically complicated molluskan groups due to its extremely high morphological variability , and 60 specimens from northeastern China, in order to shed light on the phylogeographic patterns of this species. We aimed to evaluate (1) the impact of a known history of sea-level changes during the Pleistocene on the present-day phylogeographic pattern of this chiton species, (2) population structure for a chiton species, which belongs to a marine molluskan taxon characterized by lecithotrophic development and limited planktonic larval duration; and (3) gene flow among the three regions and the influence of ocean currents on contemporary population structure.In this study, we determined the sequences of two mitochondrial genes [cytochrome oxidase subunit I (COI) and 16S ribosomal DNA (16S)] for 180 NWP individuals of A. cf. rubrolineata individuals were sampled from 11 localities in Korea, Japan, and China, and about 20 specimens per population except for the Japanese populations were sequenced for mitochondrial COI and 16S . We merged the two mitochondrial segments for each individual using the software FasParser  suggested there was no incongruence between the two segments, and therefore the concatenated sequences were used in subsequent analyses. Haplotypes were defined using DnaSP v5 (Hd), nucleotide diversity (Nd), and mean number of pairwise differences (Pd) were calculated for each of the 11 populations in ARLEQUIN v3.5  was then conducted with 10,000 permutations in ARLEQUIN v3.5 to estimate the partitioning of genetic variation. Since the HKY model was not available in ARLEQUIN, the most similar model, that of Tamura-Nei  were calculated with 10,000 permutations in ARLEQUIN v3.5.For population structure analyses, population clusters were first estimated for 10 populations  among the groups defined in SAMOVA and effective population size (Θ) of each group based on maximum-likelihood estimates. Several short runs were performed to check for convergence of chains, and appropriate upper bounds were set for each parameter after the test runs. Three long chains were run with 2,000,000 steps and sampled every 100 steps, and the first 20,000 steps were discarded as burn-in. A static heating scheme was set at four temperatures of 1, 1.5, 3, and 100,000. Five replicates were run to check the consistency of the estimates, and stationarity of the Markov chain Monte Carlo (MCMC) was assessed by examining the effective sample size (ESS). We transformed the migration rate to the number of migrants per generation (Nm) using the formula Nm = Θ*M/x   .D  based on the minimum splitting time between Caribbean and Eastern Pacific presumed geminate species pairs of the Isthmus of Panama (http://beast.bio.ed.ac.uk/Tracer). The 16S data set was not used here because it is uninformative and there is no reported molecular clock yet for this species or any other Acanthochitona species.Neutrality tests were applied to each group defined in SAMOVA and the entire population  to infer historical demography. Tajima’s D  and Fu’sD  to 1.000 . Nucleotide diversity ranged from 0.0046 (in Nomomachi) to 0.0098 (in Kinaisemen), and the mean number of pairwise differences ranged from 5.333 to 11.333 . The TCSFCT value occurred at K = 3 with a grouping arrangement of , , and , which geographically aligns with the three studied countries. Under this scenario, AMOVA analysis showed that both variation among groups (ΦCT = 0.151) and variation within populations (ΦST = 0.154) were statistically significant , while variation among populations within groups was not significant . Variation within populations accounted for the largest percentage (84.91%) of the total variation, followed by variation among groups (15.38%), whereas a slightly negative variance value (−0.29%) was estimated for among populations within groups , and all of these were observed between populations of different groups : 2.331 with 95% confidence interval (CI : 0.547–3.293) and Japan group [Nm(C→J): 2.079 (CI : 1.378–3.762)]. For the other four directions, the estimated values were relatively smaller and had a 95% CI that overlapped with zero, a signal that suggests impeded gene flow between pairwise groups. Effective population size with a 95% CI for each group revealed the smallest Θ in the China group [ΘC = 0.0038 (0.0010–0.0066)], with comparable values for the Korea [ΘK = 0.0419 (0.0178–0.0828)] and Japan [ΘJ = 0.0597 (0.0220–0.0996)] groups.For the gene flow analyses in MIGRATE-N, the effective MCMC sample size of each parameter was >2000, indicating stationarity of all the chains. The results revealed asymmetric gene flow among the three groups : signifiD test, all the analysis showed negative values, but P values were not significant in the China or Japan groups . Fertilized chiton eggs are known to hatch out after some period that mostly depends on temperature into trochophore larvae  and then become increasingly benthic and soon metamorphose into a juvenile form within a few days to about 12 days after fertilization depending on chiton species (A. crinitus) that undergoes metamorphosis within 3-4 days after fertilization , likely preventing long-distance dispersal  of A. cf. rubrolineata populations across our three studied regions. A short larval stage would likely affect population structure among the three regions, especially considering their geographic separation and habitat discontinuity, and the general lack of stepping stone habitats between them.A most striking pattern in nd Japan . Pairwist groups . Estimatt groups . These rt groups . Larval t groups . Thus, i species . From thlization , A. cf. A. cf. rubrolineata may be limited by the warm current system in the YS. The trifurcated branches of the Kuroshio Current  flow northeastward into their downstream pathways that separate the three regional groups. This oceanographic surface flow may act as a physical barrier that, albeit not completely, limits the opportunity for gene flow via larval dispersal and/or occasional rafting of the individuals across the three regions. Note that, as inferred from the MIGRATE-N results, this barrier effect may be weak for the directions from China to both Korea and Japan. Similar population structuring resulting from hydrographic patterns has also been reported in other marine species in the NWP  and 100 kya (Korea group)  , 4B, afta group) . Abrupt a group) . Considea group) . Differelar rate . PopulatA. cf. rubrolineata populations in the NWP. In contrast to the lack of phylogeographic pattern commonly revealed for other coastal species in the YS, we observed significant population structure  in A. cf. rubrolineata, which may be attributed to both life-history features  and the warm current system. Nevertheless, within each of our three regions, we also observed genetic homogeneity, which might be maintained by local surface currents and habitat continuity. The phylogeographic pattern revealed in A. cf. rubrolineata in the present study underscores the interplay of historical and contemporary oceanography (both geography and hydrology) and life-history features on historical demography and population structure in the NWP. Further population genetic studies using multi-locus genetic data are needed to assess the relative importance of biotic and abiotic factors that could more generally influence the phylogeographic patterns of marine species in the YS.Chitons represent one of the ancient groups of marine mollusks but have so far attracted little research effort in marine phylogeography but see . In this10.7717/peerj.8794/supp-1Figure S1The observed and expected mismatch distributions are indicated by a dashed blue line and solid red line, respectively.Click here for additional data file.


Petrogale xanthopus xanthopus, a threatened macropodid which inhabits rocky outcrops across the disconnected mountain range systems of the southern arid biome. We use 17 microsatellite loci together with mitochondrial control region data to determine the genetic diversity of populations and the evolutionary processes shaping contemporary population dynamics on which to base conservation recommendations. Our results indicate the highly fragmented populations have reduced diversity and limited contemporary gene flow, with most populations having been through population bottlenecks. Despite limited contemporary gene flow, the phylogeographic relationships of the mitochondrial control region indicate a lack of structure and suggests greater historical connectivity. This is an emerging outcome for mammals across this arid region. On the basis of our results, we recommend augmentation of populations of P. x. xanthopus, mixing populations from disjunct mountain range systems to reduce the chance of continued diversity loss and inbreeding depression, and therefore maximize the potential for populations to adapt and survive into the future.Little genetic research has been undertaken on mammals across the vast expanse of the arid biome in Australia, despite continuing species decline and need for conservation management. Here, we evaluate the contemporary and historical genetic connectivity of the yellow-footed rock-wallaby,  Habitat fragmentation, the division of natural habitat into small isolated patches, is associated with a myriad of problems for biodiversity worldwide. These include the interacting effects of small effective population size, isolation, contracted geographic distribution, novel species interactions, edge effects, and reduced landscape connectivity (see ). In parFragmentation of natural landscapes has increased through anthropogenic impact, especially in the last 500 years, causing population and species decline and associated long-term genetic consequences which require active management to counteract . While gVulpes vulpes and the feral cat Felis catus), as well as changed fire regimes and habitat degradation by introduced herbivores ; [∆K)  implemenK]; [∆K) . We also ran a principal coordinate analysis (PCoA) in GenAlEx to assess genetic structuring of the populations. This analysis used a covariance matrix with standardization based on genetic distances. This was conducted using the entire dataset, as well as smaller regional datasets  using a hierarchical approach to determine more fine-scale structuring.To assess the pattern of relatedness within and among populations we calculated the average pairwise relatedness in GenAlEx . First, CR haplotypes, phylogenetic analysis was undertaken comparing the sequences from SA with published CR data from P. x. xanthopus in NSW [P. x. celeris from QLD [P. penicillata) and black-footed rock-wallaby  were used as outgroups . In addition, a haplotype network was estimated using TCS v1.21 [CR haplotypes, as low genetic variation can be challenging in resolving nodes in phylogenetic analyses. Sequence divergence among populations (Dxy) was also calculated using DnaSP. To assess relationships among mtDNA s in NSW  and P. xfrom QLD ,54. Partfrom QLD  was usedfrom QLD . Analysifrom QLD , with 10STΦ) among populations was estimated and tested for significance in Arlequin v3.5.1.2 [FST calculated in Arlequin based on 110 permutations. An analysis of molecular variance (AMOVA) was conducted in Arlequin to examine the extent of population structuring within and among regional populations in SA , significance was assessed using 1000 permutations. Pairwise differentiation of mtDNA haplotypes  and 644 bp when outgroup indels removed) were identified across P. x. celeris (seven haplotypes) and P. x. xanthopus (21 haplotypes), see P. x. xanthopus, this comprised 14 haplotypes in the Flinders Ranges, three in the Olary Hills, three in the Gawler Ranges, and one from NSW. There was a total of 42 polymorphic sites and three indels among P. xanthopus haplotypes. Overall haplotype diversity within P. x. xanthopus was 0.843 and nucleotide diversity was 0.013. Haplotypic diversity ranged from 0.000 to 1.000 and nucleotide diversity ranged from 0.000 to 0.0218 , except for Pa593 (Sandy Creek), Y151 (Sandy Creek), Me15 (Eregunda), Y112 (Wilkawillina South), G26-4 (Wilkawillina North), and Pa595  after sequential Bonferroni corrections. However, there was no consistent pattern in loci across populations, except for Pa595 which was removed from further analyses. Sandy Creek was out of HWE at four loci; however, we kept this population in the analysis. There was no consistent evidence of LD with only 1.3% of pairwise comparisons significant after sequential Bonferroni correction. Locus Pa55 was monomorphic in most (six) populations and so was removed from further analyses. Four other loci were monomorphic in some populations, Pa385 and Y105 in Mt Stuart, Y175 in Mt Friday, and Y148 in Aroona Dam. Remaining loci were polymorphic across all populations. All loci in all populations were in HWE (IS) was detected for populations, with values ranging from −0.234 in Mt Stuart to −0.027 in Wilkawillina South  in the Flinders Ranges and 44.6%  in the Gawler Ranges. The mean number of alleles per locus (A) ranged from 2.7 in Yandinga to 6.1 in Wilkawillina South . Unique na South . L(K)) or two (maximum ΔK) populations were present in the 10 sampled sites. At K = 9, CLUMPAK indicated a major cluster (9/10) supporting separation of all sites, excluding Mt Stuart which was shown as highly admixed, perhaps as a result of its small sample size (n = 3). Admixture was evident between Wilkawillina North and South, as well as between Sandy Creek and multiple other Flinders Ranges populations . On the basis of the K = 2 conundrum  as the most differentiated population, with Aroona Dam (reintroduced Flinders Ranges) and Mt Friday (Gawler Ranges) also forming a distinct grouping . PC1 accP < 0.02), ranging from 0.185 in Wilkawillina North to 0.644 in Yandinga (P < 0.03), with r values being more related than expected by chance. However, the mean fell within the null hypothesis bounds for Homestead Range for both males and females, and Sandy Creek for males  except between Homestead Range and Mt Stuart. Values ranged from 0.00 to 1.00 (FST) with values ranging from 0.050 between Wilkawillina North and Wilkawillina South in the Flinders Ranges to 0.498 between Yandinga and the reintroduced Aroona Dam population  is between populations within groups, with only 28.2% variation among Gawler Ranges, Flinders Ranges, and Olary Hills. Within populations, there was 10.2% variation which was not significant as compared with the variation within and among populations and groups.P = 0.001) were detected for P. x. xanthopus, indicating isolation-by-distance among populations. The Mantel test estimated a moderate correlation (Rxy = 0.666) based on the entire dataset. When males and females were analyzed separately, the correlations were significant (P = 0.001), with much higher correlations for females (Rxy = 0.812) than for males  ≥ Rxy from the data, CR sequence data (see P = 0.009) and Olary Hills (P = 0.049). Tajima’s D results were significant for Sandy Creek and Olary Hills suggesting evidence of deviations from neutrality  and simulations were significant for these two populations and Eregunda under a constant population size model. Tests of neutrality were unable to be computed for Aroona Dam, Yandinga, and Homestead Range due to a lack of polymorphisms in these populations and Mt Stuart due to the small sample size.Tests of neutrality (Tajima’s D) and expansion (Fu’s Fs and R2) provided little evidence for selection or expansion across populations based on the data see . SimulatThe Wilcoxon’s heterozygosity excess test revealed multiple populations had significant deviations from drift/mutation equilibrium, evidence of recent genetic bottlenecks. Genetic bottlenecks were detected for Aroona Dam, Eregunda, Homestead Range, Mt Friday, Wilkawillina North, Wilkawillina South, and Yandinga based on the TPM model . HoweverA total of 13 putative first-generation migrants were detected in three populations: Wilkawillina South, Wilkawillina North, and Sandy Creek . Five inP. x. xanthopus populations across southeastern Australia has found strong fine-scale contemporary structuring both within and between mountain range systems  [P. lateralis  [FST values between populations for P. x. celeris from QLD at equivalent distances  [P. brachyotis, FST = 0.027–0.059, <67 km apart [P. wilkinsi, STF = 0.085, 1.2 km apart [FST values between more distant populations is in accordance with the isolation-by-distance results which support greater genetic differentiation with distance. Such isolation-by-distance results, particularly among females, are a common finding among rock-wallaby species [Microsatellite and mtDNA analyses found strong fine-scale contemporary genetic structuring both within and between mountain range systems. Genetic clustering of microsatellite data indicates the presence of nine distinct groupings that largely comprise the sampled populations. Populations within the Gawler Ranges were distinct, however, the PCoA results indicate some overlap of Olary Hills with populations from the Flinders Ranges  and somem apart)  and P. lm apart) . They ar0–70 km) . They arkm apart  and P. wkm apart . The AMO species ,85. CR (STΦ) (STΦ) within range systems was 0.75 to 0.89  as compared with average genetic differentiation between range systems . The lowest levels of differentiation are between geographically closer populations  but overall maternal gene flow is limited. Strong female philopatry has been reported in other Petrogale    and is chyotis, ,91. Thisanalysis . CR haplotypes were found between Homestead Range, Mt Stuart, and Sandy Creek within the Flinders Ranges (n = 3) for this population and associated lack of genetic information to form a distinct cluster  did show evidence of recent connectivity (shared mtDNA haplotypes and admixture of microsatellite genotypes), with first generation migrants detected between these sites. Shared s Ranges  and evids Ranges  , suggess Ranges . The mix cluster . Interesapped in . Some ofn = 91, P. penicillata and P. lateralis [IS, P. penicillata) have been shown to mitigate inbreeding through mate choice and sex-biased dispersal, with female philopatry and male-biased dispersal documented to assist in inbreeding avoidance [The microsatellite diversity, although similar to other remnant populations of rock-wallabies , was stateralis ,33,84,88voidance ,88,93. CR haplotypes, despite up to 100 individuals being sampled. Evidence of bottlenecks across populations . The haplotype numbers varied from one to six, with most populations only having one or two distinct ulations  may haveSTΦ) indicate significant contemporary differentiation between most populations  both revealed similarities in historical connectivity across the landscape, highlighting the need to maintain genetic diversity across the landscape to enable resilience of species across this heterogeneous environment. Comparison and analysis across diverse organisms would assist in identifying core refugial areas where species have persisted across the landscape through past climatic cycles and improve our knowledge of biodiversity hotspots, as well as highlight areas of importance for conservation to establish evolutionary resilience  demonstrates how individuals with weak signatures of inbreeding depression can have fitness declines and strong lifetime effects in reproductive success [n = 3) for example, the sample represents the entire extant remnant population at the time. Overall, our results highlight the need for focused and broad action for genetic management of P. x. xanthopus, to improve genetic diversity and rebuild connectivity, at least within mountain range systems. Furthermore, where possible in the future, additional genetic sampling coud aid in conservation management.Our results highlight the need for conservation management of  success . Despite success ). LikewiP. x. xanthopus . Augmenpus see ,118,119),119P. x.Although our sampling and analysis has demonstrated a lack of contemporary gene flow between most sampled populations, recent (post 2012) evidence of further population growth and expansion in the Gawler and Flinders Ranges (Lethbridge pers. comm.) highlights the need for ongoing genetic sampling, ideally incorporating more widespread population sampling, to enable augmentation to be fine-tuned. If, for example, the recent population growth and dispersal has enabled gene flow between previously isolated populations within range systems to be successfully re-established, then augmentation would only be necessary at a broader scale . Given the evidence from population viability analysis that some modeled translocations scenarios have no long-term impact on kinship coefficients , furtherP. x. xanthopus. Given our results of recent genetic divergence and the similarity in environments across the semi-arid zone we support moving forward with genetic rescue, with the notion that additional information from these resources be incorporated in the future. Populations are still persisting in situ, therefore, an approach with the least disturbance is the most favorable. Augmentation enhances the adaptive potential of populations and the evolvability of the species as a whole. We note, however, that genetic management needs to be undertaken in combination with effective control of exotic predators ,121, andP. x. xanthopus there is a very low risk of outbreeding depression , the risk of outbreeding depression, where offspring from genetically distant individuals have a lower fitness, needs to be considered. There is strong support for restoring gene flow for small inbred populations isolated by anthropogenic impacts within the last 500 years if the outbreeding risk is low (see ). Followsion see . These pO than most other populations in the Flinders Ranges. However, long term, individuals should be moved broadly from across populations. Detailed modeling of genetic compatibility and adaptive fitness would assist with augmentation planning moving forward.According to the available data, we suggest prioritizing augmentation of Gawler Ranges populations with individuals from the Flinders Ranges. The augmentation of some Flinders Ranges populations, particularly Aroona Dam and Sandy Creek which have the lowest diversity, should also be considered. In addition, we propose moving individuals from the Flinders Ranges into Olary Hills to boost the genetic diversity within this population. We suggest initially sourcing individuals from the Flinders Ranges, as this is the more proximal population and reflects the greater connectivity detected from mitochondrial data and genetic differentiation results. On the basis of current data, individuals for augmentation are likely best sourced from Wilkawillina North and Wilkawillina South, as they have the highest allelic richness, allelic diversity, and higher HP. x. xanthopus and potentially important for addressing concerns of future adaptability in the face of environmental and climatic change. In addition to evaluating genomic regions under selection, predictive climatic modeling would also provide valuable insight into any local environmental differences between populations and assist in interpreting adaptive variation between populations, as well as physiological and behavioral assessments. Such landscape genomic approaches are showing great promise in conservation biology . Identiy . P. xanthopus. Applying spatial population viability analyses using approximate Bayesian computations would also be a useful decision-planning tool for exploring the outcomes of various management scenarios, as they allow for more complex models to be assessed, incorporating adaptation and selection (see ). With iP. x. xanthopus and highlight how contemporary fragmentation has restricted connectivity among populations. Inference over deeper evolutionary timescales indicates greater connectivity among populations, thus, making the contemporary genetically depauperate populations suited to genetic rescue via augmentation. Incorporation of genetic data in conservation decision making is imperative if management aims to maximize the ability of populations to adapt to future threats and environmental changes. The arid biome of Australia is under-explored and improving our understanding of broad genetic structure across the landscape not only aids single species recovery but also would improve future broad-scale landscape management planning.Here, we examine the genetic effects of recent declines in