One thousand sixty-one individuals were sampled from the cities of Anbar, Baghdad, Basra, Diyala, Najaf, and Wasit in Iraq and typed for 15 forensic STRs to explore the genetic structure of Iraq and develop a forensic DNA database. The total number of alleles that were identified was 203.Analyses of molecular variance (AMOVA) were then conducted Baghdad provides a good representation of the rest of the country, while Anbar is the most genetically distinct. The average heterozygosities of these loci was 0.779, homozygosities was 0.221, polymorphism information content was 0.77, power of discrimination was 0.927, and power of exclusion was 0.563. At these loci, a matching genotype will occur, on average, in 1 in 8.152 × 1017 individuals. For paternity tests, the average paternity probability for a matching profile is 99.9997%.These loci are appropriate for use in forensic and paternity testing for this population. Iraq is similar to other countries in the Middle East, particularly Iran and Turkey, and is more similar to Europe than either Asia or Africa. Short tandem repeats, also known as microsatellites, are repeated sequences of DNA, usually consisting of 2 to 6 bases. They are highly polymorphic and distributed throughout the genome. These sequences vary in the number of repeats, resulting in them being multiallelic . ConsequThe collection of samples used in the research  was approved by the Center’s Scientific Research Ethics Committee before starting work. The volunteers signed a written consent to participate in this study.n = 139), Anbar (n = 132), Wasit (n = 120), Najaf (n = 119), Baghdad (n = 354), and Basra (n = 198). The samples were collected as Buccal swabs from patients and laboratory workers at private laboratories. These samples were used to study the population genetic diversity in Iraq. The population genetics of Iraqis is important due to their ethnic diversity. Several studies have been conducted to analyze the genetic diversity of Iraqi populations, including studies on the distribution of Y chromosome haplotypes, 23-YSTR markers, and 15 STRs. These studies aimed to analyze the genetic structure of different cities in Iraq and to create a forensic DNA database for the country.A total of 1061 unrelated individuals were sampled from six Iraqi cities: Diyala , and their DNA content was quantified with NanoDrop .Fifteen autosomal STR markers (the 13 CODIS core loci and D19S433 and D2S1338) were genotyped along with the amelogenin locus on the X and Y chromosomes using the Applied Biosystems AmpFiSTR® Identifiler™ kit (3). Approximately 1ng of template DNA was amplified for each sample following the protocols described in the user’s manual (Applied Biosystems). The samples were amplified with an Applied Biosystems Veriti® PCR System (Applied Biosystems).TM-4 (Applied Biosystems) was used for higher-resolution separations.Amplification products were diluted 1:15 in Hi-Di™ formamide and GS500-LIZ internal size standard (Applied Biosystems) and analyzed on the 16-capillary ABI Prism® 3100 Genetic Analyzer. On a 36-cm array, POPData collection was performed with Data Collection v. 2.0 software (Applied Biosystems), and samples were analyzed with GeneMapper v. 3.2 software (Applied Biosystems) at the Forensic DNA Center for Research and Training of Al-Nahrain University.Allele frequencies for each locus, heterozygosities, homozygosities, polymorphic information anthropology, and content (PIC) measure the formativeness of a genetic marker, indicating how well it can distinguish between different alleles in a population. PIC = 1−∑ (pi^2) PIC ranges from 0 to 1, with higher values indicating greater allelic diversity and formativeness of the marker.Matching probability (MP), powers of discrimination (PD) is a measure of how well a genetic marker can discriminate between individuals within a population.i-th and j-th alleles at the locus, summed over all possible pairwise allele combinations.Pi and pj are the frequencies of the PD also ranges from 0 to 1, with higher values indicating better discriminatory power. PD = ∑ (pi * pj).Powers of exclusion (PE) measures the probability that two individuals randomly chosen from a population will have different genotypes at a specific locus.PE = 1–P (same genotype)P (same genotype) is the probability that two individuals chosen at random will have the same genotype at the locus.PE can also range from 0 to 1, with higher values indicating a higher probability of distinguishing between individuals.p values were calculated using Monte Carlo methods with 100,000 permutations. The Holm-Bonferroni method [And typical paternity index (TPI) was calculated at each locus using Power Stats v1.2 . The sigi method  was usedi method .A total of 203 alleles were identified in this study. The distribution of these alleles across the 15 short tandem repeat (STR) loci is detailed in Table The city of Baghdad boasted the largest sample size, comprising 354 individuals. Interestingly, five alleles were exclusive to Baghdad and were not found in the other cities or neighboring countries. Three of these five alleles were absent from the other five Iraqi cities and all countries used for comparison, including Turkey, Iran , Syria , Kuwait An additional rare allele, allele 22 at D13S317, was identified in the city of Wasit, with a frequency of 0.4%, but was not observed in any other Iraqi cities or countries in the comparison set [p values for the HWE test [To assess whether the observed genotype frequencies adhere to the expected frequencies, the population’s adherence to the Hardy-Weinberg equilibrium (HWE) was examined . Table 2HWE test , 20, whiHWE test .Table 2Details concerning each locus are provided in Table Exploring STR analysis within Iraqi populations has been somewhat limited, and the studies that have been conducted employed distinct commercial STR kits, posing challenges for direct comparisons with the current study’s outcomes. Nevertheless, there is an overlap of the 15 STR markers between the aforementioned studies and neighboring countries, as outlined in Table Among these 15 common STR markers, D2S1338 demonstrated the highest Polymorphic Information Content (PIC) value within this study, while FGA exhibited the highest PIC value among Jordanians, succeeded by D19S433 in Turkey, D18S51 and FGA in Palestinians , and D19In forensic investigations, CMP is frequently expressed as the likelihood that one individual in a certain population subset possesses a genotype matching the composite value. With the global population approximately at 7.5 billion, an average MP of 0.173 or lower across all 15 loci is necessary for the CMP to be interpreted as 1 in 7.5 billion or greater. The dataset's mean MP across 15 loci is 0.073. In paternity reports, the probability of paternity is determined by CPI/(CPI + 1), where a CPI of 100 or higher indicates a probability of 99.0% or higher. For the average TPI to reach 1.43 for 15 loci, the average TPI over 1.5 locations was 2.36 .The findings of this investigation highlight the utility of these 15 autosomal STR loci as valuable markers for forensic and paternity testing within the Iraqi population. Within certain loci, the presence of a wide range of repeated sequences suggests possible population admixture dynamics. To elucidate the origins of the less common alleles discovered in this study, it may be beneficial to conduct further allele frequency analyses within specific ethnic groups in Iraq.Lastly, the comparative evaluation of forensic genetic efficiency parameters across diverse populations, including the Middle Eastern region, is vital for comprehending genetic diversity and potential intermingling among these populations. To achieve a more accurate comparison, it is strongly recommended to conduct comparative studies employing the same STR kit across these populations, considering that different STR kits have been used previously to define forensic efficiency parameters.Looking at the values within each population, you can observe that some alleles have consistent frequencies across populations, while others vary more. This variation could be due to a variety of factors, including historical migrations, genetic drift, and natural selection.The range of allele frequencies across the different populations indicates the genetic diversity present in these regions. Higher diversity might be indicative of a more mixed or heterogeneous population.The findings from this study highlight the effectiveness of the 15 autosomal STR loci as markers for forensics and paternity testing within the Iraqi population. It is worth noting that certain loci exhibit a wide range of repeats, which may be indicative of population admixture. To gain insight into the origins of the less common alleles identified in this study, further investigations should focus on analyzing allele frequencies within specific ethnic groups in Iraq.A comprehensive assessment of forensic genetic efficiency parameters is crucial for understanding genetic diversity and admixture among different populations, including those in the Middle East. To ensure a robust comparison, it is strongly recommended that comparative studies utilize the same STR kit across these populations. Previous studies have employed disparate STR kits, which has affected the description of forensic efficiency parameters.The study discussed in the given text highlights the effectiveness of 15 autosomal STR loci as markers for forensics and paternity testing within the Iraqi population. The CMP represents the likelihood that an individual in a specific population subset possesses a genotype matching a composite value. With an average Match Probability (MP) of 0.073 across all 15 loci in a global population of 7.5 billion, the CMP can be interpreted as 1 in 7.5 billion or greater The presence of a wide range of repeated sequences within certain loci suggests possible population admixture dynamics. To understand the origins of less common alleles, further allele frequency analyses within specific ethnic groups in Iraq are recommended. Conducting comparative evaluations of forensic genetic efficiency parameters across diverse populations, including those in the Middle East, is essential for comprehending genetic diversity and potential intermingling among these populations. Using the same STR kit for these studies is crucial for accurate comparisons. The study also highlights that some alleles have consistent frequencies across populations, while others exhibit more variation, which could be attributed to factors such as historical migrations, genetic drift, and natural selection.


Micropogonias furnieri is an important fishery resource on the southwest Atlantic coast. Despite being heavily exploited, there are a few uncertainties regarding the population structure in the transition area between the tropical and warm temperate zones (South Brazilian Bight). In the State of Rio de Janeiro (the northern part of this area), local environmental conditions, such as an upwelling phenomena and large estuarine bays, together with contributions of continental drainage and anthropogenic activities, could determine different croaker populations. The aim of this study was to assess the fine-scale population structure of this species in three localities in Rio de Janeiro State (Brazil) and to compare it with previous studies that used different approaches. Through the combined use of genetic markers (nuclear microsatellites) and otolith signatures (morphometry and chemistry), two genotypic (North + Center/South) and three phenotypic (North + Center + South) populations were found. These results could contribute to a better understanding of the M. furnieri population dynamics and allow a rational management of this important fishing resource. The whitemouth croaker Micropogonias furnieri) is one the most heavily exploited fishing resources in this area, but there are still some doubts about its population structure. In this study, through combined analyses using nuclear genetic markers and morphological and geochemical signatures of otoliths, a divergence of individuals between two populations was identified using microsatellites, while a finer spatial structure with three populations  was found based on otolith shapes and elemental signatures. This regional population structure may have direct implications for rational fisheries management and conservation of the species.The inshore area of the Southwestern Atlantic between 22 °S and 29 °S (South Brazilian Bight) is a transitional climatic zone, where the tropical and warm temperate provinces mix. In its northern part, i.e., in the coastal waters of Rio de Janeiro, Brazil, local oceanographic conditions, such as upwelling in the north, and great bays with different degrees of anthropogenic influences in the center and south can determine the population structure of several fish stocks. The Whitemouth croaker ( Micropogonias furnieri, one of the most important fishery resources in Brazil, has been overfished since 1960 [M. furnieri along the South American coast. Nevertheless, the results have been somewhat contradictory [The whitemouth croaker nce 1960 ,3. In thadictory ,5,6,7.M. furnieri has been previously suggested [M. furnieri in the three studies [Regarding the main fishing area, the existence of one, two, or even three distinct population units of uggested ,10,11,12uggested , alloenzuggested  and mitouggested ,10  and otolith analyses , the microscale population structure of The Southern Brazilian Bight (SBB) is the region along the southwestern Atlantic coast between the latitudes of 22 °S (Cabo de São Tomé–Campos dos Goytacazes/Rio de Janeiro) and 29 °S (Cabo de Santa Marta–Laguna/Santa Catarina). It is a transitional region between the tropical and temperate provinces. There are several upwelling areas that increase productivity across this area; one in the north of Cabo Frio (22.7 °S); another in the south near Cabo de Santa Marta (28.6 °S), and a third, less conspicuous, is near the Ilha Bela coast (23.8 °S). In these areas, seasonal upwelling takes place mainly between September and April, resulting in cold water temperatures that reach 18 °C .Rio de Janeiro is situated in the north of the SBB and has different influences along its 650 km coast extension, encompassing different coastal systems determined by geomorphological and ecological characteristics. In the present study, three localities along the Rio de Janeiro coast, separated by approximately 400 km, were studied: (1) Macaé, located in the north of Cabo Frio (22.7 °S), is the region most influenced by the up-welling; (2) Itaipú (23 °S), located in the center, has the influence of Guanabara Bay, an anthropized estuarine system; and (3) Ilha Grande (23.2 °S), located in the south, has the most environmentally preserved system .M. furnieri from an unpublished work [n = 18), Center (n = 45) and South (n = 37) were collected between August 2002 and May 2006 . A muscFor the otolith analyses, ninety individuals were collected between September and October 2016: 30 from the North, 30 from the Center and 30 from the South. All sampled fish were adults (TL > size at first maturity) and ranged between 29.5 and 39.5 cm of TL (to 0.1 cm). Sagittal otoliths were carefully removed, measured  and weighed . Left otoliths were photographed for shape analyses and right otoliths were preserved clean and dry for elemental chemical analyses.For both molecular and otolith analyses, fish were collected from the artisanal fishermen over several consecutive days until the desired sampling size was achieved, ensuring that the fishing area corresponded to the landing location.M. furnieri were used [2, 50 μg acetylated bovine serum albumin (BSA), 0.5 mM of each primer and 0.5 mM of the fluorescent tail, 1 unit of Taq polymerase , 1X PCR buffer, and 30 ng of template DNA in a final volume of 15 μL per reaction. PCR thermocycling conditions were one cycle of 94 °C for 5 min followed by 30 cycles of 94 °C for 45 s, 60 °C for 45 s, 72 °C for 45 s. After these first amplification steps, eight more cycles at 94 °C for 45 s, 53 °C for 45 s and 72 °C for 45 s were performed to incorporate fluorescent-labeled primers (tail) into the amplified products. Genotyping, using GeneScan Liz 500  as size standard, was performed in an automated sequencer ABI 3130 at Plataforma Genômica Multifuncional e Multiusuária (IBRAG/UERJ). It is noteworthy to highlight that samples from the North were genotyped and compared to genotypic data generated under the same conditions from a previous study [DNA from muscle tissue samples was extracted using the saline method . Microsaere used . Amplifius study .http://genepop.curtin.edu.au/ (accessed on 4 October 2021)) was used, with 10,000 iterations. A linkage disequilibrium analysis was also performed in Arlequin 3.5 (Bern Switzerland), which sought to identify the presence of associated alleles at a higher frequency than would be expected at random. To calculate the number of alleles of each loci, expected and observed heterozygosities, deviations from the expected Hardy-Weinberg equilibrium and inbreeding coefficient estimates (FIS) in the three populations, GeneAlex 6.5  was used [ST and Analysis of Molecular Variance (AMOVA). Genetic structuring was also evaluated with a Bayesian analysis using the STRUCTURE software  [5 with 5 independent replicas. To identify the presence of relatedness between individuals  within each location group, ML-RELATE software  was used [p < 0.05 [To evaluate the occurrence of problems inherent to genotyping, such as the presence of null alleles and drop-out alleles, the Micro-Checker 2.2   was usedwas used ,50. The was used  was alsoIL, USA) . This anIL, USA) . The ΔK IL, USA)  from theIL, USA)  using thIL, USA) . A correIL, USA) . MaximumIL, USA) ,59. Thiswas used . This sop < 0.05 .For the shape analyses, the otoliths were photographed on a black background with a Leica 205M stereomicroscope with a MC 170 HD camera and the software Leica Application Suite (LAS V2.0) . Multivariate analysis of otolith shape was performed using elliptical Fourier descriptors (EFDs) according to a shapeR package routine . The seq2O2, Fluka TraceSelect) for 15 min, followed by 1% ultrapure nitric acid  for 10 s, and then double-washed with ultrapure water  for 5 min, and then dried in a laminar flow chamber [3 to a final volume of 15 mL. The whole otolith samples were analyzed by inductively coupled plasma atomic emission spectrometry (ICP-OES) equipped with a cyclonic spray chamber and a Burgener MiraMist nebulizer, under the following conditions: forward power 1000 W, argon flow plasma 12 L/min, sheath gas 0.8 L/min. Otoliths were randomly analyzed to avoid sequence effects. The calibration curve was constructed using six standards made by successive dilutions with the Inorganic Venture multi-element LCA pattern, Christiansburg, VA, USA. The elements to be analyzed were pre-selected based on a previous otolith microchemistry study on this species [42Ca, 88Sr, 137Ba, 55Mn, 24Mg, and 63Cu) were consistently detected above the limit of detection limit. A certified reference material (FEBS-1) was used for quality analytical control [42Ca (100), 88Sr (4), 137Ba (20), 55Mn (20), 24Mg (20) and 63Cu (20). Geochemical signatures of otoliths were tested for normality  and homogeneity of variances , and data Log(x + 1) transformed if necessary (Ba:Ca and Mn:Ca). To prevent any size/age differences in individuals from influencing the microchemistry results among locations, thereby confounding the spatial variation, an analysis of covariance (ANCOVA) was performed [p < 0,05). Multivariate analysis of variance (MANOVA) and linear discriminant function analysis (LDFA) were used to explore the variation of multi-elemental signatures among locations [p < 0.05 was adopted.For the chemical analyses, the otoliths were decontaminated with 3% ultrapure hydrogen peroxide , Mfur04 (North and South), Mfur07 (South), Mfur10 (South), Mfur12 (North and South), Mfur17(North), Mfur25 (South) and Mfur26 (South) were detected. However, none of the linkage disequilibrium analyses showed significant values, demonstrating independence between the data.p < 0.05) was detected. The number of alleles per loci ranged from 6 at the Mfur20 loci to 33 at the Mfur03 loci with a mean value of 11.722 ± 1.105. Summary statistics for the microsatellite dataset is provided in The suspicion of null alleles in markers Mfur02 in the Southern population, Mfur10 in the Center population, and Mfur12 in the North population (ST comparisons were significant (p < 0.05), with higher values in comparisons between the north and the other locations , 0.21% among populations within groups, and 88.9% within populations.All Pairwise Focations . Bayesiaocations . This scocations . AMOVA aGene flow analysis showed an asymmetric migration pattern between pairs of locations . Theta ep < 0.05). In the Center, the mean k was 0.042, with one parent/offspring relationship, 13 full sibling relationships, and 12 half-sibling relationships confirmed by the hypothesis test . In the South, k was 0.031, with all 13 half-sibling relationships significant . All other pairs of relationships were considered unrelated .The mean relatedness (k) in the North was 0.035. Only one parent/offspring relationship, one full sibling relationship, and eight half-sibling relationships were confirmed by the hypothesis test . These differences are reflected in the mean shape at different points as the rostrum and excisura  and Mg:Ca in the North . The Ba:Ca , Mn:Ca  and Cu:Ca  ratios were higher in the Center compared with the other two locations (p < 0.001). LFDA depicted a clear separation of the individuals among the 3 locations. Overall, 99% of the individuals were correctly re-classified to the original location by the Jackknife classification matrix, ranging from 97% in the North to 100% in the Center and South  and a re-classification success of 100% for the entire dataset was achieved (LDA). Despite the result being similar between EFDs and ER, the three groups seem to be better separated using EFDs + ER  is somehow acting as a barrier for the species gene flow, causing a genetic differentiation between the populations at the Northern and Central/Southern areas. The differentiation between these populations in Rio de Janeiro may be due to the intersection of currents, where tropical waters of the Brazilian current meet cold waters of the sub-Antarctic current, giving rise to waters of subtropical convergence or Central Atlantic South Waters (ACAS) . These wMacrodon atricauda in the southeastern region of Brazil, which differs from the M. ancylodon species through shape analysis [Mugil liza with a differentiated population unit in Rio de Janeiro inferred from microsatellites [Sardinella brasiliensis identified by shape and multi-elemental analyses was found in Rio de Janeiro [Atractoscion aequidens and Argyrosomus inodurus were also recorded between the northern and southern Benguela regions, an area known to have a consistent seasonal upwelling mechanism [Little is known about the permeability effect of the barrier for species distributed across the region, but Cabo Frio in the north of Rio de Janeiro is considered a border of congeneric dividing currents and species populations ,69. Howeanalysis ,71, the tellites , and, mo Janeiro . Geneticechanism ,74,75. Techanism ,73,75,76Otoliths were efficient tools in ichthyology and can give us clues as to whether fish live in different environments, experience different feeding regimes or have different genetic backgrounds ,79,80,81The data recorded in fish otoliths showed higher concentrations of Mg:Ca in the North and Sr:Ca in the South. Such elemental concentrations present in otoliths are generally imposed by the water surrounding the environment, which constantly responds to variations caused by hydrogeological processes, tidal regimes, precipitation patterns and upwelling phenomena . UpwelliSeveral studies showed that the incorporation of vestigial and trace elements in the aragonite matrix of otoliths could be also influenced by physiological variables such as feeding regime, growth and ontogenetic differences and sex-specific variations . That isM. furnieri adults can perform regular migrations from estuarine areas to continental shelf waters and should not suffer from such limitations. However, the upwelling occurs in the same period of the year in which the spawning of the species takes place in the state of Rio de Janeiro. This species spawns during spring, summer and autumn, coinciding with the upwelling of Cabo Frio in northern area of Rio de Janeiro [The microsatellite dataset corroborated the feeling that individuals found in Rio de Janeiro coastal waters are not genetically homogenous. The big challenge now is to identify what kind of barrier separates these populations . The role of the upwelling phenomena as an oceanographic geographical barrier is well accepted for eggs and larvae that are passively dispersed through water currents ,21,76. H Janeiro . This co Janeiro ,76. Furt Janeiro ,95.M. furnieri individuals.Among the obstacles that could cause constraints to the gene flow in marine environments, upwelling cells, environmental waters transitions, and sea surface temperature gradients are known to be incomplete/permeable barriers, leading to the divergence of allopatric populations and species ,96,97. TM. furnieri. Indeed, a previous study found moderate to high genetic divergence among three populations of Whitemouth croaker sampled nearly 200 km apart long the Uruguayan Coast [M. furnieri. Probably the apparent incongruence among the two population markers used in the current study is likely related to the evolutionary timescale at which each marker varies. Natural tags such as genetic markers are usually more conservative at broader spatio-temporal frames, whereas otolith chemical and morphological markers are usually environmentally dependent phenotypic traits [The existence of a population structure in such a small-scale geographic area like that presented here is surprisingly, but not unprecedented, in an Coast . This hic traits . HoweverMicropogonias furnieri in the coast of Rio de Janeiro, geographically separated by Cabo Frio. However, otolith analyses have been able to discriminate the individuals of the three sampling regions in Rio de Janeiro, suggesting three populations instead. This study showed the importance of a holistic approach using genotypic and phenotypic complementary tools to study fish populations. New studies must be carried out to understand the cause behind the differentiation of these populations in this region. To continue to unravel this complex fish population structure, subsequent research should focus on the effects of the upwelling of the Cabo Frio region, the surface currents, the proximity of tropical regions or other environmental factors in its population dynamics.Nuclear markers suggested the existence of two populations of


Alnus glutinosa, a keystone species of riparian ecosystems that warrants priority conservation actions. We employed 17 nuclear microsatellite (Simple Sequence Repeat) markers and compared the genetic diversity and copy number of the captured alleles of 22 OP progeny families from this plantation, with 10 wild A. glutinosa populations, originating from the two provenance regions of the species in Lithuania. We conclude that the progeny plantation could be used as an ex situ plantation for the A. glutinosa populations from the first provenance region (represented by eight genetic conservation units (GCU)). Based on the present study’s results, we can expect that the A. glutinosa progeny plantation harbors enough genetic diversity of wild A. glutinosa populations from the first provenance region. This progeny plantation can serve as a robust ex situ collection containing local alleles present in at least one wild population with at least 0.05 frequency with 25 replications.Scientifically informed decisions for the long-term conservation of extant genetic diversity should combine in situ and ex situ conservation methods. The aim of the present study was to assess if a progeny plantation consisting of several open pollinated (OP) families and established for breeding purposes can also serve as an ex situ conservation plantation, using the case study of a Lithuanian progeny trial of  Alnus glutinosa (L.) Gaertn.) is a species that is widespread in Europe, and which can be found in all climate zones from the Mediterranean to the European north [Black alder  committed to preserve 70% of genetic diversity of crops, their wild relatives, and other economically or culturally valued species by 2020, but this goal has not been achieved. The amount of genetic diversity currently safeguarded in ex situ collections varies considerably species-wise. Most ex situ collections do not sufficiently represent the genetic diversity of wild populations [The present rapid climate change and anthropogenic impact on ecosystems has caused an unprecedented species extinction rate ,6. Scienulations . The numulations .The debate on the appropriate number of seeds or plants that should be kept in the ex situ collections to preserve species’ genetic diversity and evolutionary potential started almost 50 years ago . The firThe other aspect to consider for an appropriate ex situ collection is the number of allelic copies it contains. Alleles represented only once in the collection have a high chance of being lost . A securThe majority of the economically important tree species in Europe have wide distribution ranges . For manA. glutinosa (L.) Gaertn.  is an important, monoecious, self-incompatible, wind-pollinated keystone tree species found in riparian ecosystems in Europe [A. glutinosa, as a keystone species of riparian ecosystems, warrants priority conservation efforts. To preserve the local genetic diversity of A. glutinosa populations, ex situ conservation should also be considered in addition to in situ conservation. It has been suggested that ex situ conservation of A. glutinosa should incorporate a large enough number of genetically different individuals  to maintain a viable breeding population that could be used for conservation as well as breeding purposes [n Europe ,25. It in Europe , harborsn Europe ,30,31,32n Europe ,33. Ripan Europe . A. glutpurposes .A. glutinosa progeny plantation from Lithuania.The aim of the present study was to assess if an existing progeny trial established for breeding purposes can also serve as an ex situ conservation collection, using a case study of an A. glutinosa progeny plantation samples, we retained 17 loci. Three loci  were excluded, as they failed to amplify in 81, 64, and 70 of the sampled individuals, respectively. All loci retained for further analysis revealed no presence of null alleles in ex situ samples  to 21 (Ag35), and 7 (A2) to 23 (Ag35 and Ag25) alleles, in ex situ and GCU sample lots, respectively. The total number of alleles found in the progeny plantation was 232 (13.65 on average per locus), while in natural populations it was 272 (16.0 on average per locus). In the GCU sample lot 52 alleles were found that were absent in the ex situ sample lot, while 15 alleles found in the plantation were absent in the in situ samples . All stuA. glutinosa sample lots  are given in Genetic diversity parameters for A. glutinosa populations, presented a wide scatter peripheral to the wild populations cluster.The genetic relationships of the different OP families and populations were revealed by principal coordinate analysis (PCoA) . The 2D An important aspect in an effective ex situ conservation of genetic diversity is how well allelic diversity is represented in the ex situ collection. To evaluate this aspect in the progeny plantation, we divided the alleles of GCU populations into five categories : all allThe number of alleles in different single wild populations was very similar in all categories, with common and low-frequency allele categories prevailing . Allele When seeking to conserve species in ex situ collections, it is important to preserve as much genetic diversity as possible, representing not only single populations in a collection, but also multiple populations or (in widespread species case) the metapopulation. Therefore, in the present study, we not only analyzed single wild population alleles and their frequencies, but also those from the metapopulation. The number of alleles found in six OP progeny families originating from all mother tree populations  was 75% of the total number of alleles found in the five natural populations , and when all 22 OP families were included, the total number of alleles found reached 212, or 85% of total allele number of the five natural populations G comparH  is presented in Ho = 0.77 and 0.73, respectively), although this difference was not statistically significant. Expected heterozygosity was found to be higher in wild populations than in the progeny plantation. The progeny plantation and wild population’s inbreeding coefficients were close to zero . The allelic richness of wild populations based on 16 diploid individuals on average was found to be 7.33 [Ar was found to be 2.61 for ex situ OP families and 2.88 on average for wild populations. The low number of allelic richness might be explained by the low number of individuals it was calculated for . Genetic differentiation among wild populations was low [A. glutinosa was confirmed by the PCoA analysis, where OP families originating from different wild populations scattered around the cluster of 10 GCU populations used for comparisons in the present study  varies from 4 to 130. Based on the sampling strategy in this study, we investigated samples from 20 different stands (populations) and 15–20 progeny trees per population , and most of the low-frequency alleles with at least 5 copies each (86%), regardless of the number of wild GCU populations involved  . Based oA. glutinosa populations, representing the first provenance region of Lithuania (of two). Taking into account the much higher number of A. glutinosa plants present in the progeny plantation than in the investigated sample, it can reasonably be expected that this progeny plantation should be enough to encompass 25 replicates of local alleles present in at least one population with at least a 0.05 frequency, and five replicates of all alleles meeting the genetic diversity threshold of 95% [The Vytėnai progeny plantation could be used as an ex situ collection for the d of 95% .Our case study suggests that common forest tree species progeny or provenance trials could be worthy candidates to serve as ex situ collections for economically important widespread forest trees. Progeny trials most often represent a rather limited species distribution area , and in case of a severe but not widespread biotic outbreak or natural disaster, they could serve as a good source of genetic material to restore devastated stands or populations. The elevated need for immediate establishment of ex situ collections in forest trees as a safety net for climatic change is hampered by the absence of pertinent funding and long-term planning in the conservation of forest genetic resources. Progeny trials may have originally been established for breeding purposes, but they can be used to meet, at least in part, the above goal, as this study has shown.A. glutinosa progeny plantation, established in 1998. Progeny plantation no. 09JBZ001 is located in the Dubrava Regional Branch of the State Forest Enterprise (SFE), Vytėnai forest district   . The plaThe black alder GCUs investigated in the present study are part of Lithuanian national forest seed base objects, which comply with minimum size requirements for dynamic in situ GCUs of forest tree genetic diversity ,41. FiveA. glutinosa that were delineated based on climatic regions, stand productivity, and phenological and morphological data analysis in accordance with species distribution in the country. In 2020, the delineation of provenance regions was specified based on molecular data.In Lithuania, there are two provenance regions for For this study in the Vytėnai progeny plantation, we sampled 22 OP families and 15–20 plants per OP family—420 plants in total . All OP A. glutinosa GCUs . The resulting sawdust was placed in 2 mL labeled Eppendorf tubes and kept at −20 °C until DNA extraction. The drill between the samples was sterilized using ethanol and flame to avoid sample cross-contamination. For the young regenerating trees, three or four leaf samples per tree were collected.® version 2022.1.1. Scored microsatellite loci were checked for the presence of null alleles and scoring errors with MicroChecker software [The molecular analysis followed the methodology described in Verbylaitė et al. . Prior tsoftware . The maisoftware ,53,54. Asoftware . The PCoFor comparing genetic diversity parameters and allele numbers, we conducted analyses on different sample lots using allele frequency averages. The comparison scheme is presented in The next step in this study was to analyze the copy number of alleles present in the progeny plantation. This analysis helped determine how strong the Vytėnai progeny plantation is in the sense of ex situ collection robustness. The prediction of the allele number in the progeny plantation (76 OP families) for the different allele frequency categories was based on the best-fitted trendline  for the average number of alleles in the different number of examined OP families .


Heleobia atacamensis, an endangered species scattered in isolated and semi-isolated but dynamic habitats of the Atacama Saltpan. We focused on snails obtained from Peine and Tilomonte, two peripherical localities, which were compared with topotypes specimens. The range extension of the species also allowed us to reassess its conservation status. Molecular analyses showed that snails from Peine and Tilomonte belong to Heleobia atacamensis. We also discovered genetic structure in the saltpan, represented by six genetic clusters, besides morphological differences between populations. Since the observed pattern is common for other freshwater species restricted to desert aquifers worldwide, our results represent findings applicable to analogous systems. The species, listed as Critically Endangered at regional level in 2014, was reassessed as Endangered. For an eventual management plan, we suggest incorporating the genetic information obtained here.In this study, we implemented a multifaceted approach, coupling phylogenetic, phylogeographical and demographic analyses together with a morphological characterization to depict the genetic patterns of Heleobia atacamensis, endemic to the Atacama Saltpan, revealed a strong interdependence of genetic patterns with climatic fluctuations and landscape physiography. The species is currently listed as Critically Endangered at regional scale and as Data Deficient on the International Union for Conservation of Nature (IUCN) Red List. Here, we studied genetic diversity and demographic history of several populations of the species occurring on a connectivity gradient, including snails from new peripherical localities (Peine and Tilomonte), which were compared with topotype specimens. In addition, we reassessed the conservation status using the IUCN Red List categories and criteria considering species-specific idiosyncrasy. Phylogenetic and phylogeographical analyses indicated that snails from Peine and Tilomonte belong to H. atacamensis. We discovered significant differentiation in shell morphology, which was generally greater in geographically isolated populations. We also inferred six genetic clusters and a demographic expansion congruent with the wet periods that occurred at the end of the Pleistocene. Considering the highest risk category obtained, H. atacamensis was reassessed as Endangered at regional scale. Future conservation plans should consider the genetic assemblages as conservation units.Evaporitic ecosystems of the Atacama Desert contain a rich endemic fauna, including mollusk species. A recent study performed in the freshwater snail  Species extinction is a recurring event throughout the history of life on Earth, and a variety of extinction-causing factors have been documented and hypothesized, some due to natural processes, others attributed to human activities ,2,3. CurThe diversity of mollusks is quite high; they are the second most diverse group after the arthropods . EstimatBiomphalaria costata (Biese), Heleobia atacamensis (Philippi) and Heleobia transitoria (Biese) [Freshwater snails are an important component of aquatic ecosystems worldwide, but also a group prone to extinction because such systems tend to be ephemeral, in addition to being affected by a series of threats produced by human activities that have increased in the last decades ,13,14. I (Biese) ,21,22.Heleobia atacamensis is a freshwater snail endemic to Tilopozo, a small water well located at the southern end of the Atacama Saltpan in northern Chile [2 in their operations [H. atacamensis populations) would also be affected by the brine pumping [H. atacamensis were based on different antecedents of the species, they were mainly established considering the single population of Tilopozo. However, a greater population of this snail has been discovered in the last decades. In a phylogenetic analysis using mitochondrial 12S and 16S ribosomal RNA gene sequences of two snails of the genus Heleobia from the Atacama Saltpan, Collado et al. [H. atacamensis while another from Tilomonte was considered as the sister group of this species. In a subsequent phylogenetic analysis using the cytochrome c oxidase subunit 1 mitochondrial gene (COI), Collado et al. [Heleobia sp. from Tilomonte as the sister group of H. atacamensis, although specimens from Peine were not included. More recently, Valladares et al. [H. atacamensis from Tilopozo and other seven undescribed Heleobia populations located inside the Atacama Saltpan, recovering the eight populations in a monophyletic group as part of a larger clade also containing other congeneric species from the Chilean Altiplano and the Atacama Desert. In addition, based on microsatellite markers, they detected a marked genetic structure and a high level of fragmentation among populations of the species. However, despite the fact that Valladares et al. [H. atacamensis within the Atacama Saltpan, the snails from the peripherical towns of Peine and Tilomonte were not included.rn Chile . The spern Chile . Howeverrn Chile . The thrrn Chile , where terations . Recent erations ,26. In a pumping . Althougo et al.  tentativo et al.  also recs et al.  used a ms et al.  significH. atacamensis using mitochondrial markers considering congeneric species present in the South American Altiplano and Atacama Desert, (2) investigate the genetic variation and historical demography of the species including all known populations, (3) evaluate the variability of shell morphology among populations including a comparison between snails from Peine and Tilomonte with topotype specimens of the species, and (4) reassess the conservation status of the species considering the identification of Evolutionary Significant Units (ESUs).Currently, knowing the distribution, genetic structure, and morphological variation of a species is a fundamental aspect in conservation biology, especially in vulnerable species, in addition to the identification of evolutionary independent lineages within the focus taxa ,33,34,35Live snails were collected in 2015 from Peine  and Tilomonte , Northern Chile . In eachCOI gene of snails from Peine and Tilomonte were obtained following Valladares et al. [H. atacamensis from the Atacama Saltpan [DNA extraction, PCR amplification conditions and sequencing of the s et al. . Both sts et al.  in the os et al. . In all  Saltpan . This daHeleobia [Semisalsa Radoman were used as sister group and Heleobops carrikeri Davis and McKee was used as outgroup (Phylogenetic relationships were examined using the maximum likelihood (ML) and Bayesian inference (BI) algorithms. In both cases, the best sequence evolution model was previously selected using PartitionFinder v2.1.1 . ReconstHeleobia ,28,29,42outgroup .COI sequences, 7 of snails from Peine, 2 from Tilomonte and 114 individuals of Heleobia atacamensis from eight localities inside the Atacama Saltpan [8 iterations and thinning every 10,000 iterations. The range was limited between K = 1 and K = 10, and a burn-in of 25%.For the phylogeographical and demographic analyses, the matrix generated included 123  Saltpan . This da Saltpan  using Po Saltpan . The Gen Saltpan  was used7 iteration sampling parameters every 1000 steps and discarding the first 25% of steps. The analysis was implemented under a strict molecular clock and assuming an evolutionary rate of 1.7% substitutions per million years for invertebrates [Historical demography was studied by comparing the observed mismatch distributions  implementebrates .For morphological comparison, 20 individuals from Peine and Tilomonte were analyzed. In addition, 10 individuals from each of the eight sampling sites located inside the saltpan were included. In each case, individuals from the upper third of the size distribution of each locality were chosen. From each photograph, five shell variables were measured: shell length (SL), shell width (SW), aperture length (AL), aperture width (AW), and body whorl length (BWL). A principal component analysis (PCA) was performed to visualize the morphological variation of different populations. Since some variables violated the assumptions of normality, we analyzed the data grouping individuals by localities using the non-parametric Kruskal–Wallis (K-W) test. Subsequently, a non-parametric MANOVA test (PERMANOVA) was conducted using 10,000 permutations to compare individuals using the multivariate data and under the same grouping scheme. All statistical analyses were conducted in R v3.6.1 software .H. atacamensis was performed using the IUCN Red List [H. atacamensis is a gill-breathing snail with direct development and therefore low vagility [2 was used. The possible categories are Critically Endangered (CR), Endangered (EN), Vulnerable (VU), Near Threatened (NT) and Least Concern (LC).Conservation status assessment of Red List  categoriRed List  and thatRed List . This stRed List . For thevagility . Geograpvagility , which pCOI sequences from Peine and Tilomonte were deposited in GenBank (OP630467-OP630474).Voucher specimens were deposited in the Museo de Ciencias Naturales y Arqueología Profesor Pedro Ramírez Fuentes (MCNPPRF–CC 148–10 to MCNPPRF 148–24), Chillán, Chile and Museo de Zoología de la Universidad de Concepción (MZUC-UCCC 44212–44214), Concepción, Chile. Additional specimens are housed at Laboratorio de Malacología y Sistemática Molecular, Universidad del Bío-Bío (UBB), Chillán, Chile. 12S, 16S and COI) used in the concatenated phylogenetic estimations produced an alignment of 1506 nucleotides in length, generating a matrix of 49 individuals. The best-fitting model of nucleotide substitution was the HKY + I + G for 12S and 16S genes; and GTR + I, HKY + I and GTR + G for the first, second and third codon positions of COI, respectively. The phylogenetic trees estimated using ML and BI were congruent, and in both analyses, samples from Peine and Tilomonte were recovered in the same clade with populations of H. atacamensis from inside the Atacama Saltpan , defining 47 haplotypes . The haplotype network . Likewise, the analysis revealed a non-significant SSD value  that did not refute the demographic model of spatial expansion for the species. The historical trends in effective population size of H. atacamensis obtained through the Bayesian skyline plot (BSP) suggest a sudden demographic expansion that occurred in the Late Pleistocene (c. 25 kya) until the Early Holocene (c. 10 kya); after this, the effective population size remained constant . The five shell variables analyzed showed significant differences among localities in all comparisons: shell length ; shell width ; aperture length ; aperture width ; and body whorl length .Together, the first two principal components explained 98.8% of the variance in shell morphology . While a2 and AOO values of 40 km2, assigning Endangered (EN) as a preliminary threat category in both cases. Moreover, considering criterion B1 < 20,000 km² (estimated 1016 km2) and criterion B2 < 2000 km² (estimated 40 km2), more to the condition (a) regarding number of locations , and condition (b) of continuing decline inferred in habitat quality (iii) [H. atacamensis is categorized as Vulnerable (VU) B1ab(iii) + B2ab(iii). On the other hand, considering the six genetic clusters of the species as six subpopulations “between which there is little demographic or genetic exchange” [H. atacamensis is Endangered (EN) B1ab(iii) + B2ab(iii).GeoCAT calculated EOO values of 1016 kmty (iii) , H. atacxchange” , under Cxchange” , the finSuperfamily Truncatelloidea Gray, 1840;Family Cochliopidae Tryon, 1866;Heleobia Stimpson, 1865.Genus Heleobia atacamensis Paludina atacamensis Philippi, 1860: Philippi [Philippi : 166, PlHydrobia atacamensis : Frauenfeld [auenfeld : 575, 66Littoridina atacamensis : Biese [): Biese : 172, 17): Biese : 64; Stu): Biese : 15; Val): Biese : 3; Vald): Biese : 70.Heleobia atacamensis : Hershler and Thompson [Thompson : 50; ColThompson : 52, FigThompson : 6–11, FThompson : 278; CoThompson : 711; CoThompson : FiguresThe morphological analysis did not provide qualitative differences between Tilopozo, Peine and Tilomonte snails; therefore, a general description is provided for the three locations.Shell small, conic-elongated A–F, thinTilopozo , Atacama Saltpan, Northern Chile type locality.New distributional record: Peine , Salar de Atacama basin, oasis located approximately 21 km northeast of Tilopozo; Tilomonte , Salar de Atacama basin, oasis located approximately 13 km east of Tilopozo. These two ecosystems correspond to small streams that normally do not flow into the water mirror of the saltpan, drying before reaching the coast.Heleobia atacamensis was collected in soft substratum or macrophytes.Tebenquiche, Chaxa, Puilar, Quelana, Salada, Peine, La Punta, La Brava, Tilopozo, Tilomonte, Atacama Saltpan basin, northern Chile.Heleobia atacamensis in Peine and Tilomonte, previously hypothesized using rDNA sequences, although the last population was considered a candidate species of Heleobia [COI phylogenetic analysis, H. atacamensis integrated a clade together with sequences of Heleobia ascotanensis (Courty), Heleobia chimbaensis (Biese), Heleobia deserticola Collado, Heleobia peralensis Collado, Fuentealba, Cazzaniga and Valladares and Heleobia transitoria, all of them from northern Chile. Our results are also consistent with those of Valladares et al. [H. atacamensis in the southern Altiplano. Several freshwater species within different taxa such as snails of the genus Biomphalaria Preston, fishes of the genus Orestias Valenciennes and frogs of the genus Telmatobius Wiegmann, co-distributed in the area, have also been recovered as a monophyletic group [The phylogenetic estimations confirm the presence of Heleobia . These ts et al.  regardinic group ,66,67. TH. atacamensis is consistent with studies performed in other desert spring systems, where poorly dispersing species such as snails and crustaceans show highly geographically structured populations and lineages [H. atacamensis populations were highly genetically structured, with little evidence for migration between isolated localities as Tilopozo, Tilomonte and, to a lesser extent, Quelana. On the other hand, geographically close populations  showed strong evidence of connectivity. The pattern detected in La Punta is also interesting since in this population, we recovered highly divergent haplotypes. This suggests that this population had some degree of gene flow with La Brava and Salada populations. This evidence was also reported in H. atacamensis using microsatellite markers [Heleobia (pers. obs.). A second explanation for this genetic similarity is a historical connection between the populations.The genetic divergence within lineages ,69,70,71 markers , which i markers , we hypoH. atacamensis in the phylogeographic analyzes generated two main results. Firstly, we obtained evidence that the evolutionary history of the species would be circumscribed not only to the events that occurred within the limits of the Atacama Saltpan. Therefore, to understand the evolutionary processes and evaluate possible threats to the conservation of the species, it is necessary to carry out studies that consider all the populations of the saltpan, including Peine and Tilomonte. Secondly, the population of Tilopozo  would correspond to a population differentiated from the rest, which partially differs from the results obtained by Valladares et al. [The inclusion of two new populations of s et al.  since thH. atacamensis would have experienced a population expansion at the end of the Pleistocene, agreeing with the data indicating that wettest perennial lake interval (26.7–16.5 ky) and climatic fluctuations occurred in the Atacama Desert in that period [Heleobia are strictly aquatic, the impact of water availability on the resilience of species that inhabit desert systems is of particular concern. This is even more important in H. atacamensis considering that in the saltpan, in addition to brine pumping, freshwater is extracted for mining [Population demographic parameters indicate that t period ,73,74. Tr mining , which hH. atacamensis. However, morphometric analysis of the shell showed significant differences among populations, although many of them overlapped in the multivariate space. On the other hand, some populations including snails from Peine, Tilomonte and Tebenquiche formed cohesive clusters in bounded sectors of the morphospace. This result is noteworthy since these populations are geographically isolated and, in the case of Tilomonte, present exclusive haplotypes . Tilopozo samples also formed a morphological assemblage with Chaxa and Quelana, although less evident than in the previous examples. In this case, the genetic differentiation of the first population was also evident in all the analyzes performed, consistent with previous studies [No conspicuous qualitative differences were observed in shell morphology when comparing snails from Peine and Tilomonte with topotypes of  studies .Heleobia atacamensis can be easily distinguished through the penis morphology of H. ascotanensis, H. chimbaensis, H. deserticola, and H. transitoria, but it is rather similar to that of Heleobia loaensis (Biese), H. opachensis (Biese) and H. carcotensis Collado, Valladares and Méndez, maybe reflecting evolutionary stasis since they are closely related species. However, the organ has been widely used to describe and recognize species of Heleobia, being of taxonomic usefulness in most cases [H. atacamensis and snails from Tilomonte that led to suggest the presence of a candidate species of the genus supported by a sister group relationship [Heleobia can also generate morphological differences in the shape of the penis [st cases ,81,82,83tionship  would behe penis .H. atacamensis as Endangered (EN), corresponding to a higher category than Data Deficient (DD) previously listed for the species according to the IUCN Red List [H. atacamensis are of concern, it is necessary to implement a conservation program for the species. At present, there are environmental interpretive trails in some localities of the saltpan, with restricted access, which are under the care of the native population of the place. Apart from this, we suggest monitoring the different occurrence localities periodically, in addition to creating micro-reserves, which have been successful in other taxonomic groups [Heleobia species, which are small, can be kept in small containers without major complications. However, a comprehensive management should consider the genetic structure of the snail, especially regarding the six genetic populations identified in the saltpan, in addition to prioritize areas for conservation based on the genetic diversity, gene flow and connectivity between systems.In this paper, we reassessed the conservation status of Red List , but lowRed List . Howeverc groups . Captivec groups ,87. HeleH. atacamensis, although more surveys inside and outside of the saltpan basin that could lead to the discovery of new populations of the species are required.For poorly investigated and endangered species, it is essential to know the number of populations and range extent they present. The new records reported in the present study are a significant finding for H. atacamensis were identified in the Atacama Saltpan basin, constituting a baseline for an eventual management plan of this snail. We also detected morphological variation and genetic structure within the new range of the species, with signs of relatively large-scale internal migration. Further studies are needed to determine whether there are other populations of H. atacamensis within the basin and in other basins of the Atacama Desert and southwestern Altiplano. Heleobia atacamensis meets the IUCN criteria to be listed as Endangered (EN) at regional scale.In this study, two new populations and six genetic clusters of


Aedes aegypti poses a significant threat to disease control. One form of resistance, caused by kdr mutations in the NaV gene, hinders vector control efforts in Brazil. Despite genetic differences typically accumulating among isolated populations, this mosquito can actively and passively disperse through human transportation. Our study investigated the genetic structure and spread of kdr mutations in Ae. aegypti populations across six localities in Amapá State, Brazil, within the Amazonian Forest. Using 12 microsatellite loci and qPCR methods, we assessed genetic structure and identified three common kdr mutations . High prevalence of kdr alleles was observed in all localities, indicating widespread distribution in Amapá State. Microsatellite analysis revealed differentiation among mosquito populations, dividing them into two distinct clusters supported by Bayesian and DAPC analyses. Oiapoque, located along the northern border with French Guiana, exhibited the highest kdr frequencies and genetic differentiation compared to other localities. Our findings suggest genetic structure in Ae. aegypti populations in Amapá State, with some passive gene flow between clusters. The study underscores the need for continuous surveillance of Ae. aegypti populations to monitor the spread of insecticide resistance and inform effective vector control strategies.Insecticide resistance in  Arthropod-borne virus) infections, such as dengue, Zika, chikungunya and yellow fever are causing severe impacts on global health, especially in tropical low-income countries1. In Brazil, dengue is considered as hyperendemic, with regional and seasonal circulation of the four serotypes (I–IV), but other newly emerged viruses such as Zika (ZIKV) and chikungunya (CHIKV) are also circulating2. In 2022, Brazil reached the highest number of deaths caused by dengue fever (1016 deaths) ever recorded in the country3, and the recent increase in yellow fever cases in regions bordering urban centers4 highlights the imminence of re-urbanization of this disease5. As there are no effective vaccines available for most of these arboviral diseases (excluding yellow fever), controlling the vector—the mosquito Aedes aegypti —is the method of choice for reducing the risk of arbovirus transmission.Arboviruses  is seen as one of the most important threats for the control of diseases caused by viruses transmitted by Aedes, as it may reduce the efficacy of chemical-based vector control interventions11. In Brazil, nationwide insecticide resistance monitoring (MRI) has reported strong resistance of Ae. aegypti to all pesticides used by the Ministry of Health, such as the larvicide temephos (organophosphate), the insect growth regulator pyriproxyfen, and the adulticides deltamethrin (pyrethroid) and malathion (organophosphate)13.Chemical control is still the cornerstone of any vector borne-diseases control programme worldwideNaV), which cause resistance to the knockdown effect of pyrethroids, and are therefore called the kdr mutations (knockdown resistance)14. In Brazil, at least three kdr mutations have been reported in Ae. aegypti: a substitution of Valine to Isoleucine at position 1016 (V1016I), a Phenylalanine to Cysteine at position 1534 (F1534C), and a Valine to Leucine at position 410 (V410L)16. These mutations were classified as NaVR1 and NaVR2, where NaVR1 is a haplotype containing the F1534C kdr mutation only while NaVR2 exhibits F1534C + V410L + V1016I mutations17. These alleles give pyrethroid resistance under a recessive trait, and the NaVR2 causes the highest level of resistance to deltamethrin18. Other SNPs, such as V1016G, S989P and T1520I, are common in Asian populations of Ae. aegypti20 but they are absent in Brazil and neighboring countries.Among the genetic alterations involved in IR, the most common are single nucleotide changes in the voltage-gated sodium channel  resistance have been reported in Aedes aegypti with at least three well defined clustered regions within the country16. The Amapá State (AP), located in the Northern part of the country, bordering the French Guiana, is an important gateway between Brazil and the Caribbean to arboviruses and vector populations with distinct characteristics, including insecticide resistance24. By the way, this was one of the entrance doors for the CHIKV virus in 2015 in Brazil25. In addition, the geographical aspects of the region raise relevant questions for population genetic studies since locations infested with Ae. aegypti are relatively isolated from each other’s due to the vast Amazonian vegetation. In AP, Ae. aegypti from Oiapoque (the city bordering the French Guiana) is among the Brazilian mosquito populations exhibiting the highest levels of resistance to deltamethrin, temephos and malathion, while the Ae. aegypti population from the AP State capital, Macapá city, is far less resistant27.These cities are 577 km distant, connected by a road (BR-156) that crosses the dense Amazon Forest. The frequency of kdr alleles is distinct between them, with a predominance of kdr NaVR1 and kdr NaVR2 in Macapá and Oiapoque, respectively24. The presence and frequency of these kdr alleles in other cities of the AP State is however unknown.In Brazil, differences in the genetic structure and spatial distribution of pyrethroid , kdr NaVR1 (VVC) and kdr NaVR2 (LIC) alleles, and two additional. The genotype R2X1 (LL + VI + CC), composed of kdr NaVR2 and the herein called kdr NaVX1 (LVC) alleles, was found in OIA and CAL. We could not accurately determine the allelic composition of the genotype Y (LL + VI + FC), because it may be composed of LIC/LVF or LVC/LIF. This genotype was exclusively found in OIA. We named the possible alleles LFV and LIF as kdr NaVX2 and kdr NaVX3, respectively (Supplementary Tables We genotyped 272 Ae. aegypti populations was the wild-type homozygous SS (VV + VV + FF), only observed in MAC (2.3%). The most frequent genotype was R1R1 (VV + VV + CC), ranging from 2.2% in OIA to 65.1% in MAC. All populations showed high frequencies of other “resistant genotypes” , whereas their sum ranged from 63% in OIA to 100% in PGR. MAC was the only population without the homozygous R2R2 genotype (R1R1 + R1R2 = 76.7%). The genotype R2X1 was observed in CAL (8.7%) and OIA (2.2%). The genotype Y (LL + VI + FC) was found only in OIA, although at a relatively low frequency (28.3%)  and the lower kdr NaVR2 (9.3%) frequencies, while OIA showed the lower kdr NaVR1 (15.2%) and the higher kdr NaVR2 (52.2%) frequencies  and R2R2 (4 OIA) mosquitoes. The IS6, IIS6 and IIIS6 sequences of R1R1 samples were similar to previously published sequences . The R2R2 sequences of samples from OIA, IS6, IIS6, and IIIS6 fragments were also similar to known sequences .We amplified and sequenced the corresponding IS6, IIS6 and IIIS6 Ae. aegypti divided into six populations from the Amapá State. We observed a total of 60 alleles, varying from two (AC4 and B2) to 17 (Ag2)  to 3.91 (TTG), the average number of effective alleles (Ne) from 2.33 (FGO) to 2.62 (OIA), the number of private alleles per population (Np) from 0 (CAL and PGR) to 0.25 (MAC), and the number of allelic richness (R) from 3.56 (CAL) to 3.88 (TTZ). The genetic diversity of a mosquito population usually is positively related to the expected heterozygosity (He) and observed heterozygosity (Ho) values. Here the He varied from 0.510 to 0.567 and the Ho varied from 0.528 to 0.576 , respectively (Supplementary Table p > 0.031).Regarding the genetic differentiation as measured by the Fst, the values of the pairs ranged from 0.004 (PGR-TTZ) to 0.084 (OIA-PGR) Table . Except Ae. aegypti is most likely divided into two genetic clusters (K = 2) , the populations from clusters 1 and 2 presented the r 2 Fig. .Figure 3kdr genotypes and the genetic structure of Ae. aegypti populations along a South-North transect of the Amapá State, starting from the capital city Macapá to Oiapoque, located on the border between Brazil and French Guiana. The Amapá State is particularly isolated from the other States and covered by > 90% by the Amazon Forest. Our study showed that the dengue vector Ae. aegypti is divided into two well-defined genetic clusters in this State, and the kdr allelic composition roughly followed this clustering. We also found evidence of new kdr genotype arrangements in at least two of the six mosquito populations analyzed. Better understanding of the genetic structure of Ae. aegypti populations is relevant not only to better understand the possible dispersion of resistance genes in this region but also to guide decision-making for vector control and insecticide resistance management.In this study, we investigated the frequency of 12. However, malathion was never utilized in the Amapá State , and until recently, only pyrethroids were employed. The first cases of chikungunya in Brazil were recorded in 2014 in the cities of Oiapoque (Amapá State) and Feira de Santana (Bahia State), from two distinct events25. As a result, the use of pyrethroid insecticide (deltamethrin) was intensified in Oiapoque, which likely contributed to the increase in the selection pressure on resistance genes. Indeed, Ae. aegypti populations collected from Amapá State in 2014/2015 showed high resistance to deltamethrin with estimated RR50 (resistance ratio) of 46.4 and 143.9 in Macapá and Oiapoque, respectively26. Recently, Cielo has been used in ULV applications since 2019, and Spinosad was introduced in Amapá State in 2021 . In French Guiana, deltamethrin is the only adulticide authorized in vector control campaigns since 201128.Historically in Brazil, the organophosphate malathion replaced pyrethroid insecticides around 2006–2009Ae. aegypti populations from Amapá presented high frequencies of kdr alleles, considering the three important SNPs in the voltage-gated sodium channel gene (NaV): V410L, V1016I and F1534C. Interestingly, we found two additional kdr genotypes composition or arrangement, LL + VI + CC in Calçoene and Oiapoque, and LL + VI + FC in Oiapoque. These distinct genotypes were probably present in previous surveys; however, they were not detected since the V410L had not been investigated in mosquitoes from Amapá16. The LL + VI + CC (R2X1) was previously observed in populations from Peru , Colombia29, Mexico30, and USA31. The LL + VI + FC (Y) was also previously observed in populations from the countries mentioned above, and in distinct Brazilian regions, such as Natal, Salvador, Vitória and Brasília16. Regardless of the location, their frequencies were generally low: varying from 1.1% in the USA to 5.8% in Colombia (R2X1), and from 0.2% in Peru to 2.5% in the USA (Y). In Amapá the frequencies were 28.3% (Y) and 2.2% (R2X1) in Oiapoque, and 8.7% (R2X1) in Calçoene. Complementary studies conducted by our research team suggest that these genotypes may result from gene duplications, hence maintaining distinct copies of the NaV gene in the same chromosome16. Their relationship with pyrethroid resistance requires further investigation.Here, we observed that all kdr NaVR2 allelic frequency remained very high in Oiapoque  and it increased significantly in Calçoene (from 7.8% in 201816 to 35.9% in 2020). While the kdr NaVR2 was absent in Macapá in 2014/201526, it has been found at low frequency (5.6%) in 201816, and at 9.3% in 2020, as shown in this study. The homozygous R2R2 is still absent from Macapá but it has been reported in Calçoene for the first time. In the localities of Ferreira Gomes, Porto Grande and Tartarugalzinho kdr NaVR2 varied from 42.4 to 48.9% and the R2R2 genotype ranged from 21.7 to 31.1%. It is worth noting that all localities along the BR-156 road had higher kdr NaVR2 frequency than Macapá, which is the State capital and therefore highly connected to these localities. Considering the genetic cost of the kdr NaVR2 under insecticide-free environment18, we assume that the insecticide pressure by pyrethroids might be lower in Macapá compared to other cities, hence prevailing the introduction and establishment of the kdr NaVR2 in Ae. aegypti in the State capital.We also showed that the NaVS allele in all populations is of great concern. In Macapá, the frequency of the S allele was 15.7% in 2014/201526, 17.8% in 201816 and 12.8% in 2020. We also showed that the wild-type allele NaVS is now absent in Porto Grande and less than 13% in other populations tested. It is worth noting that the NaVS allele was not detected in any Ae. aegypti population from the Amazonian region in the previous nationwide surveillance (2018)26.In addition, the decrease of the wild-type kdr alleles nucleotide composition, we showed that the sequences of R1R1 and R2R2 (homozygous) samples from Macapá and Oiapoque were similar to the ones previously described33. Evidence shows that at least two haplotypes emerged independently with the 1534C kdr mutation and one haplotype with the kdr mutation 1016I17. The 1534C is present in the herein called kdr alleles NaVR1 (VVC) and NaVR2 (LIC) and the 1016I in the kdr NaVR2 in Ae. aegypti Brazilian populations16.Regarding the kdr NaVR2 and the occurrence of rare kdr genotypes, as well as to the higher expression of detoxifying genes. Indeed, Ae. aegypti populations collected in the transborder city of Saint Georges de Oiapoque in French Guiana showed marked amplification of several CYP6s and CYP9Js playing a role in pyrethroid resistance34. We then cannot discard the possibility that these genes may be present in the Oiapoque population. Indeed Oiapoque (AP-Brazil) and Saint-Georges (French Guiana) are separated by a river which probably does not avoid the intense gene flow of Ae. aegypti between both sides, facilitated by the transportation of people and goods, which directly impacts the genetic structure of the mosquito24. Further work is needed to assess the detoxification pathway and genomic changes underlying the resistance mechanism of Ae. aegypti in the transborder area between Brazil and France that exhibits extremely high levels of resistance to all public health insecticides.Overall, we suspect that the high resistance to pyrethroids in Oiapoque is associated with a higher frequency of Ae. aegypti from Amapá using 12 microsatellites markers. We showed that the Fst values among the six populations of Ae. aegypti were higher in the pairs with Oiapoque, which was isolated in a single genetic group in the Bayesian analysis (k = 2) and clearly isolated in the DAPC. Calçoene, the closest city to Oiapoque, was also represented in an isolated group in the DAPC. The isolation by distance (IBD), confirmed by the Mantel test, and the dense Amazon Forest remain the most likely explanation for the genetic structure and kdr frequency differences among the populations alongside the BR-156 Road. Interestingly, Oiapoque presented the lowest Fst and the highest Nm with Macapá, that are yet distant from 580 km. This can be explained by the fact that Macapá is the State capital, and the flow of people and goods between those cities is very intense, hence facilitating the passive transportation of Ae. aegypti.Finally, we analyzed the genetic structure and gene flow between Ae. aegypti to human activities, particularly vehicular traffic on highways36, as well as boat38 and airplane39 transportation. A prior study identified the spread of resistance and kdr mutations within Ae. aegypti populations along a major road in central Brazil. The movement of people and goods between the state capital and other cities was claimed as the factor facilitating the dispersal35. A mitochondrial DNA based study of Ae. aegypti populations from Argentina suggested that this mosquito passively commuted the long distance of approximately 60 km in a year36. In the Peruvian Amazon, the Ae. aegypti gene flow was higher between localities with heavy boat traffic compared to those with less intense movement37, similarly to our findings, however with the transportation in the road encased within a dense forest.Our findings align with other studies that support the hypothesis attributing the long-distance dispersal of Ae. aegypti populations in Brazil after the eradication programme of the 1970s may have come from at least two distinct genetically differentiated groups, i.e. one from Venezuela to northern Brazil, and one from the Caribbean populations to the southeast of Brazil41. From this perspective, the Amapá State should have been invaded by Ae. aegypti from the two genetically distinct groups: in the north  by the ‘Caribbean group’ through French Guiana, and in the south  by the ‘Venezuelan group’ through other Amazonian Brazilian locality, probably Pará State.Based on the use of microsatellite markers, some studies concluded that the reinfestation of kdr and microsatellite markers, to address distinct questions. The kdr SNPs were genotyped to obtain their genotypic frequencies in the populations. As kdr is under strong selection pressure, it is not an accurate marker to infer the population genetic structure, which was achieved by neutral microsatellite genotyping. By combining these two analyses, we observed that kdr alleles are spreading, likely by the Ae. aegypti ability to disperse itself and its eggs passively, however, without disrupting the overall genetic structure of the isolated populations.In this study, we used two types of molecular genotyping, kdr genotyping analyses evidenced genetic differences among Ae. aegypti populations relatively isolated by a dense forest, connected by an only principal road. We found that rare kdr genotypes are increasing in frequency in some regions of Amapá State and this relationship with pyrethroid resistance deserves further investigation. More additional work is also needed to better understand the environmental and landscape determinants involved in the evolution and spatial distribution of insecticide resistance including kdr mutations in the dengue vector Aedes aegypti in Amapá.Microsatellites and Laboratório de Entomologia Médica do Instituto de Pesquisas Científicas e Tecnológicas do Estado do Amapá (IEPA) installed 50 eggtraps in each of the six Amapá cities: Oiapoque (OIA) , Calçoene (CAL) , Tartarugalzinho (TTZ) , Ferreira Gomes (FGO) , Porto Grande (PGR)  and Macapá (MAC)   and October/2020 (MAC). The eggtraps in CAL, FGO, PGR and TTZ were randomly distributed in the whole city. In MAC and OIA, the eggtraps were preferentially placed on the sidelines of BR-156 road. The eggs were stimulated to hatch in IEPA laboratory, and the resulting adults (F0) were packed in silica gel and shipped to Laboratório de Biologia, Controle e Vigilância de Insetos Vetores (LBCVIV) at Instituto Oswaldo Cruz (IOC/Fiocruz) for further analysis.The field team of 42. The DNA of each sample was quantified in a Nanodrop One C spectrophotometer (Thermo Scientific) and aliquoted at 20 ng/µL in ultra-pure water. About 45 individuals from each population were genotyped for the kdr SNPs V410L, V1016I and F1543C through real-time TaqMan qPCR approach, essentially as described elsewhere16 (See Supplementary Table www.graphpad.com). We considered the three SNPs (410 + 1016 + 1534) to determine the genotype of each sample20.We extracted DNA from male mosquitoes from the F0 generation (from egg collection), individually titrated with TNES buffer, as described elsewhereNaV segments of elected samples to determine the respective kdr haplotypes circulating in Ae. aegypti populations from Amapá. We selected nine samples genotyped as homozygous to kdr NaVR1 or kdr NaVR2 alleles (see Results) from MAC and OIA. For the PCR amplification, we used the kit Phusion High-Fidelity PCR , containing Phusion Buffer 1X, DMSO 3%, the respective primers pair44 , according to a protocol standardized by Brown et al.46, using multiplex primers, originally proposed by Schuelke48, using an M13 tail at the primers 5' end and marked with FAM or HEX fluorescence  to obtain the genotype callings of all microsatellite loci, which were exported in a .csv file. We tested the loci for Hardy–Weinberg equilibrium (HWE) and linkage disequilibrium in the Genepop v4.7.5 software, with significance levels adjusted by Bonferroni correction50. The presence of null alleles in each locus was verified with Microchecker Software v2.2.351. The genetic diversity parameters: average number of different alleles (Na), number of effective alleles (Ne), private allelic richness (Np), expected heterozygosity (He), observed heterozygosity (Ho), endogamy coefficients (Fis) and the number of migrants (Nm) for each population were estimated using the software Genetic Analysis in Excel  v.6.50352. This same software was used for analysis of molecular variance (AMOVA). The allelic richness (R) was calculated with the HP-Rare v1.1 software53. We estimated the magnitude of genetic differentiation with paired Fst values using Arlequin v3.5.254 and Freena55, respectively, both with 10,000 permutations. To test the assumption of isolation by distance (IBD), we run the Mantel test, with Arlequin v3.5.2 with 10,000 permutations54, correlating genetic and geographic data .We used the software 56 to assess the population's genetic structuring based on the number of genetic clusters (K). The best K value was obtained based on ten independent runs with 500,000 Monte Carlo chains (MCMC) iterations, excluding 20% of the initial chains (burn-in). The output data was analyzed in the Structure Harvester 2.3 software57 to determine the best number of genetic clusters (K) based on an analysis of the ΔK chart58. Finally, we submitted the output files to Clumpp v1.1.259 and Distruct V1.160 software to plot the genetic structure of the evaluated populations. A multivariate statistical analysis  was performed with the Adegenet package62 in the R platform63.We also used the Structure v2.3.4 softwareSupplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.