Labeo catla (catla) is the second most commercially important and widely cultured Indian major carp (IMC). It is indigenous to the Indo-Gangetic riverine system of India and the rivers of Bangladesh, Nepal, Myanmar, and Pakistan. Despite the availability of substantial genomic resources in this important species, detailed information on the genome-scale population structure using SNP markers is yet to be reported. In the present study, the identification of genome-wide single nucleotide polymorphisms (SNPs) and population genomics of catla was undertaken by re-sequencing six catla populations of riverine origin from distinct geographical regions. DNA isolated from 100 samples was used to perform genotyping-by-sequencing (GBS). A published catla genome with 95% genome coverage was used as the reference for mapping reads using BWA software. From a total of 472 million paired-end (150 × 2 bp) raw reads generated in this study, we identified 10,485 high-quality polymorphic SNPs using the STACKS pipeline. Expected heterozygosity (He) across the populations ranged from 0.162 to 0.20, whereas observed heterozygosity (Ho) ranged between 0.053 and 0.06. The nucleotide diversity (π) was the lowest (0.168) in the Ganga population. The within-population variation was found to be higher (95.32%) than the among-population (4.68%) variation. However, genetic differentiation was observed to be low to moderate, with Fst values ranging from 0.020 to 0.084, and the highest between Brahmani and Krishna populations. Bayesian and multivariate techniques were used to further evaluate the population structure and supposed ancestry in the studied populations using the structure and discriminant analysis of principal components (DAPC), respectively. Both analyses revealed the existence of two separate genomic clusters. The maximum number of private alleles was observed in the Ganga population. The findings of this study will contribute to a deeper understanding of the population structure and genetic diversity of wild populations of catla for future research in fish population genomics. Labeo catla   . In the ion USD) . An extr to 1994  in India to 1994 . Wild poHowever, quite limited genetic information on studies pertaining to genetic variation in wild populations of catla is available using microsatellites , random Being the most abundant molecular marker in the genomes of various vertebrate organisms, SNPs have been the markers of choice for a variety of applications, including population genomics. Over the last two decades, next-generation sequencing (NGS) technology has evolved, enabling the rapid and inexpensive identification of genome-wide SNP markers in any organism without access to prior genomic data. Furthermore, genotyping-by-sequencing (GBS) has become a potential genome-wide genotyping tool that employs an enzyme-based complexity reduction method to simultaneously discover and genotype molecular markers across the entire genome, with or without a reference . The genL. catla by genotyping-by-sequencing of six riverine catla populations from distinct geographical regions of India. The results of this study will enhance our knowledge in understanding genetic diversity and population structure and help in the conservation and management of wild populations of catla.In the present investigation, genome-wide SNPs were identified and population genomics analysis was performed in Water Quality Index (WQI) of the Godavari River was found to be fair , Krishna (KRN), Godavari (GOD), Mahanadi (MAH), Brahmani (BRM), and Ganga (GAN) . The wat be fair  and less be fair . Samples be fair  along wi be fair .Samples were collected from adult individuals ranging from 1 to 2 kg in weight. Natural hybrids of catla are known to be rare in the riverine populations. Even then, all sampled individuals were checked phenotypically, considering the shape of the head and mouth . As the High-molecular weight (MW) genomic DNA (gDNA) was extracted from the fin clips using a “DNeasy Blood and Tissue Kit” , according to the manufacturer’s protocol. The integrity and concentration of the DNA were checked by 0.8% agarose gel electrophoresis and using a Nanodrop Biospectrometer (Eppendorf India Pvt. Ltd.). Samples with an adequate concentration (≥100 ng/μl) and quality (intact high-MW DNA) were selected. One GBS library was prepared using 100 samples.Using The quality of the data was checked with FastQC v0.11.8  and Multe) and observed heterozygosity (Ho), average inbreeding coefficients (FIS), nucleotide diversity (π) with their standard errors (SE), mean frequency of the most frequent allele (P), Hardy–Weinberg equilibrium (HWE) estimates, and pairwise Fst values across the six populations of catla. The pairwise Fst heatmap was constructed with Heatmapper software  was carried out using the R tool v3.5  with the2) for the six catla populations with a sliding window size of 50. A non-linear regression curve was used to estimate LD decay, and the distance between SNPs at which the average r2 drops to 50% of the maximal LD value was used to compute the LD decay distance.TASSEL v5.0  softwareCyprinus carpio and L. catla in the NCBI NT database, demonstrating the reliability of the reads. After removing reads with low base quality (Q < 30), on average, 96.91% of filtered reads were successfully mapped to a catla reference genome with approximately 278 million clean data  ranged from 0.162 (GAN) to 0.20 (GOD) and observed heterozygosity (Ho) varied between 0.053 (GAN) and 0.06 (BRM). The nucleotide diversity (π) varied from 0.168 to 0.205 across the six populations, with the GAN population having the least value. The mean frequency of the most frequent allele (P) varied from 0.87 to 0.89 among the six populations of catla. The inbreeding coefficients (FIS) of the six populations of catla ranged from 0.382 to 0.637 and were highest in the GOD population (p < 0.05). Maximum numbers of private alleles were observed in the GAN population in comparison to other studied wild populations. Ne values were successfully estimated for 10,485 SNP markers with the highest value of 1.2 in the KRN population, whereas the lowest value was 0.8 in the CAU population  was found between the BRM and KRN populations. In contrast, the CAU and GOD populations had the least genetic difference (Fst = 0.0208). Furthermore, to study the genetic variations across the populations, AMOVA  that was identified in this research falls within the range that has been reported in earlier studies involving freshwater fishes  of the Godavari River was found to be fair and less environmental impact was noted , and biological invasion . Similaras noted . Accordias noted  along wias noted . Also, tas noted . Dam-indas noted . Habitatas noted . An obseas noted , which ias noted  and biolas noted  are alsoIS) of six populations of catla indicate increased homozygosity in GOD, as compared to other studied wild populations . However, this is not always true in populations in which genetic diversity is altered by external influences. For example, despite decreases in Ne (0.9) in the GOD population, there was no decline in the nucleotide diversity (0.205) and observed heterozygosity (0.059). Despite having a higher FIS value (0.637), genetic diversity seemed to be maintained through immigration and other events, such as the recent artificial interlinking of rivers. A similar observation was obtained in Atlantic salmon   and lower pollution levels (o (0.053), and the lowest nucleotide diversity (0.168) with a moderately higher inbreeding (0.428) value. Similar observations were seen in the MAH population, which had a relatively lower Ne (0.9) and a lower nucleotide diversity (0.174) and Ho (0.054). The observed reduction in heterozygosity could be attributed to the enormous pollution due to industrial activities, sand mining, and the release of agricultural runoffs in the Ganga River (Effective population size (Ne), usually less than the census population size, influences genetic diversity in a population. Our results showed lower levels of Ne ranging from 0.8 to 1.2, and similar observations were reported by o salar) . In addin levels , indicatga River . In addiga River . Thus, GL. catla species taken from distinct river basins in the current study had a similar ancestral gene pool, for which at least historical connections existed across different rivers  was found between the BRM and KRN populations. In contrast, the CAU and GOD populations had the least genetic difference (Fst = 0.0208). The Fst value revealed that KRN samples were significantly different from the BRM, GAN, and MAH samples, whereas BRM samples were observed to be substantially different from GAN. Smaller genetic distance and high gene flow might be the possible reasons for reduced genetic differentiation between the CAU and GOD populations. Our sample of SNPs, which shows low-to-moderate molecular marker differentiation across rivers, revealed minimal genetic differentiation because of adaptive selection or drift and suggested that there had been continuous gene flow amongst the analyzed river basins  revealed minimal genetic variation among populations (The pairwise Fulations . The maxr basins . As withof India . Additioof India , Sahoo eof India , and Dasof India , and is of India . This kiof India , as seenulations .2 < ∼ 0.2 as it approaches equilibrium. This, along with LD decay at r2 = half decay distance, is a widely used criterion in the literature (In this study, no significant pairwise linkage disequilibrium was observed among the loci, which is similar to the findings obtained by terature . An admiL. catla. Low genetic diversity was observed with low-to-moderate levels of pairwise genetic differentiation in the six catla populations. Most of the genetic variations were found within populations. Both Bayesian and multivariate techniques divided L. catla into two clusters. The results of this study will help in the conservation and management of wild populations of catla.In the present investigation, 10,485 genome-wide SNPs were identified using the GBS method for genome-scale analysis in six riverine populations of


Genome-wide association studies (GWAS) have identified many genetic variants associated with alcohol consumption in Europeans and East Asians, as well as other populations. However, the genetic homogeneity and heterogeneity between these populations have not been thoroughly investigated, despite evidence of varying effect sizes of variants between ethnicities and the presence of population-specific strong signals of selection on loci associated with alcohol consumption. In order to better understand the relationship between Europeans and East Asians in the genetic architecture of alcohol consumption, we compared their heritability and evaluated their genetic correlation using GWAS results from UK Biobank (UKB) and Biobank Japan (BBJ). We found that these two populations have low genetic correlation due to the large difference on chromosome 12. After excluding this chromosome, the genetic correlation was moderately high (The online version contains supplementary material available at 10.1186/s12864-023-09766-8. Rapid social progress is improving the overall quality of life, with a significant shift in dietary habits. Alcohol, which has traditionally been an important part of human diet, has become a major public health concern in recent years as the relationship between drinking and health issues are better understood. Studies have shown that alcohol consumption is linked to many diseases, including alcohol dependence, cardiovascular and cerebrovascular diseases, digestive system diseases, and other non-communicable diseases and mental diseases. These conditions can seriously impact physical health, quality of life, and even lead to a higher mortality and disability rate than smoking or hypertension –4.The InPrevious genetic association studies have identified genetic loci associated with alcohol consumption and related health issues. For example, Hnrnph1, a novel regulator of alcohol reward driving alcohol consumption and high-dose alcohol aversion, is potentially relevant to the neurobiology of alcohol abuse and alcoholism . StudiesAlthough genetic studies on alcohol consumption have largely focused on European populations, studies have also been conducted on individuals of other ancestries, including Asian and African , 11. LarOn the other hand, studies have showed evidence that some of the genetic variants associated with alcohol consumption have different effects between populations. For example, strong associations have been identified at ALHD2 in multiple Asian populations , 13, 14,Despite evidence that genetics play a role in alcohol consumption and that its effect varies by ethnicity, 13, the2, sex, age * sex, age2 * sex, and 20 PCs. BBJ is a GWAS based on ~ 165,000 individuals of Japanese ancestry from Biobank Japan. These individuals were genotypes on either the Illumina HumanOmniExpress Exome BeadChip or a combination of the Illumina HumanOmniExpress and HumanExome BeadChips, and phased and imputed with a reference panel constructed from 275 unrelated EAS haplotypes from 1000 Genomes. After adjusting for age, age2, sex, and the status of the 45 target diseases registered in BBJ, a GWAS was conducted on drinker status with ~ 83,000 controls and ~ 81,000 cases, with a linear mixed model to control for cryptic relatedness and population structure. SNP positions are according to GRCh37 (hg19).We obtained publicly available GWAS summary statistics from two populations: UK Biobank (UKB) and Biobank Japan (BBJ). UKB is a GWAS based on ~ 360,000 individuals of white British ancestry in the UK Biobank. These individuals were genotyped with either the Affymetrix UK BiLEVE Axiom Array or the Affymetrix UK Bio-bank Axiom Array and imputed to whole-genome sequencing data for ~ 13.8 M variants. Association testing was done on drinker status with ~ 11,000 cases (Never drinker) and ~ 350,000 controls (Ever drinker) correcting for age, ageWe employed LD Score Regression (LDSC) , 20to esThe genetic correlation of drinker status between European and East Asians was computed using Popcorn . It estiWe used the tool PESCA  to deterWe conducted a multi-ancestry meta-analysis of UKB and BBJ using the tool MAMA . CompareTo confirm the signals found in MAMA, we did a secondary meta-analysis using METAL . To combThe heritability of drinker status (Never versus Ever drinker) was assessed in two populations, those of European descent in the UKB and those of East Asians descent in the BBJ. The heritability in the UKB was 0.1279 , which is higher than the estimate of 0.0846 provided at the UKB website which assumed a proxy population prevalence equal to the prevalence rate in the GWAS dataset. The heritability in the BBJ was 0.2355 . It’s bigger than the estimate of 0.1237 in the original study , but thiWe then used the Popcorn method to measure genetic correlation between UKB and BBJ. The genome-wide genetic correlation of effect sizes was low (Through the PESCA analysis, we found 0.30% of all common SNPs were inferred to have nonzero effects in both EUR and EAS; 0.28% and 0.14% were inferred to have population-specific nonzero effects in EUR and EAS, respectively. In other words, approximately 41.9% of SNPs inferred to be causal were shared between EUR and EAS. We inferred the posterior expected number of population-specific/shared causal SNPs in each of the 1,368 independent LD blocks by summing all SNPs in the block the per-SNP posterior probabilities of being causal in a single or both populations. High-posterior SNPs for both the population-specific and shared causal configurations were spread nearly uniformly across the genome (Fig. Given that most causal variants were shared between Europeans and East Asians, we conducted a meta-analysis of UKB and BBJ using MAMA. The analysis successfully identified new signals that were originally missing in one of the populations or both Fig. . One proRecently GWAS using common variants have reported signals of association between many genes and alcohol-related traits, including those involved in alcohol metabolism, such as ADH1B, ADH1C, and ADH4 –31. HoweWe observe a predominantly shared genetic architecture for alcohol consumption between Europeans and East Asians, except for chromosome 12, where East Asians show significant enrichment of genetic effects compared to Europeans. This discrepancy aligns with evidence of strong natural selection at the ALDH2 locus on chromosome 12 in Japanese populations . These fGiven the moderately high genetic correlation and the large proportion of causal variants shared between populations, we performed a GWAS meta-analysis on BBJ and UKB. By combining GWAS signals from multiple populations, MAMA successfully identified new signals of association with alcohol consumption that were previously missed in initial GWAS. One such example is the CADM2 gene located on chromosome 3. The role of CADM2 is increasingly being recognized in the behavioral and metabolic traits. Recent studies have found that the CADM2 gene is significantly associated with a wide range of traits, including cognitive, risk taking, and dietary traits –35. CADMSince the majority of genetic studies are conducted in European populations, it is imperative to conduct a more systematic and comprehensive assessment of genetic homozygosity and heterozygosity across different populations. This is essential for enhancing genetic findings and understanding of the transferability of polygenic risk score in underrepresented populations . Our finBelow is the link to the electronic supplementary material.Supplementary Material 1


QST) and neutral markers (FST) to plant biology and summarize conservation applications derived from QST–FST comparisons, particularly on how to capture most AGV and NGV on both in‐situ and ex‐situ programs. Based on a review of published studies, we found that, on average, two and four populations would be needed for woody perennials (n = 18) to capture 99% of NGV and AGV, respectively, whereas four populations would be needed in case of herbaceous perennials (n = 14). On average, QST is about 3.6, 1.5, and 1.1 times greater than FST in woody plants, annuals, and herbaceous perennials, respectively. Hence, conservation and management policies or suggestions based solely on inference on FST could be misleading, particularly in woody species. To maximize the preservation of the maximum levels of both AGV and NGV, we suggest using maximum QST rather than average QST. We recommend conservation managers and practitioners consider this when formulating further conservation and restoration plans for plant species, particularly woody species.Increased access to genome‐wide data provides new opportunities for plant conservation. However, information on neutral genetic diversity in a small number of marker loci can still be valuable because genomic data are not available to most rare plant species. In the hope of bridging the gap between conservation science and practice, we outline how conservation practitioners can more efficiently employ population genetic information in plant conservation. We first review the current knowledge about neutral genetic variation (NGV) and adaptive genetic variation (AGV) in seed plants, regarding both within‐population and among‐population components. We then introduce the estimates of among‐population genetic differentiation in quantitative traits  and adaptive (AGV) genetic variation on both in‐situ and ex‐situ programs. To capture 99% of NGV  and AGV , two and four populations would be needed for perennial trees, respectively. For annuals, we would need four and six populations, respectively, whereas four populations would be needed for herbaceous perennials for both AGV and NGV. On average, QST is approximately 3.6‐, 1.5‐, and 1.1‐fold greater than FST for woody plants, annuals, and herbaceous perennials, respectively. For both AGV and NGV, it is recommended to use the maximum QST rather than the average QST to best preserve the maximum level. For example, for the widespread tree Populus balsamifera in North America, two populations  would be needed to capture 99% of NGV, whereas up to 25 populations would be targeted to maintain sufficient AGV (QST = 0.832 for the bud set). Conservation managers and practitioners are encouraged to consider this when formulating further conservation and restoration plans for plant species, particularly woody species.One of the conservation applications derived from  Plant evolutionary biologists, foresters, and conservation geneticists have long been interested in the genetic differences among populations and the degree to which these may reflect local adaptation (see Table %P), allelic richness (AR), or gene diversity —is considered a poor “proxy” of levels of AGV in quantitative traits  to the conservation and restoration of plant species have been somewhat controversial due to the assumed evolutionary neutrality of used markers and their limitations to be informative about the adaptive potential  and QST  and theoretical studies of NGV and AGV within and among populations in a variety of organisms are abundant in the literature  covering most AGV and NGV based on the analyses of molecular and quantitative trait data.With this in mind, we first introduce the current knowledge about within‐population genetic variation and among‐population differentiation both in NGV and AGV in seed plants to highlight the distinction between the approaches used to identify the two types of genetic variation. Next, we introduce the known general application of 2He and h2 at the population level. These two estimates are determined by somewhat different processes.As neutral genetic markers reflect demographic processes (including past demographic histories) within local populations, they are informative for management and conservation purposes. Small populations are generally susceptible to the loss of NGV and less adaptive to novel environments due to the loss of AGV through genetic drift , indicating that neutral marker‐based measures only explain 4% of the variation in quantitative traits  published datasets, μ), low‐statistical power, environmental effects on quantitative characters, and impact of regulatory variation. In addition, various forms of natural selection affecting the level of neutral polymorphism at linked sites may also contribute to the lack of a relationship between NGV and AGV. The most dramatic effect on neutral variation occurs when beneficial alleles at loci contributing to AGV spread into a population, a process known as a “selective sweep”  versus FST estimates  are substantially lower than migration rates  are not recommended to be used in QST–FST comparisons  selection. Second, if QST ≈ FST, trait differentiation is indistinguishable from the effects of drift, and thus, there is no evidence for selection  and that QST > FST (p < .001), confirming the main conclusions of Merilä and Crnokrak  a sampling bias due to the deliberate selection of populations from contrasting environments to be investigated, as well as focus on populations previously known to be phenotypically divergent; (ii) a publication bias favoring studies reporting QST > FST outcomes, possibly because of difficulties interpreting QST ≈ FST and QST < FST patterns. QST < FST could be due to canalization, which refers to a process or tendency in which “species genetic backgrounds share the same genetic constraints” . A weak but positive overall relationship between pairwise QST and FST values  QST reflects both neutral forces and natural selection caused by environmental differences and FST only measures neutral processes including genetic drift and gene flow, (ii) QST and FST estimates are based on the same (among‐population) partition of total genetic variation, differing only in the data used in estimation—quantitative adaptive loci (the former) and neutral loci (the latter), and (iii) divergent selection that causes QST could also lead to the increase of FST by restricting gene flow , suggesting that isolation by distance plays an important role in adaptive evolution. The authors' meta‐analysis suggests that plant species are generally differentiated by natural selection in various types of traits . For example, the authors detected a larger QST–FST difference values for non‐fitness traits than for fitness traits, confirming the expectation that the former respond, in general, faster to directional selection than the latter , but the difference was not significant. This can be viewed to be at odds with the prediction , several researchers have provided information on the evolution of invasiveness and the adaptive potential of invasive plant species  could be sourced from a few populations distributed across the species' range, whereas seeds of rare herbaceous species  should be taken from many populations to capture the highly localized genetic diversity. Based on a small body of available data on seed plant species N for NGV, where PNGV = proportion of NGV captured by sampling, N = number of populations , where PAGV = proportion of AGV captured by sampling. However, one should be aware that if there are more than two alleles per locus for the neutral markers, then QST and FST are on different scales, and the formulae PAGV = 1 – QSTN and PNGV = 1 – FSTN cannot be interpreted in the same way . For multi‐allelic markers, it depends on μ whether this is problematic—for bi‐allelic single nucleotide polymorphisms this does not constitute a problem. Since ФST, the ratio of the among‐population variance component to total variance obtained based on genetic distances among alleles for the neutral markers, is conceptually similar to QST, it is advisable to use ФST rather than GST, FST, or θ , to capture 99% of NGV and AGV for woody perennials, only two and four populations would be needed using the abovementioned formulae, respectively. On the other hand, four populations of herbaceous perennials would be needed to secure 99% of NGV and AGV, respectively, because the average difference between QST and FST is small (0.032). For annuals, four and six populations would be needed to secure 99% of NGV and AGV, respectively.Population(s) to be protected in situ or to be sampled for seed banking purposes could be estimated using the following formulae: PNGV = 1 – Populus balsamifera  obtained from 13 ecophysiological and phenological traits originating from 20 populations across North America. Two populations from this tree species would be needed to capture 99% of NGV using the above formula. When we apply the mean QST value to the formula, at least six populations would be necessary to capture the same level of AGV. However, the value of QST depends on the trait under consideration: for traits with a high QST, more populations should be sampled than for traits with a low QST. Application of too low values of QST for this equation would lead to an underestimation of the number of populations needed to preserve the desired level of genetic variation. Given this, it would be wiser not to use the average QST but the maximum QST. Thus, as in the case of the P. balsamifera QST = 0.832 for the bud set, then up to 25 populations would be needed to be targeted to maintain enough AGV. Of course, this does not mean that NGV is not important; there is probably a reservoir of genetic variation in every population that is neutral now but that may become selectively important if environmental conditions change. Furthermore, NGV can be very informative about the populations' past demography which is often of interest in conservation biology  species. Following the reasoning laid out above, managers, or practitioners should design restoration and conservation strategies by knowing that, on average, QST is about 3.6, 1.5, and 1.1 times greater than FST in woody plants, annuals, and herbaceous perennials, respectively.The application of the above formulae to plants with different life forms, as well as the example of FST appears to be more closely related to AGV than within‐population genetic diversity metrics , the former should be considered as a more predictable parameter for plant conservation and restoration purposes; estimating the value of FST  is important for prioritizing populations for both in situ and ex situ collection and for identifying appropriate sources for reintroductions  in conservation management cannot be overstated, particularly when it comes to annuals and herbaceous perennials.As 6He are often correlated with fitness  of quantitative traits (AGV). As discussed above, the relationship of He to h2 or H2 is often very weak, while the relationship between FST and QST is comparatively stronger; thus, FST could be considered a weak proxy of QST. However, whenever logistically possible, common garden and/or transplant studies are strongly recommended to quantify patterns of adaptive genetic variation and differentiation , quantitative traits, and/or quantitative trait loci coding for traits and contemporary genome‐wide association approaches in the context of a common garden experiment, and environmental variation  are needed to gain comprehensive insights into conservation of herbs and trees ; funding acquisition (lead); project administration (lead); writing – original draft (supporting); writing – review and editing (supporting). Juha Merilä: Conceptualization (supporting); writing – review and editing . Yuseob Kim: Writing – review and editing . Kangshan Mao: Writing – review and editing . Jordi López‐Pujol: Writing – review and editing . Myong Gi Chung: Conceptualization (lead); project administration (lead); writing – original draft (lead); writing – review and editing (lead).This work was supported by the research fund of Chungnam National University, the Republic of Korea to MYC.All the authors state that there is no conflict of interest.


Plasmodium falciparum erythrocyte binding antigen 175 (PfEBA-175) plays essential role in erythrocyte invasion by the parasite and is a leading vaccine candidate. However, its genetic diversity in global isolates is a concern in developing an universal vaccine incorporating this protein. This study aimed to investigate genetic polymorphisms and natural selection of pfeba-175 region II (RII) in Myanmar and Vietnam P. falciparum isolates. Vietnam pfeba-175 RII displayed a low genetic polymorphism, while Myanmar pfeba-175 RII showed high levels of genetic diversity across the region. Point mutations, deletion, and recombinations were main factors contributing to genetic diversities in P. falciparum populations. Global pfeba-175 RII revealed similar, but not identical, genetic polymorphisms and natural selection profiles. Despite profiles of amino acid substitutions differed among populations, five major amino acid changes  were commonly detected in global pfeba-175 RII populations. Haplotype network and genetic differentiation analyses of global pfeba-175 RII populations demonstrated no geographical relationships. Non-neglectable level of genetic diversity was observed in global pfeba-175 RII populations, emphasizing the need to consider this when designing an effective vaccine based on this protein. This study underscores the importance of the continuous monitoring of genetic diversity of pfeba-175 RII in the global P. falciparum populations. Among five species of human-infecting Plasmodium species, P. falciparum is the deadliest species contributing to the majority of malaria deaths. Recent spreading of resistance strains against antimalarial drugs has been a priority concern for effectively controlling and eliminating malaria2. Thus, alternative methods, including effective vaccine development, are urgently needed to cope with falciparum malaria.Despite a recent decline in morbidity and mortality of global malaria due to improved control measures, this disease remains a major public health burden in Africa and Southeast AsiaPlasmodium parasites is the first step that is crucial for parasite growth and malaria pathogenesis. Initial attachment of merozoite to erythrocyte is mediated by merozoite surface proteins (MSPs). Tight interactions are then formed between ligands of the merozoite and specific surface receptors on the host’s erythrocytes. Two classes of protein families, Duffy binding-like (DBL) and reticulocyte binding-like (RBL) proteins, have been identified to be critical ligands involved in the formation of initial tight interaction3. Among them, erythrocyte binding antigen 175 of P. falciparum (PfEBA-175) has gained attention as a leading blood phase vaccine candidate because antibodies against this protein can inhibit its binding to host receptor glycophorin A (GPA) and eventually block invasion of merozoites in vitro4. PfEBA-175 protein comprises seven separated regions, namely RI to RVII5. The RII of this protein consisting of two cysteine-rich domains (F1 and F2) plays an important function by interacting with GPA directly followed by inducing erythrocyte membrane deformability essential for successful merozoite invasion7. PfEBA-175 RII is a promising target for vaccine development as antibodies against this protein have been detected in individuals with naturally acquired immunity8. In addition, it can induce host immune responses9. Moreover, antibodies that block the binding of PfEBA-175 RII with GPA can potentially neutralize parasite invasion12. However, pfeba-175 RII identified in P. falciparum wild isolates is genetically diverse, suggesting that monitoring genetic variations of this gene is necessary to provide helpful information for developing an effective vaccine based on this protein15.Erythrocyte invasion by 1. However, malaria elimination in the GMS is still challenging due to various existing and emerging matters such as changing malaria transmission patterns and spreading of antimalarial drug resistance17. High levels of genetic diversity of major vaccine candidate antigens such as MSPs, apical membrane antigen-1 (AMA-1), and circumsporozoite surface protein (CSP) have been reported in P. falciparum population of GMS countries24, suggesting remarkable genetic heterogeneity of P. falciparum population circulating in the region, which hampers the design of effective vaccines. Similar to these vaccine candidate antigens, PfEBA-175 also shows genetic polymorphisms among P. falciparum populations25. However, information about the genetic diversity of pfeba-175 in P. falciparum population in the GMS is limited. Thus, this study aimed to analyze genetic diversity and natural selection of pfeba-175 RII in P. falciparum isolates from two GMS countries, Myanmar and Vietnam, to understand the genetic structure and evolutionary aspect of the pfeba-175 gene in the population.The Greater Mekong Subregion (GMS) covering Cambodia, China (Yunnan and Guangxi Provinces), Lao People's Democratic Republic (Lao PDR), Myanmar, Thailand, and Vietnam is a malaria hot spot. Malaria burden in this region has decreased continuously in recent years due to constructive efforts of all GMS countries toward malaria eliminationpfeba-175 RII in blood samples was successful. Sizes of amplified products were approximately 1.9 kb on agarose gel. No size variation was detected among isolates. A total of 33 and 59 sequences of pfeba-175 RII were successfully obtained from P. falciparum isolates from Myanmar and Vietnam, respectively. These sequences were subjected to further analyses.Amplification of pfeba-175 RII covering 616 amino acids was highly conserved among isolates. Compared with the 3D7 reference sequence (GenBank No.: XM_001349171.2), only eight nonsynomous single nucleotide polymorphisms (SNPs) were detected (Supplement File 1: Table pfeba-175 RII sequences were classified into five haplotypes (H1 to H5), none identical to the reference sequence of 3D7. H1 harboring K286E, K478N, and R664S was the most dominant haplotype. H2 was the haplotype showing the highest polymorphic amino acid changes at seven positions.Vietnam pfeba-175 RII sequences exhibited high levels of polymorphisms. A total of 21 nonsynomous SNPs (Supplement File 1: Table pfeba-175 RII sequences were classified into 22 distinct haplotypes (H1 to H22), different from the reference sequence of 3D7. H1 was the most prevalent haplotype with a frequency of 21.2% (7/33).Myanmar pfeba-175 RII were comparatively analyzed. The global pfeba-175 RII population displayed polymorphic characters presenting amino acid changes at 32 positions, including 31 amino acid substitutions and a deletion of two-amino acids in the region  and one trimorphic amino acid substitution, K405M/E. The F2 domain displayed 9 dimorphic changes  and one trimorphic substitution (Q584K/E). These amino acid substitutions were not evenly detected in the global pfeba-175 RII. Myanmar pfeba-175 RII showed amino acid changes in the largest number of positions (21 positions). High levels of amino acid changes were also identified in Kenya, Thailand, and Nigeria pfeba-175 RII at positions of 19, 18, and 17, respectively. Meanwhile, lower amino acid changes were detected in Vietnam, Colombia, and Peru pfeba-175 RII with 8, 10, and 10 positions, respectively. Only four amino acid changes  were commonly found in all 12 pfeba-175 RII populations, albeit their frequencies differed by country. High frequencies of R664S were detected in 11 populations except the Equatorial Guiana population, suggesting that it was also a common amino acid substitution found in global pfeba-175 RII populations. N348T, K400Q, and E450G were uniquely found only in Myanmar pfeba-175 RII. Deletion of two-amino acids (I401 and S402) observed in the Myanmar population was also observed in Thailand, Nigeria, and Kenya populations with different frequencies. No close geographical relationship of these amino acid substitutions was detected among populations.Amino acid polymorphism patterns of global pfeba-175 RII sequences from different countries, the same length portion (nucleotide positions from 565 to 2019 of the full-length pfeba-175 gene) available from 11 countries, including Myanmar and Vietnam, was applied for comparative analysis of genetic diversity and natural selection of global pfeba-175 RII. Equatorial Guiana sequences were excluded from further analyses because they lacked a large part of the C-terminal region of pfeba-175 RII. Global pfeba-175 RII showed similar patterns of genetic diversity and natural selection in global P. falciparum populations  test  exhibited a statistically significant increase compared to the ratio observed between P. falciparum and P. vivax , as well as between P. falciparum and P. knowlesi . These results suggest that pfeba-175 RII is subject to positive balancing selection. The assertion was also supported by high neutrality scores . The site-wise selection pressure was also tested by FUBAR test. Positive selection signals (posterior probabilities > 0.9) were predicted in 16 codon sites, including 38, 86, 87, 91, 98, 148, 200, 202, 215, 217, 290, 293, 389, 396, 404, and 476  value of 0.146. Recombination also was likely to affect the genetic diversity of global pfeba-175 RII. The estimated minimum number of recombination events between adjacent polymorphic sites (Rm) in global populations ranged from 0 to 6  analysis, but no significant association between the loci was found  to 0.593 (between Vietnam and French Guiana). The values between Africa populations except French Guiana were generally low or moderate, suggesting high levels of gene homogeneity within the populations. Interestingly, the values of Vietnam and French Guiana populations against other populations, including populations within the same continent, showed strong genetic differentiations.To investigate pair-wise genetic differentiation between global ted Fig. . Fst valpfeba-175 RII (n = 331) and the 3D7 reference sequences were clustered into 62 different haplotypes (H1 to H62) connected with a complicated network   Fig. . Vietnampfeba-175 RII in Myanmar and Vietnam P. falciparum isolates and performed comparative analyses of genetic diversity and evolutionary aspects of pfeba-175 RII in global populations. Vietnam pfeba-175 RII was relatively well-conserved with only limited numbers of amino acid changes, while Myanmar pfeba-175 RII was highly polymorphic harboring amino acid changes at 21 positions. Global pfeba-175 RII also showed polymorphic characters, of which the F1 domain was more polymorphic than the F2 domain. However, close geographical relationship of these amino acid substitutions was not detected. These amino acid changes were not evenly distributed among global pfeba-175 RII, but five amino acid changes  were commonly detected in global pfeba-175 RII populations with different frequencies by country. Interestingly, a deletion of two amino acids (I401 and S402) was also detected in several populations including Myanmar, Thailand, Nigeria, and Kenya, showing different frequencies. The two residues are likely to be located in the C-terminal region of the F1 domain, which is connected to the linker domain6. It is currently unclear whether this deletion could induce significant changes in PfEBA-175 RII structure to alter interaction with GPA. This merits further study.PfEBA-175 RII plays a pivotal role in erythrocyte invasion by merozoite. Thus, it has gained attention as a potential vaccine candidate for falciparum malaria. In this study, we analyzed the genetic diversity of pfeba-175 RII showed different levels of π values. French Guiana pfeba-175 RII had the lowest value of 0.00215 ± 0.00016, while Myanmar pfeba-175 RII showed the highest value of 0.00449 ± 0.00028. However, the slide window plot of π across the gene revealed that global pfeba-175 RII shared highly similar patterns of π, consistent with a previous report15. Positions of five major peaks of π well corresponded to positions of five common amino acid substitutions, suggesting these amino acid changes were major factors contributing to the genetic diversity of global pfeba-175 RII. The dN–dS values of global pfeba-175 RII suggested that weak positive natural selection might have influenced global populations, but it was not statistically sifnificant. Values of Tajima's D, Fu and Li’s D, and Fu and Li’s F were positive for most populations, suggesting that balancing selection mainly acted on the gene. Only one exception was the French Guiana population, which showed a negative value of Tajima’s D, implying a weak purifying selection or an expansion in its population size. The balancing positive selection on the global population was also proven via the MD and FUBAR tests. The French Guiana population showed high frequencies of amino acid changes at positions where amino acid substitutions were found, which might have increased genetic homogeneity of the population. Patterns of Tajima’s D in the sliding window plot of the global pfeba-175 RII were matched well with patterns of π, suggesting these five major common amino acid changes were also major forces affecting the neutrality of global populations. Deletion of two amino acids (I401 and S402) and high recombination events observed in some populations might have also acted as driving forces contributing to the genetic diversity of pfeba-175 RII. Interestingly, the deletion was only found in Asia and Africa pfeba-175 RII, not in America population. The overall value of recombination events was also higher for Asia and Africa populations than for America populations. These collectively suggest that the genetic diversity of pfeba-175 RII is much higher in Asia and Africa P. falciparum populations than in America populations.Global 6. It has also been reported that antibodies against the F2 domain can alter the binding affinity of merozoite to host erythrocytes10, indicating the F2 domain could be a major drug or vaccine design target. Recently, it has been reported that recombinant PfEBA-175 RII F2 protein carrying Q584E and E592A favors GPA interaction than recombinant protein with wildtype Q584 and E59213. High frequencies of Q584K/E and unneglectable levels of E592A found in global populations suggest that pfeba-175 RII is evolving to improve its binding ability to GPA. Besides, K481I, N577K, and R664S were also major common amino acid changes detected in the F2 domain in global pfeba-175 RII. As they are predicted to be located in the interface of GPA binding site8 or the dimer interaction site6, high levels of polymorphic characters of these residues in global pfeba-175 RII proposed a hypothesis that pfeba-175 RII needs these variants to adapt to the host GPA, or the remaining positions receive stronger selection resulting in variants not being retained. This emphasizes the necessity to investigate biological effects of these mutations on structural and functional properties of PfEBA-175. However, considering that limited numbers of sequences from restricted geographical origins were applied in this study, larger-scale of genetic analyses of larger numbers of P. falciparum isolates from diverse geographical areas are necessary.The critical function of the F2 domain has been reported to be in GPA binding. Amino acid residues in the F2 domain prominently line channels of PfEBA-175 dimer and make the majority of glycan contacts, emphasizing their priority roles in the interaction between PfEBA-175 and GPApfeba-175 RII populations were investigated by estimating Fst values and constructing a haplotype network. Interestingly, Vietnam and French Guiana populations revealed different trends of genetic differentiation from other populations. They showed higher Fst values against other populations, including those in neighboring countries of the same continent, implying that Vietnam and French Guiana populations had strong genetic differentiation from other populations. Haplotype network analysis also supported low genetic relationships of these two populations with other pfeba-175 RII populations. Most haplotypes of Vietnam and French Guiana populations were clustered into a few unique haplotypes separated from other haplotypes. The reason why these two populations showed such distinct patterns of genetic differentiation and is currently unclear. Such different genetic profile of Vietnam P. falciparum population compared to the populations from other neighboring countries in the GMS, including Myanmar and Thailand, has been reported previously18. These findings may indicate that the distinctiveness of genetic composition of Vietnam P. falciparum was not limited to the pfeba-175 gene alone but encompasses the other genes in the Vietnam P. falciparum population. The geographical features of Vietnam may serve as a contributing component to this phenomenon. The Annamite range (Truong Son) running across the length of Vietnam may act as a natural barrier that separates Vietnam from west Indochina. The height and climatic fluctuations within the range can exert an influence on malaria transmission. Areas at lower altitudes on the windward side may exhibit more favorable conditions for the proliferation of mosquitoes and subsequent malaria transmission, whereas higher-altitude regions on the leeward side may be less conducive to these phenomena. These could affect variations in the prevalence and transmission of malaria on different sides of the range. Furthermore, the geographical characteristics of the range can also exert an influence on patterns of human migration and genetic exchange of malaria parasites across distinct groups. It needs to be clarified in the future.Genetic differentiation between P. falciparum population compared to populations from the other GMS countries have been reported previously27. Further analysis is necessary to understand the high genetic heterogeneity in Myanmar P. falciparum population and its evolutionary aspect.One more interesting finding of this study was that the Myanmar population showed low genetic differentiation with Africa populations but not with populations from neighboring GMS countries such as Vietnam and Thailand. Considering the great geographical distance between the two countries, it is not easy to deduce the answer. One possible reason for this strange association of Myanmar and Kenya could come from the high sequence similarity of these 2 populations, rather than a biological exchange of individual migrants. Higher genetic diversities of diverse vaccine candidate antigens in the Myanmar pfeba-175 RII were similar to those in previously reported global populations. However, Myanmar pfeba-175 RII showed greater nucleotide diversity and larger amino acid changes than the global population. Meanwhile, Vietnam pfeba-175 RII showed lower genetic diversity and different trend of genetic differentiation compared to populations of neighboring GMS countries, Myanmar and Thailand. Comparative genetic analyses of Lao PDR and Cambodia pfeba-175 RII populations would contribute to interpreting these genetic differences among GMS countries. Amino acid substitutions, natural selection, deletion, and intragenic recombination might be main forces generating genetic diversity of global pfeba-175 RII. High or unneglectable level of genetic diversity observed in global pfeba-175 RII populations, emphasizing the need to consider adaptive selection in P. falciparum populations when designing an effective vaccine. The limitation of this study was small and unequal sample size of P. falciparum isolates from each country and different sampling periods to generate the sequence data. The global pfeba-175 RII sequences that were gleaned from the previous studies also span a range of chronological periods. Thus, it is conceivable that the results of this study might not precisely depict the current genetic characteristics of the global pfeba-175 RII population. These could limit generalizability of the findings in this study. However, results of this study provide an evidence on genetic heterogeneity of global pfeba-175 RII and the necessity of continuous monitoring the genetic diversity of the gene in the global P. falciparum population for developing an effective vaccine based on PfEBA-175.In summary, overall patterns of nucleotide diversity and natural selection in Myanmar and Vietnam P. falciparum-infected blood samples used in this study were collected from Myanmar and Vietnam  were collected from the patients in Upper Myanmar  from 2013 to 201528. In Vietnam, blood samples (n = 59) were collected from malaria patients residing in four districts in Dak Lak province in the Central Highlands region from 2018 to 201929. The malaria infection was confirmed by species-specific polymerase chain reaction (PCR) targeting the 18S ribosomal RNA (rRNA) gene31. Monoclonal infection of P. falciparum was also confirmed in previous studies31. The study protocol was approved by the Ministry of Health, Myanmar (97/Ethics 2015) and the Ministry of Health, Institute of Malariology, Parasitology, and Entomology Quy Nhon, Vietnam (No.: 386/VSR-LSDT). All methods were performed in accordance with the relevant guidelines and regulations by the institutions. Informed consent was obtained from all participants and/or their legal guardian(s).nam Fig. . Myanmarpfeba-175 RII  was conducted by using nested PCR. The primers for the first round PCR were 5'-ACAACAATTATCAATCATTTTTATCGA-3' and 5'-TTTCCTCAGGAACTGCTTCTTGCGAA-3'. The thermal cycling parameters for primary PCR were as follows: one cycle of initial denaturation at 95 °C for 5 min, 30 cycles of 94 °C for 1 min, annealing at 58 °C for 2 min, and extension at 72 °C for 2 min, followed by a final extension at 72 °C for 5 min. The nested PCR was performed with primers, 5'-ATCGACAAGTTCATTAATAAAGCAAAA-3' and 5'-GCGAAGTTTGTTCATTATTTCTTATTA-3', at similar amplification conditions, except the annealing temperature of 53 °C. Ex Taq DNA polymerase  with proofreading activity was used in all PCR steps to minimize incorrect nucleotide incorporations during the amplification steps. Each PCR product was separated by electrophoresis on 1.5% agarose gel, and the corresponding amplified product was extracted from the gel and cloned into a T&A cloning vector . Each ligation mixture was transformed into Escherichia coli DH5α competent cells, and positive clones with appropriate inserts were selected by colony PCR with the nested PCR primers. The nucleotide sequences of the cloned pfeba-175 RII were analyzed by the automatic Sanger method with M13 forward and reverse primers. To increase the sequencing accuracy in the middle overlapping areas, two internal primers (5'-GCATGGAGAATTTTTGCTTGAAAG-3' and 5'-TTTTCCTACTAATTTTCTATTGCTC-3') were also applied in the sequencing process. Plasmids from at least two independent clones from each transformation mixture were analyzed to confirm the sequence accuracy. The nucleotide sequences of pfeba-175 RII obtained in this study have been deposited at GenBank under the accession numbers for Myanmar (OQ680031–OQ680063) and Vietnam (OQ680064–OQ680122).DNA was extracted from the dried blood spots using the QIAamp DNA Blood Kit  following the manufacturer’s protocols. Amplification of the pfeba-175 were examined using Editseq and Seqman in the DNASTAR package . The pfeba-175 sequence of the 3D7 strain (GenBank ID: XM_001349171.2) was used as the reference. The values of the number of segregating sites (S), haplotypes (H), haplotype diversity (Hd), nucleotide diversity (π), and the average number of pair-wise nucleotide differences within a population (K) were calculated with DnaSP ver. 5.10.0032. The rates of synonymous (dS) and non-synonymous (dN) substitutions were calculated and compared using the Z-test (P < 0.05) with the MEGA6 program33 using Nei and Gojobori's method34 with the Juke and Cantor (JC) correction of 1,000 bootstrap replications. Based on the acquired values, the dN–dS value was calculated. A positive value for dN–dS implies positive natural selection, whereas a negative value suggests negative or purifying natural selection35. Statistical analyses of Tajima’s D, Fu, and Li's D and F were performed using DnaSP ver. 5.10.0032 to evaluate the neutral theory of natural selection.The nucleotide and deduced amino acid sequences of Myanmar and Vietnam pfeba-175 RII were analyzed. The sequences of global pfeba-175 RII available in the public database were used in this study  and P. knowlesi strain H (GenBank ID: XM_039113751.1) were used as out groups. The numbers and parameters of recombination event were also estimated by DnaSP ver. 5.10.0032. To investigate site-wise influence of the natural selection in the common region of the global pfeba-175 RII population, FUBAR analysis was performed using the HyPhy distribution (http://www.hyphy.org/). In addition, to check the associations between the indentified SNPs in the global pfeba-175 RII population, linkage disequilibrium analysis between these SNP positions was performed and plotted by SRplot (https://www.bioinformatics.com.cn/en). The ARLEQUIN ver 3.5.2.2 program36 was used to investigate the molecular variance (AMOVA) to evaluate fixation (Fst). The haplotype network for the global pfeba-175 RII population was analyzed using the PopART program37 with the Median Joining algorithm38. The ancestry of the global pfeba-175 RII was analyzed by STRUCTURE software with a Bayesian method39. Three iterations for the number of clusters (K) from two to ten were initiated, each with a burning period of 10,000 steps and 20,000 Markov chain Monte Carlo iterations. The best K number was determined as previously described39 with the aid of Pophelper R Package41.The genetic diversity and natural selection of the global The study was approved by the Ministry of Health, Myanmar (97/Ethics 2015) and the Ministry of Health, Institute of Malariology, Parasitology and Entomology Quy Nhon, Vietnam (No.: 386/VSR-LSDT).Supplementary Table S1.Supplementary Table S2.Supplementary Table S3.Supplementary Table S4.Supplementary Figure S1.Supplementary Figure S2.Supplementary Table S5.Supplementary Legends.


Drosophila subobscura is considered a good model species for investigation of a population’s ability to adapt and cope with climate changes. Decade long research has shown that inversion frequencies change in response to environmental factors indicating their role in adaptation to novel environments. The mechanisms behind organisms’ responses to temperature are complex, involving changes in physiology, behavior, gene expression and regulation. On the other hand, a population’s ability to respond to suboptimal conditions depends on standing genetic variation and population history. In order to elucidate the role of local adaptation in population response to the changing temperature, we investigated the response to temperature in D. subobscura individuals originating from two different altitudes by combining traditional cytogenetic techniques with assessing the levels of Hsp70 protein expression. Inversion polymorphism was assessed in the flies sampled from natural populations and in flies reared in laboratory conditions at three different temperatures after five and sixteen generations and Hsp70 protein expression profile in 12th generation flies at the basal level and after heat shock induction. Our results indicate that local adaptation and population history influence population response to the changing temperature. Drosophila species, most of them having in common that due to the absence of recombination in heterokaryotypes, each inversion captures specific alleles in a beneficial combination, and as such, their frequency changes in response to the environment.Chromosomal inversions are traditionally described as adaptive structural mutations that become advantageous in populations by capturing favorable allele combinations . Since DDrosophila subobscura is particularly well documented, with numerous evidences of long-, mid- and short-term changes in their frequencies correlating with environmental variables. Parallel latitudinal distribution in inversion clines [D. subobscura natural populations [Regardless of mechanism, environmental variables found to influence changes in inversion polymorphism are many. Climatic factors such as temperature, humidity, precipitation and isolation are the most common factors correlated with shifts in inversion distribution in natural populations ,11,12, bn clines  and incrn clines ,15,16, in clines ,18. Despn clines ,20, withDrosophila subobscura is a Palearctic, temperate region species, with a thermal range between 6 and 26 °C, but it is generally considered a cold-adapted species with a thermal optimum of 18 °C [Drosophila genus [D. subobscura the Hsp70 family consists of two intronless and closely spaced Hsp70 genes arranged as a head-to-head inverted repeat (Hsp70IR), and that this gene family has identical genomic organization in Ost and O3+4+7 karyotypes [D. subobscura strains revealed that O3+4 flies have higher basal expression levels of Hsp70 proteins not boosted after heat shock and that Ost individuals show a different pattern [3+4, O3+4+7 and Ost files from different geographical regions [Drosophila species adaptation to colder climates strongly influences the magnitude of that response, indicating that changes in regulatory regions may be a target of selective pressure. Other experiments have demonstrated genome-wide changes in inversion polymorphism in response to temperature [D. subobscura associated with thermotolerance dispersed across all chromosomes has revealed that breakpoints do not disrupt any candidate gene directly associated with temperature adaptations, thus further corroborating the genetic complexity of thermal traits [By origin, of 18 °C . With mola genus ,23,24. Sla genus ,18,25. Tla genus , is consla genus ,26,27 asla genus , but allla genus ,30. The la genus . Sequencryotypes ,32. Expe pattern ; however regions . Howeverperature  and diffl traits ,39,40,41Although molecular genetic analysis has provided deeper insights into genes and mechanisms involved in temperature adaptation in this species, many evolutionary issues still remain unresolved. Despite the power of sequencing technology for the characterization of inversion polymorphisms, the cytogenetic technique, although with lower resolution, still remains a valuable tool for the analysis of the adaptive significance of this structural chromosomal polymorphism.D. subobscura individuals originating from two different altitudes by combining traditional cytogenetic techniques with assessing the levels of Hsp70 protein expression. Inversion polymorphisms were assessed in the flies sampled from natural populations and after 5 and 16 generations in laboratory conditions at three different temperatures. The Hsp70 protein expression profile was performed on the 12th generation at the basal level and after heat shock induction. Laboratory temperature regimes were chosen as stressful but not lethal for D. subobscura, with 16 °C prolonging development time, 19 °C considered as optimal, and 25 °C as a temperature which may induce male sterility [Our previous work on the effect of population history and different laboratory thermal conditions, performed with populations from different altitudes, indicated that both factors shape responses to stressful conditions . In the terility ,25. The Wild caught females were sampled in mid-August on Stara planina mountain, in Serbia, from two different altitudes: 1080 m and 1580 m . After establishing isofemale lines (IF) in the laboratory, their progeny  was mixed to make two mass laboratory populations that differ in sampling location: high altitude (marked as H) and lower altitude (marked as L). The first generation progeny of these two laboratory mass populations was used to establish three experimental groups per population which differed in their maintenance temperature: 16 °C, 19 °C and 25 °C. There were six experimental groups in total, reared on standard Drosophila medium , at approx. 60% relative humidity, light of 300 lux and 12/12 h light/dark cycles.D. subobscura males or F1 males of individual females from natural populations and with those from F5 and F16 generations, in all experimental groups from different temperature regimes. Each unique cross between 4 and 5 virgin females from the Küsnacht laboratory strain and between 25 and 35 males from each sample was performed. The Küsnacht strain was used to identify chromosomes with inversions in the progeny since it is homokaryotypic for the standard chromosomal configurations on all five chromosomes . The sample size of the studied populations is given in Inversion polymorphism analysis was performed with wild Experimental groups established twelve generations earlier were maintained at the above-mentioned temperature regimes and conditions. Their female and male freshly emerged progeny was collected and separated in order to prevent mating. For each assay, approximately 50 ♀ and 50 ♂ virgin flies were used per experimental group. Five flies were placed into empty falcon tubes (50 mL volume) with moistened plugs to prevent desiccation and placed in an incubator; set to 37 °C for 90 min. Flies were allowed to recover at room temperature for 30 min and then frozen at −80 °C for further analysis. To measure basal levels of Hsp70 expression, the same number of flies from each experimental group were collected and frozen in the same way.A total of six biological replicates per group were analyzed. All protocols were performed according to the manufacturer’s instructions. Homogenizations of five flies per experimental group and per sex  were performed in 1.0 mL of a suitable mix  following the protocol in . The totO) and expected (HE) heterozygosity, were calculated using Arlequin ver. 3.5.2.2 software under the Hardy–Weinberg equilibrium model [STF values and Nei’s distances. The statistical significance of all performed tests was assessed with 10,000 permutations. The matrix of pairwise population STF values was visualized using a multi-dimensional scaling method (nonmetric MDS) implemented in the PAST 4.03 software [Population genetic analysis was based on the frequencies and distribution of different inversions. For the analysis of differences in individual chromosome arrangements we used Z statistics. Observed  we used karyotypes of each individual. This method consists of performing the linear discriminant analysis (LDA) on the principal components analysis (PCA)-transformed matrix. LDA was performed on the first 14 PCs, cumulatively conserving 98.9% of the total variance. Additionally, the PCA-transformed matrix was used to find the optimal number of clusters using Ward’s method. We tested 2–50 clusters, and the optimal number of clusters was chosen using BIC statistics using the “diffNgroup” method. The relative expression of Hsp proteins was analyzed using the full factorial general linear model (GLM) procedure and Fisher’s post hoc test in the STATISTICA ver. 12 with fixed factors: Population, Rearing temperature, Treatment and Sex.O) was detected in the H natural population, with a general trend of higher HO in natural compared to laboratory populations, and a weaker trend of higher HO in laboratory populations derived from H then its L counterparts. Inversion polymorphism frequencies are shown in 3+4 is the most frequent arrangement for the O chromosome, but with significantly lower frequency in the L population. Complex arrangements in the O and E chromosomes are mostly absent from the L lab populations but retain small frequencies in the H lab populations regardless of temperature conditions. It is interesting to note that arrangement frequencies in the J chromosome behave differently in the H and L populations. While Jst in the H natural population has a much lower frequency than J1, its frequency rises in laboratory conditions, but the opposite trend is observed for the L population where J1 is dominant in the natural population and preserves its frequency in laboratory conditions. In the H population, after five generations at different temperatures, the Ost is more frequent in warm conditions, but after 16 generations of standard arrangements in A, J and O chromosomes have significantly higher frequencies in both cold and optimal conditions compared to the warm. In the L population after five generations, there are no differences in Ost frequencies at different temperatures, but it is more frequent in warm conditions after 16 generations.Standard parameters of genetic diversity per group are shown in STF statistics  with an inferred number of clusters and multi-dimensional scaling (MDS) showed a similar pattern of population differentiation . The natWhen the H and L populations were analyzed separately, strong differentiation could be observed between the natural populations and corresponding groups reared in laboratory conditions, and such differentiation was more prominent in populations from the higher altitude .p = 0.000668). Besides this, individuals in the H group tended to have higher levels of expression, rising with temperature with no difference between sexes.The results of the relative Hsp expression in each experimental group are presented in Although no significant interaction was observed between the combinations of factors, it is interesting to note the results of Fisher’s post hoc analysis of population/rearing temperature/treatment . IndividD. subobscura has been in the focus of scientific research for over seven decades, as one of the first genetic variability markers linked to a species adaptation to climate change [D. subobscura is considered to be adaptive [Abundant inversion polymorphism e change . Althouge change ,48, recee change . Naturaladaptive ,13,18,47D. subobscura from two altitudes in constant laboratory conditions under three thermal regimes, and assessing inversion polymorphism change over generations. Furthermore, we assessed whether adaptation to different thermal regimes in laboratory conditions influences the expression of the Hsp70 protein, both on a basal level and after heat shock.We attempted to experimentally validate the decade-old assumption that temperature is the main force shaping the distribution of chromosomal inversion frequencies across latitudes and climates by maintaining two distinct populations of 3+4 increasing in warm conditions. In laboratory groups derived from the L population, no such pattern could be detected, and Ost frequency was elevated in warm conditions. For other chromosome arrangements associated with thermal response [D. subobscura [Under the assumption that temperature was the main selective factor forming latitudinal clines ,25,47, cresponse , no cleabobscura ,20, suggbobscura ,25,52,53bobscura ,55,56,57bobscura ,59,60. STF values. A similar pattern was observed at the chromosomal level by [1 in L groups, irrespective of the thermal regime. Higher heterozygosity may be associated with the distribution of differentially expressed genes dispersed across all chromosomes [Further evidence that population history may have an effect on how the population responds to temperature changes is observed in differences in population structure. Our chosen natural populations from different altitudes across the mountain slope of Stara planina showed initial differences in inversion polymorphism, and that differences are preserved across generations and laboratory conditions with high level by , where tlevel by . In addiomosomes , providiDrosophila species is not ubiquitous [D. subobscura there is clear evidence of a plastic response to thermal variation [The observed changes in the Hsp protein expression patterns are in line with the observed differences between the H and L populations in laboratory conditions based on inversion polymorphism. Although not significant, the observed increase in Hsp70 levels with rising temperature as well as different patterns of expression in the H and L population further confirm the influence of population history. Expression levels are lower in H groups without temperature stress, but after heat exposure, all H groups have higher expression levels compared to their L counterparts, indicating a better ability to alleviate the influence of the extreme rise in temperature. Moreover, thermal shock induces higher protein expression, but significant difference between the basal- and temperature-induced levels could be observed only in H groups reared in cold and optimal conditions. Hsp protein expression is a balance between their protective role in various stressful conditions ,63 and tiquitous ,76,77,78ariation .D. subobscura showed the presence of population variation in evolutionary potential for long-term thermal adaptation suggesting that environments with higher thermal variation and pronounced thermal extremes may enhance reproductive success at higher temperatures [Recent studies with eratures . Our resD. subobscura populations, pushing them to their upper thermal limits, but the contribution of some other factors, such as population history, has to be considered. The results obtained in our study reveal that local adaptations significantly shape the populations’ response to the changing temperature as one of the climate parameters. Combined with phenotypic assays and functional expression of relevant proteins, chromosomal inversions and frequency changes of gene arrangement seem to be a relevant marker for inferring population structure, differentiation, and ability to withstand environmental challenges, such as temperature stress.Climate change and rising temperatures strongly influence changes in the genetic structure of natural


Mesembriomys gouldii), aiming to provide insight into the timing and extent of population declines across a large region with a dearth of long-term monitoring data. By estimating recent trajectories in effective population sizes at four localities, we confirm widespread population decline across the species’ range, but find that the population in the peri-urban area of the Darwin region has been more stable. Based on current sampling, the Melville Island population made the greatest contribution to overall allelic richness of the species, and the prioritisation analysis suggested that conservation of the Darwin and Cobourg Peninsula populations would be the most cost-effective scenario to retain more than 90% of all alleles. Our results broadly confirm current sub-specific taxonomy, and provide crucial data on the spatial distribution of genomic diversity to help prioritise limited conservation resources. Along with additional sampling and genomic analysis from the far eastern and western edges of the black-footed tree-rat distribution, we suggest a range of conservation and research priorities that could help improve black-footed tree-rat population trajectories at large and fine spatial scales, including the retention and expansion of structurally complex habitat patches.Genomic diversity is a fundamental component of Earth’s total biodiversity, and requires explicit consideration in efforts to conserve biodiversity. To conserve genomic diversity, it is necessary to measure its spatial distribution, and quantify the contribution that any intraspecific evolutionary lineages make to overall genomic diversity. Here, we describe the range-wide population genomic structure of a threatened Australian rodent, the black-footed tree-rat ( The Earth is experiencing its sixth mass extinction event , a semi-arboreal rodent that has experienced an estimated 30–50% decline in population size throughout the 2000s , and apply a range of analyses to identify patterns of genomic differentiation, population genomic structure, and historical demography. We also use a set of analyses to inform strategies for conserving the genomic diversity of the species. This includes comparison of levels of genetic diversity  and recent trends in effective population size which may indicate which populations are most at-risk from loss of genomic variation and are potentially candidates for management. It also includes a quantitative prioritisation analysis to identify the most important populations for conservation of the species’ genomic diversity and to quantify the contribution of each sampled population to the species’ genomic diversity. Conservation planning considers ecological, social, cultural and economic factors in addition to genomic data, but our strategy here is to present the genomic analyses independent of any other considerations or constraints.We hypothesise that (1) multiple evolutionary lineages will be present in this species, likely representing differentiation between mainland populations and offshore islands, and (2) conservation management will need to consider multiple populations and lineages in order to conserve >90% of the genomic diversity of the black-footed tree rat as a species. We also suggest that demographic trajectories inferred from genome-wide SNP data will broadly reflect observations of decline severity from ecological research, while also providing further insight into the timing and extent of population declines.Eucalyptus tetrodonta and E. miniata), crowns of the arborescent monocotyledon Pandanus spiralis , and habitat degradation by feral herbivores , inhabiting tropical open forests and woodlands across the northern end of the continent Friend . The speWe assembled a collection of 83 tissue samples from 11 distinct localities across the distribution of the black-footed tree-rat  ligation with one of 48 unique inline barcoded adapters compatible with the restriction site overhang, (3) manual sample pooling, (4) DNA purification (QIAquick PCR Purification Kit followed by SPRIselect paramagnetic beads), (5) size-selection targeting fragments of 280–375 bp in size , and (6) a PCR amplification step where one of two multiplexing index primers was added. Indexed libraries were pooled together and loaded onto flow cells for 150-bp single-end sequencing on an Illumina NextSeq 500 platform.Tissue samples were prepared and extracted in plate format (Qiagen DNeasy 96 Blood & Tissue Kit) following the manufacturer’s protocol with an extended lysis (incubation at 56 °C for 2 h then reduced to 37 °C overnight). Double-stranded DNA concentrations were quantified using a Qubit 3.0 Fluorometer and normalised to 200 ng DNA in 25 µL, and samples arranged on a 96-well plate that included 12 technical replicates and a negative control. The plate was sent for double‐digest restriction‐associated DNA (ddRAD) sequencing at the Australian Genome Research Facility in Melbourne, Victoria . Reads were aligned to the broad-toothed rat (Mastacomys fuscus) chromosome-length genome assembly (https://www.dnazoo.org/assemblies/Mastacomys_fuscus) using the bwa (v0.7.17) mem algorithm  were retained after using the rithm Li . Mastacoangsd v0.93 software package  > 0.6   falls to less than 0.4 , as SNeP uses linkage-based methods for estimating effective population size.To remove SNPs in linkage disequilibrium, we used the FST statistic from an individual ancestry matrix and identified outliers using the approach implemented in the LEA package , effective number of alleles (AE), SNP observed heterozygosity (HO), SNP expected heterozygosity (HE), and Wright’s fixation index (FIS). All calculations were made using the ‘gstudio’ package  of ancestral populations, allowing for independent investigation of values for k using cross-entropy metrics. It also incorporates the spatial location of sampling, to remove bias associated with patterns of isolation-by-distance. Cross-entropy criteria were calculated for values of k between one and 10, and a cross-entropy scree-plot was output for visual interpretation. The matrices of individual admixture coefficients were then extracted and plotted as stacked bar plots to visualise hierarchical population structure.To identify patterns of hierarchical population structuring, we used the cross-entropy methods of the ‘tess3r’ package  to identify patterns of historical demography in each locality. The SNeP v1.1 software tool  and Ne . Effective population sizes for the past 100 years were plotted for each locality using the geom_smooth function of the ‘ggplot2’ package   – AR(-i) / (AR(t) – 1), where AR(t) is total allelic richness and AR(-i) is allelic richness over all localities excluding the one in question.We conducted a set of analyses to identify the contribution of the sampled populations to genomic diversity in the black-footed tree-rat across northern Australia, as has been done for the brush-tailed rabbit-rat  and Melville Island (HE auto = 0.54) populations, and lowest levels in the Darwin (HE = 0.49) and Weipa (HE auto = 0.41) populations , demonstrating a slight deficit of heterozygotes at most localities. However, the value for the Weipa population was negative (FIS = −0.04), demonstrating a slight excess of heterozygotes.We observed small differences between the patterns of SNP and autosomal heterozygosity estimates Table . AssuminFST = 0.37). Substantially lower values were found among mainland localities, although the Weipa population was strongly differentiated from populations in the Northern Territory (FST = 0.25–0.28). Localities within the Northern Territory showed considerably less differentiation (FST = 0.13) . The population of black-footed tree-rats in Darwin appears to have been somewhat more stable than other areas over the past century. The final estimate for Darwin showed a potential increase in population size; however, a lack of very recent sampling  in this locality prevented estimation of post-1980 Ne values.Investigation of historical demographics using ury Fig. . The widWhile the Darwin population had the highest allelic richness (1.46) of all localities Table , its conmarxan suggested that the Darwin population (72% of iterations) was the single most effective region to conserve for allelic diversity , although the conservation benefit of conserving the Cobourg Peninsula population (28% of iterations) was quite similar   in this declining species, the conservation of at least two mainland populations is required, with prioritisation analyses suggesting that the Cobourg Peninsula and Darwin populations are of greatest value, although further sampling of populations in Queensland and Western Australia is sorely needed. Additionally, our analysis of the trajectory of effective population sizes over the past century suggests that many populations of the black-footed tree-rat have potentially been declining for at least the last century. Much of the ecological survey data that have been used to identify small mammal declines in northern Australia come from the past three decades  and on Melville Island (M. g. melvillensis) displaying a high level of historical isolation from the rest of the mainland populations (M. g. gouldii). The population genomic structure of the black-footed tree-rat broadly aligns with other widespread mammal species across northern Australia, such as the brush-tailed rabbit-rat   . When paired with that population’s low and apparently declining Prioritisation analyses identified the locations that contribute the greatest amount to genomic diversity in the sampled populations of the species. This provides information that can be used to develop formal conservation management strategies, forecast the likely loss of genomic diversity if individual populations become extinct, and help identify combinations of populations that, as sources for reintroductions, best represent the full genomic diversity of the species. These analyses suggest that the populations most important for the conservation of black-footed tree-rat genomic diversity were those across the mainland of the Northern Territory, followed by those in Queensland or Melville Island. While the populations around Darwin and on the Cobourg Peninsula are clearly important for conservation purposes, sampling of other remnant populations across the species’ distribution might alter the results of these analyses by uncovering geographic areas containing additional genomic diversity. For example, based on our current results, conserving just the Darwin and Cobourg populations would meet our 90% target for genomic diversity; however, the loss of black-footed tree-rats from Melville Island and in Queensland would result in the loss of two subspecies (as currently described). Further analysis after additional sampling has taken place may reduce the relative importance of the Darwin and Cobourg populations, however, this is uncertain. Importantly, our prioritisation confirms that the Cobourg Peninsula should be recognised as an important reservoir of genomic diversity for mammal species in northern Australia  populations , and greater retention of old growth vegetation  in close proximity to known populations (von Takach et al. While the black-footed tree-rat persists in parts of Western Australia and north Queensland, little is known of these populations. In Western Australia, the species appears to be very rare, and possibly at high risk of local extinction, with very few confirmed records in the last 40 years. As such, obtaining demographic and genomic data from these regions should be a priority. Genomic investigation of populations in Western Australia will help clarify their level of differentiation from, and enable comparison of genomic diversity with, the geographically distant Northern Territory populations. Further, the relative importance of feral cat predation, feral herbivore disturbance, and frequent or high-intensity fires on population persistence across the species distribution is largely uncertain. While it appears as though some populations can persist in the presence of one or more of these threats, the degree to which population demography, dispersal processes, and genetic diversity are impacted is still unknown. Critically, there is a growing body of evidence suggesting that rapid and intensive management of mammal species is required across large expanses of northern Australia (Woinarski et al. Supplemental Material