Puccinia striiformis f. sp. tritici (Pst), is one of the most important diseases of wheat worldwide. To understand the worldwide distribution of its molecular groups, as well as the diversity, differentiation, and migration of the Pst populations, 567 isolates collected from nine countries  in 2010–2018 were genotyped using 14 codominant simple sequence repeat markers. A total of 433, including 333 new multi-locus genotypes (MLGs), were identified, which were clustered into ten molecular groups (MGs). The MGs and country-wise populations differed in genetic diversity, heterozygosity, and correlation coefficient between the marker and virulence data. Many isolates from different countries, especially the isolates from Mexico, Ecuador, and the U.S., were found to be identical or closely related MLGs, and some of the MGs were present in all countries, indicating Pst migrations among different countries. The analysis of molecular variance revealed 78% variation among isolates, 12% variation among countries, and 10% variation within countries. Only low levels of differentiation were found by the pairwise comparisons of country populations. Of the 10 MGs, 5 were found to be involved in sexual and/or somatic recombination. Identical and closely related MLGs identified from different countries indicated international migrations. The study provides information on the distributions of various Pst genetic groups in different countries and evidence for the global migrations, which should be useful in understanding the pathogen evolution and in stressing the need for continual monitoring of the disease and pathogen populations at the global scale.Stripe rust, caused by  Puccinia striiformis Westend. f. sp. tritici Erikss. (Pst) is a destructive disease across major wheat growing regions of the world . ipe rust . The difl states , where Pern U.S. ,43,59,60ontinent ,30,31,61f origin . In agre. ipe rusPst is a dikaryotic fungus, the heterozygosity of isolates makes a major contribution to the overall diversity. Ali et al. (2014) studied the heterozygosity of Pst populations in different countries and reported heterozygosity values of 0.28 in China and 0.35 in Pakistan [Pst populations in these two regions of China are quite different [P = 0.027), which may support the hypothesis that heterozygous isolates may have advantages in pathogen evolution because of the heterosis effect, which may be involved in adaptation to different environments and host cultivars with different resistance genes [As Pakistan . In the Pakistan , and thePakistan . HoweverPakistan . This diifferent ,62. In ace genes ,43. Pst populations in many parts of the world [Pst in China and the Himalayan region [Pst in barberry for sexual reproduction was reported at low frequencies in China [P = 0.01 level, and at the P = 0.05 level, 3 MGs  were still considered to have possible recombination. Based on the lowest rbarD value (0.006) and the highest P value (0.309), MG8 is most likely to be produced by sexual recombination. In fact, most MG8 isolates were from China and Italy. China has been shown to have sexual reproduction [Pst infection on barberry [Pst sexual recombination in some countries, especially China. Not all possibilities of recombination are through sexual reproduction, and some of them may be results of somatic recombination. Somatic recombination has been demonstrated for Pst under controlled conditions [Puccinia graminis f. sp. tritici, the wheat stem rust pathogen [Pst evolution, which is illustrated by the minimum span of the network for stepwise mutations to produce the majority of MLGs in the present study  between the molecular genotype data and the previously published virulence data ,52 was lPst isolates from 9 countries were used in this study, including 132 U.S. isolates selected to represent different races and molecular groups during the same period of collection in the other countries for the purpose of genotype comparison . The tube was incubated at 37 °C for 2 h to completely dissolve the DNA pellet. The concentration of the DNA stock solution was determined using a ND-1000 spectrophotometer , and the quality was checked in a 0.8% agarose gel. A work solution of 5 ng µL−1 was made from the stock solution by adding sterile deionized water for use as a DNA template in the polymerase chain reaction (PCR). Total genomic DNA was extracted from dried urediniospores following a universal and rapid salt-extraction method for high-quality genomic DNA  with modPst . A mixtuPst isolates shown in previous studies [Fourteen pairs of SSR primers were selected based on their co-dominant polymorphisms among  studies ,35,36,39 studies ; PstP001 studies ,70. The  studies . For eac studies . 2 (Sigma Life Science), 0.96 µL 2.5 mM dNTP (Sigma Life Science), 0.12 µL of 5 µM forward primer, 0.6 µL 5 µM reverse primer, 0.24 µL of 5 µM M13 universal primer, 0.2 µL 5 U/µL Taq polymerase (Sigma Life Science), 4 µL DNA , and 4.68 µL sterile ddH2O. The amplification cycles and conditions were 94 °C for 5 min for initial denaturation; 42 cycles of 94 °C for 30 s, 45 to 54 °C for 30 s depending on primers, and 72 °C for 45 s; and 7 min of final extension at 72 °C. The sizes of the PCR products were estimated using capillary electrophoresis on an ABI3730 Genotyper . The internal molecular weight standard for ABI3730 was Genescan 445-LIZ (Applied Biosystems). Allele sizes in base pairs were scored and analyzed using the software GeneMarker V2.2 . Amplification of PCR was performed in a Bio-Rad iCycler  following the protocol described in previous studies ,36. EachPst is a dikaryotic fungus at the uredinial stage, each isolate was scored for two alleles to determine homozygous or heterozygous for each SSR locus using GeneMarker V2.2 (https://genemarker.software.informer.com/2.2/ (accessed on 20 August 2021)). The data file generated by GeneMarker was converted to a ‘csv’ file to be analyzed with the R package. The sufficiency of markers to describe the population structure was assessed through the detection of multilocus genetypes (MLGs) plotted against the number of loci, and a graph was generated using the ‘poppr’ package in the R 4.0.3 program (https://r-for-windows.updatestar.com/ (accessed on 20 August 2021)) [poppr’. If isolates in the present study had identical alleles across all 14 SSR loci, they were assigned to the same MLG. The MLGs were named in continuation with the previous MLGs identified from the U.S. isolates of 2010 to 2017 . Since Pt 2021)) . The gent 2021)) ,74,75 anTo identify putative clusters of genetically related isolates in the present study (567 isolates), hierarchical clustering analysis based on the 14 SSR markers  was conducted using the dissimilarity values and the “ward.D2” method with the “hclust” function in the R stats 4.0.3 program . The pari2 and H = ∑PilnPi, respectively, where Pi is the observed frequency of the ith MLG in a population [Pst is a dikaryotic fungus at the uredinial stage and DNA was extracted from urediniospores, each isolate was scored for homozygous or heterozygous at each SSR locus using the software GeneMarkerV1.5 (https://genemarker.software.informer.com/download/ (accessed on 20 August 2021)). Heterozygosity in percentage was calculated for each isolate based on the number of loci with different alleles across the 14 SSR markers analyzed using the software GenAlEx 6.503 (https://biology-assets.anu.edu.au/GenAlEx/Welcome.html (accessed on 20 August 2021)) [Genotypic diversity was determined by estimating both genotypic richness (the number of observed MLGs) and evenness (the distribution of genotype abundance). Stoddart and Taylor’s index and Shannon’s diversity index for MLG diversity were calculated as G = 1/∑Ppulation ,54,77. At 2021)) . Mean hePst isolates based on countries using the function “poppr.amova” in the poppr R program version 4.0.3 [ST) among the nine countries, except for Pakistan and Egypt which had isolate numbers less than ten, were analyzed using the software GenAlEx 6.503 [adegenet” R package, was also performed to generate a scatter plot. The DAPC was carried out for all marker loci, and an α-score optimization was used to determine the number of principal components to retain [Analysis of molecular variance (AMOVA), which allows the hierarchical partitioning of genetic variation within and among countries , was conon 4.0.3 ,80. PopuEx 6.503 . A migrao retain ,82. poppr” program, and the null hypothesis rbarD = 0 was tested with 1000 permutations [P < 0.001, then the samples are likely from a clonal population [The mode of reproduction was tested by adjusted/standard index of association (rbarD) . This teutations . If an opulation .P = 0.05. To determine the correlations between the molecular data obtained in the present study with the virulence data in a previous study from our group , a distaPst collections of nine countries on five continents in recent years, and clustered them into 10 MGs. These MGs differed greatly in frequency, distribution, genetic diversity, heterozygosity, and correlation coefficient between the molecular genotypes and virulence phenotypes. Similarly, we also detected differences in these aspects among the nine country-wise populations. Although significant differentiations were observed between all possible pairwise country populations at P = 0.05, except between Canada and Mexico, the greater the distance between two countries, the greater the differentiation between the two countries’ populations. The presence of the same MGs and the detection of identical or closely related MLGs in different countries indicated frequent migrations of the pathogen among countries and continents. The analysis of the standardized index of association indicated possible sexual reproduction involved in some of the MGs, although the overall population or individual country populations were found to be clonal. The relatively low correlation coefficient values obtained for the overall population, individual country populations, and MGs suggest the need for characterization of Pst populations using both virulence phenotyping and molecular genotyping. This study provides insights for understanding the diversity, distribution, and evolution of Pst populations on a global scale, and the information is useful for continual monitoring of the pathogen populations. The detection of global migrations of Pst genotypes in the present study and virulent races in the previous studies emphasizes the importance of considering the global pathogen populations when breeding wheat cultivars with broad, durable resistance, for the sustainable control of stripe rust.In this study, we identified 433 MLGs from the


Coilia nasus to fresh water by comparing four freshwater-resident populations with their ancestral anadromous population. Linkage disequilibrium network analysis and population genetic analyses revealed two putative large chromosome inversions on LG6 and LG22, which were enriched for outlier loci and exhibited parallel association with freshwater adaptation. Drastic frequency shifts and elevated genetic differentiation were observed for the two chromosome inversions among populations, suggesting that both inversions would undergo divergent selection between anadromous and resident ecotypes. Enrichment analysis of genes within chromosome inversions showed significant enrichment of genes involved in metabolic process, immunoregulation, growth, maturation, osmoregulation, and so forth, which probably underlay differences in morphology, physiology and behavior between the anadromous and freshwater-resident forms. The availability of beneficial standing genetic variation, large optimum shift between marine and freshwater habitats, and high efficiency of selection with large population size could lead to the observed rapid parallel adaptive genomic change. We propose that chromosomal inversions might have played an important role during the evolution of rapid parallel ecological divergence in the face of environmental heterogeneity in C. nasus. Our study provides insights into the genomic basis of rapid adaptation of complex traits in novel habitats and highlights the importance of structural genomic variants in analyses of ecological adaptation.Rapid adaptation to novel environments may drive changes in genomic regions through natural selection. However, the genetic architecture underlying these adaptive changes is still poorly understood. Using population genomic approaches, we investigated the genomic architecture that underlies rapid parallel adaptation of  Understanding the mechanisms that facilitate adaptation of populations to changing environments is a long-standing central goal of evolutionary biology . AlthougCoilia nasus, exists in two distinct life history forms  and SVs in a population genomic framework to characterize how differentiation is structured across the genome, which would allow us to assess whether the same chromosomal features are implicated in divergence between the anadromous population and the four freshwater-resident populations from different lakes. The source of adaptive genetic variants for freshwater adaptation was also illustrated. The results will provide a general understanding of the genetics of rapid adaptation of complex phenotypes to changing environment in wild populations.Here, we seek to investigate genomic architecture that underlies rapid parallel adaptation to fresh water in C. nasus (EcoRI restriction sites (one per 10 kb) across 24 linkage groups (chromosomes). The RAD sequencing covered about 110 Mb (14%) of the total genome with a size of 812 Mb , which was much larger than those in the four freshwater-resident populations. In addition, the Ne estimates of Taihu Lake population  and Chaohu Lake population  were a magnitude higher than those of Hongze Lake population  and Luoma Lake population  (The published chromosome-level genome assembly for C. nasus  containsf 812 Mb  online. f 812 Mb  online af 812 Mb  online.  to 0.24  online. 160-161)  online.FST) between the anadromous population and the four freshwater-resident populations were generally low but significant, with an average value of 0.07 for all SNPs and 0.03 for neutral SNPs, indicating that the freshwater populations were recently derived from the anadromous one (K = 4 (Hongze Lake and Luoma Lake formed one cluster and other three populations formed distinct clusters) (FST (The genome-wide fixation indexes (mous one  online. lusters)  online, rs) (FST C. Princirs)  of the 1,147 candidate SNPs in all freshwater–anadromous population pairs B and C. t-test P < 2.2e-16; The FWA for 1,024 of the 1,147 candidate outlier SNPs (89%) occurred as standing genetic variation in the Yangtze River Estuary anadromous population, suggesting that standing variation was the predominant source for adaptation to fresh water. Considering the relatively low frequency of the FWA as standing variations in the anadromous population and the relatively small sample size, it was not unlikely that FWA of the other 11% candidate SNPs could also present as standing variation in the anadromous population. In the anadromous population, frequency of the FWA for the candidate SNPs  was significantly higher than the minor allele frequency (MAF) of the neutral SNPs  identified a total of five single-outlier clusters (SOCs) on different chromosomes, with median LD 31.54 Mb . Populat31.54 Mb  online. C. nasus is not very high. Moreover, we found a large “gap” in the distribution of outliers on LG22 (r2 = 0.80), implying that there might be assembly errors here. We thus excluded this gap from the inversion region on LG22, which resulted in a size of 21.97 Mb. All the following related results were based on the “gap” removed inversion on LG22.The identified putative inversion on LG6 covered about 31.54 Mb containing 1,077 genes, and the inversion on LG22 covered about 27.40 Mb with 759 genes  online.  on LG22 A, which FST between the anadromous population and the four freshwater-resident populations calculated with all SNPs located in the chromosome inversion regions on LG6 and LG22 showed elevated differentiation with an average value of 0.37, which was an order of magnitude larger than the FST values based on neutral SNPs  were reference homokaryotype (AA), whereas most of the freshwater individuals were alternative homokaryotype   online. ral SNPs  online.Annotations for 859 of the 1,147 candidate outlier SNPs were retrieved. Of these, 380 SNPs were located in genic regions with 23 in coding sequence and 357 in introns, and 479 were within 10 kb of the closest gene. For the 23 SNPs in coding sequence of 16 genes, eight were nonsynonymous mutations in six genes  online. P < 0.01; Gene Ontology (GO) enrichment analyses of genes within the two chromosome inversions in terms of biological process identified a total of 72 and 73 significantly enriched GO terms for inversion regions on LG6 and LG22, respectively , mutations at genes encoding solute carrier proteins have been associated with annual salinity variation in the Baltic Sea (Gadus morhua), which experience different salinities (KCNH4) are found on either side of one chromosomal inversion that has undergone parallel selection after freshwater invasion in three-spined sticklebacks, suggesting marine and freshwater specific isoforms  (Clupea harengus), a 7.8-Mb inversion on chromosome 12 is found both in the East and West Atlantic, which possibly underlies ecological adaptation in relation to the water temperature during gonadal maturation before spawning or the water temperature at spawning/early larval development , which may facilitate local adaptation to environmental conditions prevailing at spawning sites (Littorina saxatilis), several candidate chromosomal inversions are associated with rapid parallel adaptation, which can store shared variation that fuels rapid parallel adaptation to heterogeneous environments  . In the elopment . Recentlng sites . In the ronments . The impronments .Ne is crucial in determining the effectiveness of selection relative to drift. Large populations experience less genetic drift than small ones, which increases the efficiency of selection and the power to detect selected alleles. Indeed, the frequencies of FWA and alternative arrangement of chromosome inversions on LG22 were higher in the two populations with larger Ne (Chaohu Lake and Taihu Lake) than in the two populations with smaller Ne (Hongze Lake and Luoma Lake), suggesting a stronger selective strength in populations with larger Ne. All these conditions combined could lead to the observed strong selection in rapid adaptation of C. nasus to fresh water in different lake populations. However, the loci that showed signals of selection but not in all the four freshwater populations may reflect local adaptation.Besides the nature of the genes and genomic regions under selection, the rate of genomic adaptation is also determined by the degree of environmental change, the availability of beneficial mutations, and the efficiency of positive selection. Populations adapt to novel environments in two distinct ways: selection on preexisting genetic variation and selection on new mutations . Due to C. nasus, which contribute to the accumulating evidence that SVs form a significant part of adaptive genetic variation  using BWA MEM v0.7.15-r1140  Read pairs with adaptors were removed using cutadapt v1.16 , 2) low-15-r1140  with def15-r1140 . High-qu15-r1140  or in hoFST) and demographic analysis.For population genetic and demographic analyses that assume a set of neutral and unlinked markers, we created a reduced data set as following: First, any SNPs that were identified as outliers by either of the two detection methods  in any population pair were removed; second, we removed all SNPs on the two chromosome inversions (see Results section); third, only one SNP was kept in a 10-kb region to remove putative LD using VCFtools . This neHO) and expected (HE) heterozygosity of SNPs for each population were estimated using populations in Stacks. FST among all populations/between each pair of populations were calculated with Arlequin v3.5.2.2 and their significance was determined using 10,000 permutations (Ne) of each population was estimated using the LD method in NeEstimator v2.1  > 4) was used as the threshold after examining the Manhattan plot (FDR level of 0.005 (max = 0.007). Second, we used the R package pcadapt  values between pairs of SNPs were calculated individually on each chromosome by VCFtools using the whole data set with MAF of 0.1. The r2 matrix was then used for LDna. There are two key parameters that can be set in LDna. The minimum number of edges |E|min, which corresponds to the minimum number of connections among the vertices of a cluster. This parameter controls the minimum number of SNPs within an OC. After several preliminary runs, |E|min = 30 was applied to represent a compromise between detecting clusters large enough to represent chromosomal rearrangements and avoiding noise that result from physical linkage within chromosomes. Another key parameter φ controls the minimum LD threshold above which the median pairwise LD within a cluster is higher than the intercluster LD for the group of SNPs to be considered as an OC. This parameter was selected by initializing its value = 2 then increased the value of φ by one in each iteration until no more LD clusters were obtained for at least three times within one chromosome. Only SOCs with a minimum of 30 SNPs were retained, and clusters with a low median LD (r2 < 0.3) were also discarded. The sizes of each SOC were defined as the most extreme positions of the SNPs included in each SOC . The resulting SOCs were then examined in the downstream analyses.As clusters of loci with unusually high LD might be generated by chromosomal rearrangements, such as inversions, we detected clusters of SNPs in high LD  using the LDna as implemented in the R package “LDna” . All popSOCs can arise due to various reasons, such as chromosome inversions, spatial population structure, or local adaptation. Inversions can be identified by detecting groups of genetically distinct individuals that correspond to different karyotypes. The suppressed recombination between arrangements due to inversions will result in the presence of three distinct groups of individuals, which corresponded to three karyotypes . The second group with karyotype AB should have the highest heterozygosity relative to both the homokaryotypes that should have reduced heterozygosity. Thus, three groups will be separated along the axis of PC1 of PCA based on all SNPs within an LD cluster for inversions. Chromosome inversions were then confirmed by PCA and heterozygosity analyses for each SOC identified by LDna. The rearrangement karyotype of each individual was determined using the “k-means” algorithm as implemented in R . Based ohttps://github.com/nextgenusfs/funannotate, last accessed November 11, 2020) and BRAKER v2.1.5 (Clupea harengus (NCBI assembly accession number: GCA_000966335.1) and Denticeps clupeoides (NCBI assembly accession number: GCA_900700375.1). Thirdly, RNA-seq reads were aligned to the genome using HISAT2 v2.1.0  for the outliers were determined by Ensembl Variant Effect Predictor  with disC. nasus to the protein database of Danio rerio, then we retrieved the related GO terms from the Gene Ontology Annotation (GOA) database . Enrichment analysis for GO terms in each candidate inversion region was performed by the R package “topGO” (Gene models and annotations for genes within the two putative chromosome inversion regions were extracted using BEDtools . In orde “topGO”  using thMolecular Biology and Evolution online.msaa290_Supplementary_DataClick here for additional data file.


Habitat isolation and loss are significant factors that lead to the decline of wildlife populations worldwide, and habitat loss further leads to the shrinkage of populations, which increases the risk of inbreeding and the genetic decline of the populations. To explore the independent evolutionary characteristics of different populations, this study analyzed the genetic disparity of the introduced oriental fire-bellied toad in Beijing from a source population in Shandong Province. The results show that, despite originating from a small artificially introduced population, the toads in the Beijing region have maintained a moderate genetic diversity after 90 years of independent evolution, indicating that this species has a high capacity for survival and adaptation.Bombina orientalis) were artificially introduced to Beijing from Shandong Province in 1927, and since then, this separated population went through an independent evolution. To explore the differentiation of the introduced population with its original population, this study analyzed the genetic structure of the oriental fire-bellied toad, based on the mitochondrial genome control region and six microsatellite sites. The results showed that the haplotype diversity and nucleotide diversity of the mitochondrial D-loop region partial sequences of the Beijing Botanical Garden population and the Baiwangshan population were lower than those of the Shangdong Kunyushan population. Microsatellite marker analysis also showed that the observed heterozygosity and expected heterozygosity of the Beijing populations were lower than those of the Kunyushan population. The phylogenetic trees and network diagrams of haplotypes indicated that the three populations were not genetically separated. However, the structure analysis showed a genetic differentiation and categorized the sampling individuals into Beijing and Shandong genetic clusters, which indicated a tendency for isolated evolution in the Beijing population. Although the Beijing populations showed a decline in genetic diversity, it was still at a moderate level, which could maintain the survival of the population. Thus, there is no need to reintroduce new individuals from the Kunyushan source population.Detailed molecular genetic research on amphibian populations has a significant role in understanding the genetic adaptability to local environments. The oriental fire-bellied toads ( Bombina variegata and Bombina bombina have been widely studied in the area of molecular biology [Bombina bombina, revealing obvious genetic differences between sampling populations due to habitat fragmentation. A small-scale multilocus phylogeography analysis on Bombina variegata and Bombina bombina indicated that no genetic exchange has taken place between the two species [Habitat isolation and loss are significant factors that lead to the decline of wildlife populations worldwide, and habitat loss further leads to the shrinkage of populations, which increases the risk of inbreeding and the genetic decline of populations . Genetic biology , such as species .Bombina, including Bombina orientalis, Bombina fortinuptialis, and Bombina maxima, distributed within the territory of China, there has been little research work conducted on their genetic structure. A study on the mitochondrial structure analysis of Bombina orientalis found population genetic differentiation as a result of isolated evolution for geological change in northeast China [Bombina orientalis indicated that the genetic structure diversity was caused by historical geological activities [Five species of Bombinatoridae are found in China, and three species are deemed to be threatened . Habitatst China . The evotivities .Bombina genus living in northern China and is mainly distributed in Hebei, Heilongjiang, Liaoning, Jilin, Inner Mongolia, Anhui, Shandong, and Beijing; it is also distributed in peninsular Korea and the far east of Russia [B. orientalis individuals from Kunyushan in Yantai to Beijing and released them in the Yingtaogou of the Xiangshan Wafo Temple and in the wetland near the Western Mountain, where they have since reproduced [B. orientalis mainly focuses on its baseline biological characteristics, reproductive ecology, population status, and food composition [The existing oriental fire-bellied toad is the only species of the f Russia . In 1927produced . Currentposition ,11. At pposition . In our B. orientalis in Beijing that differ from the source population in size after long time independent evolution, and to see whether genetic diversity is related to population size or not. In order to detect the actual situation and explore the detailed independent evolutionary trajectory of this species in Beijing, we analyzed the genetic structure diversity using mitochondrial and microsatellite markers based on samples from two areas in Beijing and from the Kunyushan source population in Shangdong Province.Under the neutral model, a population’s genetic diversity depends on its effective population size and gene mutation rate; genetic diversity will increase with a larger effective population size and the decreasing effects of drift. Higher population genetic diversity in the abundant species is likely due to a combination factors . The goaSamples from Beijing were collected at the Beijing Xiangshan Botanical Garden (ZWY) in 2017 and Baiwangshan (BWS) Forest Park in 2018. Samples from Shandong Province were collected from Kunyushan (KYS) of Yantai city in 2017. A total of 63 samples were collected from the three sampling locations . The method of euthanizing was to use diethyl ether deep anesthesia. The individuals were adults with body lengths between 40 and 45 mm. The hind leg muscles were taken off and fixed with anhydrous ethanol for sampling in a centrifuge tube, then stored in a refrigerator at −20 °C for DNA extraction. The treatment of the animals followed the general protocol for experimental animal processing .Bombina orientalis (GenBank accession number AY585338) and synthesized by Ruibio Biotech Co., Ltd. . Three pairs of primers were used to amplify the mitochondrial D-loop sequence: DFLC-1 , DFLC-2 , and DFLC-3 . Different primers had overlapping fragments of at least 100 bp in the mitochondrial genome to improve the sequencing accuracy.Total DNA was extracted using the TIANamp Genomic DNA Kit . The primers were designed referring to the D-loop segment in the complete mitochondrial DNA sequence of The PCR amplifications were conducted in a total volume of 20 μL, including 10 μL of Extaq enzyme , 0.8 μL of each forward and reverse primer (10μmol/L), 2 μL of DNA, and DNase/RNase-free deionized water. The PCR program was as follows: pre-denaturing for 4 min at 95 °C, denaturing for 40 s at 95 °C, annealing for 40 s at 50 °C or 52 °C, extension for 40 s at 72 °C, followed by 40 cycles; this was extended to 72 °C for another 7 min. The PCR products were detected by 1% agarose gel electrophoresis, and sequencing was performed by Ruibio Biotech Co., Ltd.  using the ABI3730XL sequencer.The sequencing results were compiled in the Contig Express software and complemented by manual checking. Sequence alignment was performed by the ClustalX 2.1 software , and theB. bombina and B. variegata based on the reports of Guicking [The primers of 25 microsatellite sites of Guicking  and NümbGuicking  were selGuicking . A fluorThe PCR reaction system was the same as the amplification reaction of the mitochondrial control region. The PCR program was as follows: pre-denaturing for 4 min at 95 °C, denaturing for 40 s at 95 °C, annealing for 30 s at 48~52 °C, extension for 30 s at 72 °C, followed by 40 cycles; this was extended to 72 °C for another 8 min. The PCR-amplified samples were sent to Norse Genomics Research Center Co., Ltd. , for gene scanning in the ABI-PRISM310 genotyping machine, and the microsatellite allele band size was determined by the GeneMarker v2.4.0 software .Bombina bombina as the outgroup (GenBank accession number: EU115993). The phylogenetic tree was constructed in PAUP 4.0 [The haplotype diversity, nucleotide diversity, and nucleotide divergence between populations and the haplotype number of the three populations were calculated in the software Dnasp5 . The KimPAUP 4.0  based onPAUP 4.0  and the PAUP 4.0 .Fst and P values were calculated to indicate the differentiation degree among the studied populations.The population genetic structure was analyzed in the software Arlequin 3.5 , the genFst and its significance among populations were calculated using the Arlequin 3.5 software [The microsatellite allele number, effective allele number, expected heterozygosity, observed heterozygosity, gene richness, Nei genetic distance, and polymorphism information content were calculated by the GenALEx 6.5 software  and FSTAsoftware . The gensoftware .Through DNA extraction, PCR amplification, sequencing, and splicing, the mitochondrial D-loop fragment sequences of 47 samples were successfully obtained, including 24 from the botanical garden, 7 from Baiwangshan, and 16 from Kunyushan. In the 1122 bp sequence, there were 23 nucleotide polymorphism sites, 17 monomorphism sites, 6 simple information sites, and 14 haplotypes, which were defined accordingly . One haplotype (BWS–ZWY) was shared by the populations of Baiwangshan and the Botanical Garden, the haplotype BWS–KYS was shared between the Baiwangshan and Kunyushan populations, and the haplotype ZWY–KYS was shared by the populations of the Botanical Garden and Kunyushan. The Botanical Garden and Baiwangshan populations had the unique haplotypes of ZWY1 and BWS1, while the population in Kunyushan had nine unique haplotypes (KYS1–KYS9).Based on the calculation of Dnasp 5.2, the overall genetic diversity indexes of the Beijing populations were obviously lower than those of the Shandong population . The nucThe genetic distance between the 14 haplotypes was between 0.001 and 0.011, with an average of 0.002, according to MEGA 5.2 software calculation, indicating that there was a low genetic difference among the three populations.p < 0.01). The Fst between the Beijing and Shandong populations was 0.124 (p < 0.01). Based on the AIC standard, the most appropriate nucleotide substitution model was the HKY+I model. The NJ tree and ML tree based on partial sequences of the mitochondrial D-loop region showed that the haplotypes between the Beijing and Shandong populations had not yet formed a significant lineage structure , indicating that there was a moderate degree of genetic differentiation between the two populations based on microsatellite markers. Based on the Structure software calculation, when K = 2, the average value of ln-likelihood was higher . Therefore, this result supported dividing the sampling B. orientalis individuals into two populations (genetic clusters) of Beijing and Shandong  and the nucleotide diversity was 0.00182 (95%CI: 0.0014-0.00246) [B. bombina  and B. variegata  [B. orientalis population also showed a moderate genetic diversity. In addition, the mitochondrial haplotype diversity, nucleotide diversity, observed heterozygosity, and expected heterozygosity in microsatellites were all higher in the Shandong population  might be due to there being fewer haplotypes in the Beijing population. However, there exists a slight genetic differentiation based on the analysis of microsatellite markers. According to the Bayesian analysis of the Structure software, the B. orientalis individuals were divided into two clusters. This genetic discrepancy trend was supported by the differentiation coefficient based on microsatellite markers , showing the two populations that appeared with significant genetic differentiation. This result indicated that the Beijing population has shown an independent evolutionary trend.Based on 14 haplotypes of mitochondrial genes, the phylogenetic NJ tree and ML rootless tree showed no obvious lineage structure between the Beijing and Shandong populations , so the B. orientalis population in Beijing was lower than that in Shandong Province, it is still at a normal level which could maintain the survival of the introduced population. A significant genetic differentiation has been formed between the two populations, as the geographic distance between the two populations is great and there are no chances of natural gene exchange between the two populations; therefore, we suggest that the Beijing population should be regarded as a separate management unit, and currently there is no need to reintroduce new animals to improve the genetic diversity of the populations in the Beijing area. This is due to the fact that aimless reintroduction can affect the genetic structure of the local population and lead to genetic drawbacks through crossbreeding. Originating from a small artificially introduced population, the B. orientalis in the Beijing region have maintained a moderate genetic diversity after 90 years of independent evolution, indicating that this species has a high capacity for survival and adaptation.Although the genetic diversity of the


Glycyrrhiza uralensis is a valuable medicinal legume, which occurs widely in arid and semi-arid regions. G. uralensis demand has risen steeply due to its high medical and commercial value. Interpret genome-wide information can stimulate the G. uralensis development as far as its increased bioactive compound levels, and plant yield are concerned. In this study, leaf nutrient concentration and photosynthetic chlorophyll index of G. uralensis were evaluated to determine the G. uralensis growth physiology in three habitats. We observed that G. uralensis nutrient levels and photosynthesis differed significantly in three regions (p < 0.05). Whole-genome re-sequencing of the sixty G. uralensis populations samples from different habitats was performed using an Illumina HiSeq sequencing platform to elucidate the distribution patterns, population evolution, and genetic diversity of G. uralensis. 150.06 Gb high-quality clean data was obtained after strict filtering. The 895237686 reads were mapped against the reference genome, with an average 89.7% mapping rate and 87.02% average sample coverage rate. A total of 6985987 SNPs were identified, and 117970 high-quality SNPs were obtained after filtering, which were subjected to subsequent analysis. Principal component analysis (PCA) based on interindividual SNPs and phylogenetic analysis based on interindividual SNPs showed that the G. uralensis samples could be categorized into central, southern, and eastern populations, which reflected strong genetic differentiation due to long periods of geographic isolation. In this study, a total of 131 candidate regions were screened, and 145 candidate genes  were identified by selective clearance analysis based on Fst and θπ values. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed significant enrichment of 110 GO terms including carbohydrate metabolic process, carbohydrate biosynthetic process, carbohydrate derivative biosynthetic process, and glucose catabolic process (p < 0.05). Alpha-linolenic acid metabolism, biosynthesis of unsaturated fatty acids, and fatty acid degradation pathways were significantly enriched (p < 0.05). This study provides information on the genetic diversity, genetic structure, and population adaptability of the medicinal legumes, G. uralensis. The data obtained in this study provide valuable information for plant development and future optimization of breeding programs for functional genes. Glycyrrhiza uralensis) is a widely consumed medicinal plant. It is a type of leguminous perennial herb that is widely distributed in many arid and semi-arid regions of the world, including China  to geographical differences in habitats, including light factor, temperature factor, and water factor  of plant genomes can detect an array of DNA, such as single nucleotide polymorphisms (SNPs), insertions and deletions, copy number variation (CNV), and presence/absence variation (PAV) , providiG. uralensis species has increased rapidly, G. uralensis plantation number and genome-wide information have hardly progressed. This, in turn, has adversely affected the research efforts related to increasing the concentration of bioactive compounds and productivity of G. uralensis plants through molecular breeding. Thus, unraveling genome-wide interspecific differences and genetic diversity can provide insights into the genetic mechanisms of growth and development of G. uralensis. Besides, it can also help in designing effective conservation and management strategies for G. uralensis  identify the geographical differences in nutrition and photosynthesis, (2) elucidate the geographical differences in genetic structure and genetic diversity of G. uralensis population, and (3) determine the evolution adaptation of G. uralensis population.In this study, leaf samples of G. uralensis. This research site was divided based on the geographical location as central , southern , and eastern  regions , eastern (UW), and southern (YW)] were all measured using a dual-PAM-100 device  between 9: 00 and 12: 00, as described previously by In total, 0.5 cm diameter for 48 h . The resG. uralensis, and the statistically significant differences were performed by SPSS (version 19.0)  followed by Bonferroni’s statistical test for multiple comparisons, and the significance level was set to 0.05.The analysis of variance (ANOVA) was used to analyze the effects of geographical location on the leaf nutrient elements, chlorophyll content, and chlorophyll fluorescence parameters in ™, Thermo Fisher Scientific). Paired-end sequencing with 150 bp read length was performed using an Illumina HiSeq 2500 sequencer based on the manufacturer’s instructions for genome sequencing of G. uralensis on the Illumina HiSeq 2500 platform  with 10× sequencing depth. Library size evaluation (insertion size of 150 bp) with micro tip electrophoresis with Agilent Bioanalyzer 2100 system and accurately quantification effective concentration (3 nmol/L) with q-PCR  are performed before Illumina sequencing to ensure the quality of the library.The genomic DNA from all sixty leaf samples was extracted using a DNA secure Plant Kit , as per the manufacture’s instruction, and subsequently used to construct a sequencing library. Before library construction, NanoDrop2000  was used to detect the concentration and purity of the extracted DNA at A260/A280 nm, and 1% agarose gel electrophoresis was used to determine DNA integrity. The qualified genomic DNA samples were cut into 300 to 500 bp fragments using the Covaris ultrasonic crushing instrument . These fragments were repaired and spliced through PCR amplification to construct the DNA library. Sequencing libraries were constructed with Invitrogen Collibri ES DNA Library Prep Kit   with thee genome 1, and afarameter . In addiarameter  were empmeter  based on the calculation of SNPs differences in individual genomes was used to cluster individuals into different subgroups according to principal components of different traits. In genetics, PCA is mainly used for clustering analysis. In this study, based on the SNP differences between individuals, GCTA software5 was used to calculate the eigenvectors and eigenvalues. R software6 was employed to construct the PCA distribution map. Population genetic structure refers to a non-random distribution of genetic variation within a species or population. A population can be divided into several subgroups according to the criteria of geographical distribution. Individuals within the same subgroup are closely related to each other, but the relationships between different subgroups are slightly distant. The analysis of population structure could unravel the evolutionary process. PLINK software and structure software . LD analysis was performed using VCFtools (version 0.1.12b) (2 between SNPs with pairwise distances larger than 1 kbp.Linkage disequilibrium (LD) refers to the non-random association between alleles at different loci within a population. In LD, the probability of coexistence of two alleles on the same chromosome is greater than the probability of co-occurrence due to random distribution in the population. LD is denoted by its parameter (R0.1.12b) , which eG. uralensis population from different geographical locations was evaluated through the pairwise sequentially markovian coalescent (PSMC) method (Effective population size (Ne) represents a population with the same gene frequency variance as the actual population, reflecting the average homozygous rate of genes in the population genetic structure. Ne in ) method  after deThe fixed coefficient F of the population, which is a special case of the F statistic (Fst), reflects the level of allelic heterozygosity of the population. Fst was calculated using VCFtools software  with a n7, and the number of target genes mapping to each term was calculated. The P-values obtained through hypergeometric test and corrected using Bonferroni several times  were applied to identify significantly enriched GO terms for the candidate target genes  functional analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. In GO analysis, all candidate genes were queried in the GO databaseet genes . The biop-value less than 0.05 after correction)  . The impG. uralensis leaf samples from central (SW), eastern (UW), and southern (YW) regions of Xinjiang  were significantly higher than in the southern (YW) (p < 0.05). In contrast, the PTP levels in G. uralensis leaf samples from the southern (YW) were significantly higher than in central (SW) and eastern (UW) (p < 0.05). There was no significant difference in PTN and PTK levels between the three regions (p > 0.05). In addition, C: N, C: P, and N: P ratios differed significantly in G. uralensis leaf samples from different geographical locations (p < 0.05). Specifically, the ratio of C: N in the G. uralensis leaf samples from the central (SW) was significantly higher than in the eastern (UW) (p < 0.05). However, the C: P and N: P ratios in the southern (YW) were significantly lower than in the central (SW) and eastern (UW) (p < 0.05) , nitrogen (PTN), phosphorus (PTP), potassium (PTK), and other stoichiometric indices in Xinjiang . The POC < 0.05) .G. uralensis leaves samples from the southern (YW), and chlorophyll a (Chla) levels were significantly higher in G. uralensis leaf samples from the central (SW) based on ANOVA outcomes (p < 0.05). The chlorophyll b (Chlb) and total chlorophyll (ChlT) levels in the G. uralensis leaves samples from the southern (YW) were the highest than that in eastern (UW) and central (SW) (p < 0.05). However, carotenoid contents (Car) did not differ significantly between the three regions (p > 0.05) (The highest Chla levels were found in  > 0.05) .G. uralensis chlorophyll fluorescence (p < 0.05)  were significantly higher than in central (SW) and eastern (UW) samples (p < 0.05), but the geographical location did not implicate significant differences in Fv/Fm, Y (II), NPQ, Y (NO),Y (NPQ) indices (p > 0.05)  . Specifi > 0.05) .G. uralensis leaf samples. Genome coverage was 6.27× through sixty individuals. After subjecting these data to strict filtering, 150.06 Gb high- quality clean data was obtained. The sequencing data output statistics for each sample are listed in Total 150.59 Gb was obtained in the whole-genome re-sequencing of sixty Based on BWA software, 895237686 reads from 997491918 valid sequencing reads were aligned against the chromosome level high-precision genome . SupplemG. uralensis sequencing data. A total number of 6985987 SNPs were identified. After filtering, 117970 high-quality SNPs were obtained for subsequent analysis. ANNOVAR software annotation results showed that most SNPs were located in the intergenic region (84.74%), and only 2.75% of SNPs were located in the exonic region. In the exonic region, 1232 synonymous (amino acids in the coding region are not changed) and 1902 non-synonymous (amino acids in the coding region changed) transformations were observed. The ratio of non-synonymous to synonymous transformations was found to be 1.54 , eastern (UW), and southern (YW), reflecting strong genetic differentiation. This might be the outcome of long-term geographical isolation , eastern (UW), and southern (YW). When K = 4 or K = 5, the G. uralensis population could still be clearly divided into three different populations despite the presence of other colors or other genetic structures being evident  to the southern (YW) and central (SW) population. These results suggest that G. uralensis populations from different regions may share a common origin, despite the genetic differences between populations.A phylogenetic tree was constructed based on individual SNPs to evaluate the population stratification and genetic correlation between solation . In addisolation . It indi evident . When K G. uralensis populations in the Xinjiang region, we performed Ho, He, AMOVA, and π analysis. Arlequin software was employed to calculate the genetic diversity index of each population based on the filtered SNP loci. The outcomes showed that the values of Ho (0.492), He (0.324), and π (0.329) were the highest in the central (SW) population, and the values of Ho (0.436), He (0.3), and π (0.304) were the lowest in southern (YW) population  populations was higher than that in southern (YW) populations. Molecular variance analysis demonstrated 8.72% of the significant genetic differences in G. uralensis samples from the population in the three regions and 149.97% of the significant genetic differences within individuals between different populations (p < 0.05)  . It indiG. uralensis population in three regions, revealing the fluctuation of G. uralensis effective population size from 10,000 to 100,000 years ago . Higher R2 represents a stronger bond and a smaller corresponding SNP spacing. When R2 decays to half, the value of the LD decay is represented by the corresponding SNP interval. R2 values were found to be low (0.1–0.2) for central (SW), eastern (UW), and southern (YW) populations  population having the slowest decay rate and the central (SW) population having the fastest decay rate.The LD value is represented in terms of linkage disequilibrium coefficient  was obtained in the population differentiation analysis , indicatG. uralensis population  in the GO functional analysis of G. uralensis populations from three distinct geographical regions. The GO functional categories enriched included 76 biological processes, 32 molecular functions, and 2 cellular components , including carbohydrate metabolic process , carbohydrate biosynthetic process (GO: 0016051), glucose catabolic process (GO: 0006007), carbohydrate derivative biosynthetic process (GO: 1901137), monosaccharide biosynthetic process (GO: 0046364), hexose catabolic process (GO: 0019320), single-organism carbohydrate metabolic process (GO: 0044723), hexose biosynthetic process (GO: 0019319), protein glycosylation (GO: 0006486), glycoprotein biosynthetic process (GO: 0009101), galactosyltransferase activity (GO: 0008378), beta-galactosidase complex (GO: 0009341), gluconeogenesis (GO: 0006094), beta-galactosidase activity (GO: 0004565), galactosidase activity (GO: 0015925), glycolysis (GO: 0006096) (p < 0.05)  identified in the selected regions, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed for their functional enrichment. A total of 690 GO terms  were mapped, among them 110 GO terms were significantly enriched  . In addi < 0.05) .p < 0.05) (p < 0.05). In the central (SW) population, only the spliceosome (ath03040) signaling pathway was significantly enriched (p < 0.05). These results indicate that functional genes related to biochemical metabolic and signal transduction pathways play a crucial role in the population differentiation of G. uralensis from central, southern, and northeastern regions.To unravel the signaling pathways associated with the candidate genes in the three populations, KEGG pathway analysis was performed on the selected candidate genes. A total of 17 KEGG pathways  were enriched in the three populations, of which 7 KEGG pathways  were significantly enriched ( < 0.05) . As showG. uralensis samples were subjected to whole-genome re-sequencing to explore the evolutionary patterns and genomic differentiation in G. uralensis from different geographical locations. After filtering out the low-quality readings, a total of 150.06 Gb of high-quality re-sequenced clean reads and 117970 SNPs were obtained. The mutation sites in gene regions significantly impact the growth and development, stress, and synthesis of bioactive components of G. uralensis plants  and its downstream genes that play a vital role in protection against toxic xenobiotics. In this study, up to 84.7% of SNPs were in the intergenic regions, 1902 SNPs in the non-synonymous regions, and 2858 and 2251 SNPs were determined to be located in the upstream and downstream regions, respectively. Thus, G. uralensis species may have diverse phenotypes and ecological adaptations. One plausible reason might be that G. uralensis population from widely distributed habitats (both temperate grassland and desert areas) with differences in drought levels, light and heat resources and salinity , which witnessed dramatic climatic fluctuations and historic tectonic changes. It led to population differentiation and even the extinction of multiple plant species in northern China . In our K = 3, only one color was present for the eastern (UW) population, but it did not exclude possible hybridization in other regions , human development activities, and animals (birds) could be considered as the primary factors for the gene flow.Furthermore, genetic differentiation among populations is strongly influenced by genetic drift and gene flow . Analysi regions , indicatG. uralensis genetic diversity and observed that the Ho and He for G. uralensis ranged between 0.492–0.436 and 0.324–0.3, respectively  region was slightly higher than that in southern (YW) and eastern (UW) regions  , chlorophyll content  and chlorophyll fluorescence parameters (ETR (I) and ETR (II) of  < 0.05) . This su < 0.05) . This isG. uralensis population in Xinjiang, China. Based on the genomic information, we observed higher Ho, He, and π values of G. uralensis population in the central region than in the southern and eastern populations , glycyrrhizic acid  and a series of key genes, such as the chalcone synthase gene (CHS). Previous studies , whether as part of a molecule or individually, have a variety of functions in cells, from structural “building blocks” of cell membranes to providers of energy and signaling molecules . The stupulation , which iG. uralensis leaves at the physiological level. At the genomic level, the genetic structure of G. uralensis populations showed significant genetic differentiation with clearly divided into Central, Eastern and Southern. As per the outcomes of this study, many GO terms related to carbohydrate metabolic pathways and biosynthesis play an important role in the population differentiation of G. uralensis in central, southern and eastern regions. Further research will be required to characterize the mechanisms by which these GO terms are involved in the population adaptation of plant.In conclusion, this study provided new insights on population genetic diversity, genetic structure, and population adaptation of the medicinal plants. There were significant geographical differences in nutrient concentration and photosynthetic of PRJNA 730103.The data presented in the study are deposited in the National Center for Biotechnology Information (NCBI) repository, accession number XP, HD, and TZ conceived and designed the research project. HD and TZ performed methodology, data analysis, investigated and were major contributor in writing the manuscript, have contributed equally to this work, and share first authorship. YL, GL, and LZ collected leaf samples and modified the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Gypsies are a separate ethnic group living in Pakistan and some other countries as well. They are mostly known as ‘Roma’ and ‘untouchables’. They have different types of lifestyles as compared to other common people, as they always keep migrating from one place to another. They do not have proper houses; they live in tent houses and most probably work on daily wages to earn their living. Gypsies cannot be specified according to the place of residence and can only be classified according to their migration route. Previous historical and linguistic research showed the north Indian origin of Roma people. The present study collected 285 unrelated Roma individuals living in Punjab and typed with the Goldeneye Y20 system. Allelic frequencies ranged between 0.0035 and 0.5266, with haplotype diversity (HD) of 0.9999 and discrimination capacity (DC) of 0.8790. Gene diversity (GD) ranged from 0.6489 (DYS391) to 0.9764 (DYS391) (DY385ab). A total of 223 unique alleles were observed. Interestingly, the haplogroup R accounted for 40.56% and J for 22.06%. In MDS analysis, Pakistani Roma formed a close cluster with Roma from Constanta, Romania. The migration pattern of the Roma population from Pakistan, India and Europe was inferred using coalescence theory in the Migrate-n program. Overlapping Y-STR data were used to test different migration models. These migration models showed us the dominant gene flow from Pakistan to India and Europe to Pakistan. The results of our study showed that Y STRs provided substantially stronger discriminatory power in the Pakistani Roma population. In general, it is believed Gypsies or Roma people began their journey from northern India (modern-day Pakistan) through several migrations, and they were in Persia (modern-day Iran and Turkey) by the 11th century . The RomGenetic studies based on uniparental markers, such as mitochondrial DNA and Y-chromosome, established the south Asian origin of the Roma population. The prevalence of Y-chromosomal haplogroup H1a ,14 and mIn this study, we sampled 285 unrelated Roma individuals across Punjab, Pakistan, intending to investigate the possible origin of the Pakistani Roma population by utilizing the Y-chromosome short tandem repeats (STRs) to (i) shed light on the genetic makeup of the Pakistani Roma population, (ii) their relationship to its close-neighboring population and (iii) whether the European Roma people are descended from the Punjab region of Pakistan.A total of 285 blood samples were collected from different areas of Punjab, Pakistan. All participants who were included in this study were unrelated and self-declared Roma/Gypsy individuals of at least three generations. All participants gave their informed consent either orally and with a thumbprint (in case they could not write) or in writing after the study aims and procedures were carefully explained to them. This study was approved by the University of Health Sciences Lahore Pakistan (2019-CMU-1/04). All the experimental procedures were performed by the standards of the Declaration of Helsinki 1964.Axygen AxyPrep Blood Genomic DNA Miniprep Kit was used to extract genomic DNA according to the manufacturer’s protocol , and the final concentration of DNA was diluted to 1 to 2 ng/μL. DNA was amplified using GoldenEye Y20 system Kit . PCR amplification was carried out using the Applied Biosystems GeneAmp PCR System 9700 thermal cyclers. PCR amplifications were performed as recommended by the manufacturer, although using half of the recommended reaction volume (12.5 μL). After successful PCR amplification, the PCR products were analyzed by using an 8 capillary ABI 3500 DNA Genetic Analyzer with POP-4 polymer  according to the manufacturer’s protocol. GeneMapper I-DXSoftware version 1.4  was used for the genotype assignment. DNA typing was performed according to the manufacturer’s protocol using the locus panel and allele bins supplied by the manufacturer and allele designations corresponding with the allele ladder supplied by the manufacturer. Genotype nomenclature was based on the International Society for Forensic Genetics recommendations. Our laboratory participated and passed the YHRD quality assurance exercise in 2015. Haplotype data were already made accessible via the Y-chromosome Haplotype Reference Database (YHRD) under accession number YA004554.n is the male population size, and ip is the frequency of i-th haplotype. Discrimination capacity (DC) was calculated as the ratio of unique haplotypes in the samples. Match probabilities (MP) were calculated asip is the frequency of the i-th haplotype. We also evaluated these forensic genetics parameters at 9 Y STR loci of minimal haplotype , 11 loci of extended haplotype , Powerplex 12 Y (11 extended haplotype loci + DYS437), Yfiler™  and finally at 20 Y STRs . Genetic distances were evaluated using the Rst, Fst statistic and to perform analysis of molecular variance (AMOVA) tests between reference populations and currently studied populations on overlapping STRs,  were calculated by using Arlequin Software v3.556 [Allelic and haplotype frequencies were computed by direct counting method, and haplotype diversity (HD) was calculated according tohttp://www.nevgen.org (accessed on 10 January 2022)). Any haplotypes that had null alleles or duplication variants in the Roma population were excluded from the analyses.A neighbor-joining phylogenetic tree was constructed for the Roma and the reference populations based on a distance matrix of Fst using the Mega7 software . We alsohttp://www.fluxus-engineering.com (accessed on 10 January 2022). The weighting criteria for Y-STRs were set following Ref. [To define the genetic structure of the Roma population, a median-joining network was constructed. We used the stepwise mutation model and median joining-maximum parsimony algorithm  by usinging Ref. . Any hapWe used MIGRATE program version 4.2.14  by applyA total of 281 out of 285 individuals were successfully genotyped, and their genotypes are summarized in The genetic distances (Rst and Fst) between the Roma population and other relevant populations from Pakistan, India, Afghanistan and Europe are listed in Phylogenetic relationships among the Roma Pakistani population and 80 other regional reference populations were assessed using MDS analysis based on Rst distances derived from the Y-STR data . In thisAMOVA was performed on the Pakistani Roma population and other geographically and historically relevant reference populations. The results of AMOVA showed that the Hungarian Roma population has a more genetic affinity with the Pakistani Roma population. On other hand, Croatian Roma showed major genetic indifferences with the Roma population from Pakistan .Ancestry informative markers (AIMs) play an important role in genealogy. So, we used NEVGEN software to calculate haplogroups from STR haplotypes. Only six haplogroups  accounted for 89% of the total Roma population from Pakistan. The median-joining network of haplotypes  showed tThe frequency of haplogroup E was 5.69% in the currently studied populations, and it was the most frequent haplogroup in west Asia and east Africa ,32. ThisThe frequency of haplogroup I was at 3.91% in the currently studied population, and this haplogroup is predominately found in central European populations. It possibly originated in central Europe around 31–35 KYA . The freHaplogroup J accounted for 20% in the currently studied population. This haplogroup is predominately found in the Arabian Peninsula. The origin of this haplogroup is from the Middle East area known as the Fertile Crescent, comprising Palestine, Jordan, Syria, Lebanon and Iraq around 42.9 KYA . MerchanHaplogroup L accounts for 5% of the currently studied population, and this haplogroup is believed to have originated in the middle east or the subcontinent around 25–30 KYA . The sprHaplogroup R originated in the north of Asia about 27 KYA . It is the most frequent haplogroup in Europe and Russia and, in some parts, it comprises 80% of the population. Some researchers believe that one of its branches originated in the Kurgan culture . The freWe studied Roma population gene flow at two levels. At level one, it was between the subcontinent (Pakistan and India) and at level two, from Pakistan to Europe. Publicly available data from Indian Roma, European Roma and Turkish populations were used to explore the migration route. Indian populations contained a set of west Indians, south Indians, southcentral Indians, northwest Indians, north Indians, northcentral Indians and east Indians, while the European Roma population set contained Bulgarians, Hungarians, Romanians and Slovakians. We investigated the Y-chromosome diversity of Pakistani Roma to gain a better understanding of the Roma population structure in Pakistan and their genetic affinities with relevant local and regional populations. The dominance of the R haplogroup in the Roma population from Pakistan showed the west Eurasian ancestry like other Pakistani populations, in addition to their shared linguistic, cultural affinities, which may have impacted the paternal genetic landscape of Roma people across the world. The MDS and AMOVA results also showed the similarities between Pakistani Roma and European Roma populations. We have also explored the forensic population genetic parameters and found that the Goldeneye 20Y kit showed high discrimination power for Roma population of Pakistan, and this kit can be used for population studies and kinship testing. More comprehensive studies covering the whole genome are required to unfold information within the Pakistani Gypsy gene pool, enabling an extensive reconstruction of the major episodes in their demographic history.


Cingula trifasciata is herewith studied to clarify its population structure in the Northeast Atlantic Ocean and factors shaping it, with a special focus in intra-archipelagic dynamics.In the marine realm, dispersal ability is among the major factors shaping the distribution of species. In the Northeast Atlantic Ocean, the Azores Archipelago is home to a multitude of marine invertebrates which, despite their dispersal limitations, maintain gene flow among distant populations, with complex evolutionary and biogeographic implications. The mechanisms and factors underlying the population dynamics and genetic structure of non-planktotrophic gastropods within the Azores Archipelago and related mainland populations are still poorly understood. The rissoid Cingula trifasciata, supporting previously raised classification issues and detecting potential cryptic diversity. The finding of connectivity between widely separated populations was startling. In unique ways, dispersal ability, habitat type, and small-scale oceanographic currents appear to be the key drivers of C. trifasciata’s population structure in the remote Azores Archipelago. Dispersal as non-planktotrophic larvae is unlikely, but its small-size adults easily engage in rafting. Although the typical habitat of C. trifasciata, with low hydrodynamics, reduces the likelihood of rafting, individuals inhabiting algal mats are more prone to dispersal. Sea-surface circulation might create dispersal pathways for rafts, even between widely separated populations/islands.Coupling microsatellite genotyping by amplicon sequencing (SSR-GBAS) and mitochondrial datasets, our results suggest the differentiation between insular and continental populations of Our results show that gene flow of a marine non-planktotrophic gastropod within a remote archipelago can reveal unanticipated patterns, such that the understanding of life in such areas is far from well-understood. We expect this work to be the starting of the application of SSR-GBAS in other non-model marine invertebrates, providing insights on their population dynamics at distinct geographical scales and on hidden diversity. How transversal is the role played by the complex interaction between functional traits, ecological features, and sea-surface circulation in the population structure of marine invertebrates can be further addressed by expanding this approach to more taxa.The online version contains supplementary material available at 10.1186/s12862-021-01862-1. Species’ ranges are shaped by multiple biotic and abiotic factors; however, in the marine realm, dispersal ability is one of the main factors influencing the distribution of taxa, with significant evolutionary and biogeographic implications , 2. DispCingula trifasciata  is found throughout the Northeast Atlantic Ocean, being reported for the Azores Archipelago, Iberian Peninsula, up to the Bay of Biscay and British Isles ) studies –67, phyl studies , 69, pop studies –74, as w studies –78. Micr studies . This neC. trifasciata, with a special focus on the influence of sea surface circulation. This was done by applying a traditional analysis of the COI molecular marker from several populations and developing a set of primers for microsatellite markers for this non-model species, using the SSR-GBAS approach, a method that relies on amplicon sequencing with second generation sequencing techniques to determine genotypes at microsatellite containing loci. By doing so, we aim to increase the current knowledge of the widespread rissoid C. trifasciata, as well as to clarify the intraspecific genetic structure and population dynamics of this species in the NE Atlantic Ocean, with an emphasis on its behaviour in the remote Azorean islands. With this study, we intend to understand the role of habitat and small-scale surface currents among islands in shaping the genetic structure in a remote archipelago.In this work we investigate the processes shaping the genetic diversity and structure patterns of The COI dataset comprised a total of 75 sequences, with seven to 14 individuals per population. A total of 44 haplotypes were distinguished in the 658 bp alignment, none shared between populations from the Azores and Vigo. Among Azorean haplotypes, the estimates of evolutionary divergence ranged from 0.2 to 2.6%  in the phylogeny. A BLASTN search [C. trifasciata haplotypes from the Azores, but 99–100% identity with a shorter Galician sequence deposited at the database .The distribution of mitochondrial haplotypes Fig.  reflectsN search  of VIG sST levels were low and non-significant, whereas Mosteiros  shows only significant differences with Graciosa and Pico from the central group. The analysis revealed significant and high FST values in the pairwise comparisons with Flores Island. In comparison with the remaining estimates, the pairwise comparisons involving Flores, Santa Maria, and Caloura retrieved higher FST levels, suggesting higher isolation of these populations.Pairwise F-statistics between populations were estimated for the COI dataset  and 56,441 (CAL5184). After filtering the matrix and exclude the samples with more than 60% missing data across all markers, the dataset was reduced to seven individuals from Santa Maria, nine from Caloura and two from Mosteiros (São Miguel), six from São Jorge, 11 from Pico, five from Graciosa, one from Flores, and three from Vigo . As only one sample was successful in SSR-GBAS genotyping, the population from Flores island was excluded from further analyses. Although they had been successfully amplified in the single PCR test, the following markers were excluded due to failure in the multiplex step or Illumina MiSeq, resulting in data unfit for further analysis or missing data: CT6_TTGT and CT17_TTTG were excluded due to non-specific amplification; CT1_TGTT, CT3_GAATA, and CT10_GTTT were excluded for yielding over 60% missing data. Thus, a total of 26 markers and 43 samples were suitable for population genetic analyses of Ho varying from 0.235 to 0.921 and He from 0.448 to 0.956. Within each of the five populations in study, with five or more individuals  and 0.620 (Santa Maria), and He between 0.498 (São Jorge) and 0.623 (Santa Maria). Private alleles were detected in all populations.In the WAI (Whole Amplicon Information) dataset, the markers analysed had between 6 and 41 alleles . Errors in the assignment of genotypes causing deviations to HWE were discarded after checking the markers plots generated after the Allele Length calling script. The deviations were either caused by the dominance of null alleles  inferred from FreeNA and checked in the original matrix, excess of homozygotes indicated by FIS (CT33_CATT in Pico), or both (CT23_TTTA in Pico). A total of six out of 26 markers deviated from HWE in the population from São Jorge  due to excess of homozygotes suggested by the FIS levels, which led to higher frequencies of null alleles despite the absence of real null alleles. In Vigo population (data not shown), three loci failed to amplify  resulting in null alleles across all the samples of this population; the negative values of FIS in the several loci suggest an excess of heterozygotes in this population.Tests for Hardy–Weinberg Equilibrium , Mosteiros vs. Graciosa (0.119), and Graciosa vs. Pico (0.122), in congruence with the previous results of PCoA and STRUCTURE, suggesting gene flow between these populations.The AMOVA analysis Table , performCingula trifasciata in the NE Atlantic Ocean, with a special focus in the Azores Archipelago. The results indicate a divergence between insular and continental populations of this microgastropod and the existence of genetic structure within the Azores Archipelago on a small spatial scale. This indicates dispersal barriers for the non-planktotrophic species that could form the starting point of population differentiation. To our knowledge, this is the first time that the SSR-GBAS protocol generates a numerous set of de novo SSR primers for a marine microgastropod. Comparing to previous studies in other marine invertebrate taxa, a higher number of loci is herewith amplified contributing to an informative dataset even with a low number of individuals. Thus, this methodology has the potential to be applied in similar study systems, increasing the number of markers successfully developed for marine taxa. These topics will be further discussed in the next sections.With this work we address biogeography and the processes shaping the genetic structure and evolutionary dynamics of C. trifasciata were developed and characterized and genotyped using sequence information of Illumina MiSeq according to the SSR-GBAS methodology described earlier [In this study, a total of 26 SSR loci of  earlier –93. Seco earlier , 94–97.  earlier , 97. The earlier , 96–98.  earlier , 94, 97. earlier , 96, 98. earlier . Relying earlier –93 and sIn congruence with the previous studies using SSR-GBAS, a set of highly informative markers was obtained for the non-model, marine microgastropod, with moderate to high PIC values according to the classification scheme . Only loC. trifasciata possesses np-larvae with low dispersal ability, which contributes in one hand to the high genetic structure detected but also to homozygote excess in some populations. Nevertheless, this should not affect the biogeographical interpretations regarding the Azorean populations, as their prevalence in other gastropod species has been shown to not influence the population structure inferred . Hi. HiC. tred . A complete separation of the Azorean and Vigo networks at 95% connection limit  occurs between Alvania mediolittoralis (Gofas 1989) and A. formicarum (Gofas 1989), two recognized species occurring in the Azores Archipelago with distinctive morphological characters. The inclusion of these Alvania species in a calibrated molecular phylogeny of Rissoidae has shown them to have diverged quite recently, at about 0.36 million years (Ma), within a range uncertainty of 0.12–0.61 Ma [Cingula trifasciata, causing a split between the evolutionary lineages from the Azores and Vigo. The differentiation between insular and mainland (Vigo) populations is supported by the COI and WAI datasets, and evident both in clustering analyses .C. trifasciata have previously been reported [C. trifasciata to the European taxon raised two decades ago are now supported by molecular data of mitochondrial and SSR markers. These results reinforce the importance to couple morphological, ecological, and molecular data to have a broader perspective of biodiversity, especially in such an understudied environment as the marine realm. Following the results of this study and life-traits of C. trifasciata, we propose that the insular and mainland populations are currently experiencing a split, constituting distinct evolutionary lineages. As C. trifasciata is the only known representative of the genus in the NE Atlantic Ocean, further studies including more populations across the established distribution range of the species in the NE Atlantic Ocean, as well as from the putative populations in the Mediterranean Sea, might clarify questions regarding its current taxonomic classification.Morphological differences between Azorean and Iberian individuals of reported , in easiC. trifasciata within the Azores Archipelago, suggesting low dispersal among islands. For Graciosa, Flores, and Mosteiros the discordant number of individuals analysed for both datasets can be explained by older samples or poorer preservation, causing their degradation and easier amplification of the COI. The main findings regarding the relationships among Azorean populations of C. trifasciata are summarized in a geographical map of the archipelago  [C. trifasciata gathers the common characteristics of rafters [Dispersal ability, habitat type, and oceanic circulation patterns within the archipelago, appear to be the main drivers of population structure in the Azores. The finding of strong genetic structure of astropod , 124. The stages , 32, 33.e stages , 125, as rafters , 127, naC. trifasciata and other microgastropod fauna are easily found in protected sites, under boulders in enclosed tide pools with low hydrodynamics and mesotidal regimes [e.g. Cerco da Caloura, Poça da Barra, and Fajã da Caldeira do Santo Cristo [C. trifasciata has been reported in similar habitats outside of the Azores , located 215 to 225 km far, respectively. The sea-surface circulation regimes nearby the westernmost tip of São Miguel Island, where Mosteiros is located, might favour the frequent dispersal of individuals to or from the Central Group. Within the central islands, gene flow seems to be frequent and results point to recent haplotype differentiation. The distance separating the islands sampled in the central group  is probably small enough to not constitute a barrier for the occasional migration and demographic expansion, supported by the low mitochondrial differentiation in levels comparable to other gastropods [In the Azores, o Cristo , Mosteir Ireland ). This r Ireland  and Prai Ireland . The infala spp. . In our ala spp. , 6. The ala spp. , 30, 34.stropods , 106.C. trifasciata and other np marine invertebrates in the Azores Archipelago.The success of rafting episodes and maintenance of connectivity are influenced by the complex sea-surface circulation patterns and hydrological conditions around the Azores Archipelago –131. AltC. trifasciata from the Azores and Vigo suggest the existence of potential cryptic diversity within the genus. Based on genetic data and life-history traits, these constitute distinct evolutionary lineages. Deep insights into spatial genetic structure of a microgastropod in the isolated Azores Archipelago were also reached with this study, providing crucial knowledge to better understand gene flow and connectivity among islands and considerably isolated populations. Several aspects seem to work in concert to shape the genetic structure of C. trifasciata in the Azores. Its np nature hinders dispersal during larval stage, but occasional exchange of individuals must occur through processes of rafting or by extreme weather events. Our results, especially the SSR dataset, reveal a preliminary analysis of these patterns in the remote islands of the Azores. A most comprehensive interpretation can only be achieved with further studies in related research areas, namely oceanography, but also on expanding molecular approaches to other marine invertebrates. Without a thorough understanding of fine-scale circulation patterns within the Azores, unexpected connections, as the one detected between Mosteiros and Graciosa, become difficult to justify. The application of the SSR-GBAS approach to other marine invertebrates might be the key to generate comparative datasets and to determine how widespread are the patterns inferred for C. trifasciata. How transversal is the complex interaction between the larval development type, ecological traits related to the habitat occupied, and the sea-surface circulation patterns in the population dynamics and genetic structure of marine invertebrates can be further addressed by expanding the taxa studied. Understanding differentiation levels and patterns of diversity at the regional scale might be a useful proxy for other intertidal invertebrate species, and to implement conservation and management strategies of the shore habitats in a way that connectivity in the remote Azores Archipelago will be preserved.Combining mitochondrial and SSR datasets provide the opportunity to detect hidden patterns of cryptic diversity and to get a clearer perspective of gene flow among populations. Differentiation between C. trifasciata in the NE Atlantic Ocean, with a special focus on its behaviour and factors shaping the genetic structure in the remote Azores Archipelago.With this work we aim to investigate the processes shaping the intraspecific genetic diversity and population dynamics of Cingula trifasciata specimens from the Azores Archipelago  and Vigo (Spain) were used in this study, either recently collected by the authors in intertidal habitats or retrieved from collections . Permits for sampling were issued by the respective authorities in the Azores . Samples from two localities in Graciosa island and from a NE Atlantic population in Vigo were loaned, respectively, from the DBUA and CIBIO-InBIO molluscs’ collections. A total of 75 samples were considered in this study.ns Table . Fresh sDue to its minute size, total genomic DNA (gDNA) was extracted from the entire animal, removed from the shell when possible, following the manufacturer’s instructions for the column-based commercial kit PureLink® Genomic DNA (Invitrogen™). gDNA was eluted in a volume of 40 μl and its quality assessed based on the absorbance ratios measured with Nanodrop®2000. An electrophoretic run in agarose gel 0.8% at 100 V for 50 min was performed to evaluate DNA integrity.The fragment of the mitochondrial cytochrome oxidase subunit I (COI) were amplified in 25 μl volumes, containing 12.5 μl of QIAGEN Multiplex PCR Master Mix , 5 µL of each primer at a concentration of 2 μM, and 2.5 µL of gDNA. The amplification of COI was achieved with the primers LCO1490/HCO1490  or jgLCOC. trifasciata from the Azores, publicly available at GenBank [C. trifasciata were calculated in MEGA v7 [C. trifasciata haplotypes [C. trifasciata from the Azores and Vigo. Pairwise FST estimates with COI haplotypes of C. trifasciata and Rissoidae species were performed in Arlequin v3.5 [Geneious 8.1.9  was used GenBank , were in GenBank  and the  MEGA v7  to estim MEGA v7 . The out MEGA v7 , allowinplotypes  was obtaplotypes , and booplotypes , under tplotypes  , were conducted for marker development and raw reads can be accessed through the BioProject PRJNA702169, at GenBank [Two low-coverage Illumina MiSeq runs, from two  GenBank . Library GenBank , were pr GenBank  to trim  GenBank . Merged  GenBank . For theC. trifasciata. PCR reactions were conducted for a final volume of 10 µL: 5 µL of QIAGEN Multiplex PCR Master Mix , 1 µL of each primer at 1 µM, 1 µl of diluted gDNA in a 1:3 proportion, and water. The cycling profile was applied as follows: 95 °C for 15 min; 30 cycles of 95 °C for 30 s, 55 °C for 1 min, and 72 °C for 1 min; and a final extension at 72 °C for 10 min. The PCR results were visualized after an electrophoretic run in 1.5% agarose gel and 80 V. Primers that successfully generated amplicons of the expected size were combined in three mixes of eight primer pairs and one mix of seven primer pairs, each with a final concentration of 1 µM , 0.5 µL of each primer mix, and 2 µL of gDNA diluted in 1:3 proportion. Cycling conditions were the same as used in the single PCR to test the primers individually. For each sample, and following Curto et al.’s (2019) protocol, equal volumes of PCR products from different primers mixes were pooled into a final volume of 6 µL. PCR clean-up, aiming the removal of unused primers and primer-dimer products, was conducted using the magnetic bead technology offered by the Agencourt AMPure XP PCR Purification kit , applying some modifications to the standard protocol. The total volume of pooled PCR product (6 µL) was mixed with 4.3 µL of AMPure XP beads, followed by a 5 min incubation period at room temperature. The beads, to which DNA bound after the first step, were captured by an inverted magnetic bead extraction device, VP407‐AM‐N , and afterwards washed twice for 45 s in 200 µL of 80% ethanol. The beads were then dried at room temperature for 5 min and the DNA finally eluted in 17 µL of elution buffer  at 65 °C.Once purified, the multiplex PCR product underwent a second PCR, whereby designated as index-PCR, for the assignment of indexes to each sample. A unique combination of forward and reverse indexes was carefully chosen, so that each sample can be unambiguously identified after the MiSeq Run. A 10 µL PCR reaction was performed, comprising 5 µL of QIAGEN Multiplex PCR Master Mix , 1 µL of each index primer at 1 µM, and 1 µL of pooled purified PCR product. The cycling conditions were applied as follows: 95 °C for 15 min; 10 cycles of 95 °C for 30 s, 58 °C for 60 s, and 72 °C for 60 s; 72 °C for 5 min. The index-PCR product, from 5’ to 3’, entails: P5 motif for flow cell hybridization, index 1 with a length of 8 bp, P5 sequencing primer, specific forward primer, target DNA for sequence, specific reverse primer, P7 sequencing primer, 8 bp long index 2, P7 motif for flow cell hybridization. After visualizing the index-PCR products in 1.5% agarose gel stained with GelRed (Biotium), all the samples were pooled in equal volumes of 2 µL. The Illumina MiSeq run for PE 300 bp sequencing at the Genomics Service Unit at Ludwig Maximillian Universität, München, Germany, using the pooled index-PCR product as input, produced sequences to be analysed with a genotyping by amplicon sequencing (GBAS) protocol.Raw FASTQ sequence data (reads R1 and R2), automatically extracted by the MiSeq equipment based on the index combinations and each corresponding to a different sample, were downloaded from Illumina BaseSpace. These Illumina reads, containing all sequences per index, underwent the quality control and merging procedures resorting to FastQC, Trimmomatic v0.39, Usearch v11 (cf. “SSR discovery” section). Genotyping and analysis of each sample for all the SSR loci was performed with the SSR_GBS_pipeline scripts available at GitHub , 143. ScCingula trifasciata. The two populations of Graciosa island were analysed together, as no major differences were found between them. Genetic diversity indices, null alleles and deviations to Hardy–Weinberg Equilibium (HWE) in the WAI dataset were estimated only for populations with five or more individuals analysed – Santa Maria, Caloura, Graciosa, Pico, and São Jorge. FreeNA [Ho) and expected (He) heterozygosity. Although excluded from the inference of genetic diversity, monomorphic markers were kept in the dataset for downstream analyses, as they are informative [IS) were performed by marker and population resorting to GenAlEx. The remaining analyses based on the WAI dataset included information of all individuals studied, belonging to the seven populations sampled. The software Cervus v3.0.7 [ST values.Descriptive population genetics and marker variability analyses, as well as evaluation of genetic structure patterns, were performed on the codominant WAI dataset of . FreeNA  was used. FreeNA , 88 alloormative . Tests fs v3.0.7  was useds v3.0.7 , 88, ands v3.0.7 , 90 withs v3.0.7 . The onls v3.0.7  was useds v3.0.7 . A hieras v3.0.7 ), as imps v3.0.7 , 88, wasAdditional file 1: Table S1. Complete list of all SSR primers designed in this study. Includes sequence of forward and reverse primers (5’-3’), the repetition motif, and number of repeats in the original sequence used for the primer design, the primer mix in which it was included for the Multiplex PCR, and allele length range (bp). Table S2. Estimates of evolutionary divergence (raw p-distances) between COI haplotypes of Cingula trifasciata. The analysis, performed on MEGA v7 [Table S3. Pairwise FST values between populations of Cingula trifasciata, based on the COI dataset. Table S4—Pairwise FST values between populations of Cingula trifasciata, based on the complete WAI dataset. Table S5 – Estimates of evolutionary divergence (raw p-distances) between COI sequences of several Rissoidae species. The analysis, performed on MEGA v7 [Alvania formicarum and A. mediolittoralis, and between Cingula trifasciata from the Azores and Vigo. Figure S1. Genetics diversity of the SSR dataset in study. a) Variability measures estimated for each of the 26 SSR loci in study in complete whole amplicon information (WAI) dataset: number of alleles (Na) and polymorphic information content (PIC); b) Genetic diversity patterns across the five populations of Cingula trifasciata with more than five individuals: number of alleles (Na), number of different alleles with frequency over 5%, number of effective alleles (Ne), number of private alleles (Npa), expected (He) and observed (Ho) heterozygosity. Fig. S2. Genetic structure analysis of the complete WAI dataset of Cingula trifasciata. Inferred with STRUCTURE v2.3.4 [ primers ’-3’, the


Chondrosia reniformis, descript only morphologically along Tunisian coasts, was chosen to estimate the influence of natural oceanographic and biogeographic barriers on its genetic differentiation and its Phylogeography. The cytochrome oxidase subunit I (COI) gene was amplified and analyzed for 70 Mediterranean Chondrosia reniformis, collected from eight localities in Tunisia. Polymorphism results revealed high values of haplotype diversity (Hd) and very low nucleotide diversity (π). Thus, these results suggest that our sponge populations of C. reniformis may have undergone a bottleneck followed by rapid demographic expansion. This suggestion is strongly confirmed by the results of neutrality tests and “mismatch distribution.” The important number of haplotypes between localities and the high genetic differentiation (Fst ranged from 0.590 to 0.788) of the current C. reniformis populations could be maintained by the limited gene flow mN (0.10–0.18). Both haplotype Network and the biogeographic analysis showed a structured distribution according to the geographic origin. C. reniformis populations are subdivided into two major clades: Western and Eastern Mediterranean. This pattern seems to be associated with the well‐known discontinuous biogeographic area: the Siculo‐Tunisian Strait, which separates two water bodies circulating with different hydrological, physical, and chemical characteristics. The short dispersal of pelagic larvae of C. reniformis and the marine bio‐geographic barrier created high differentiation among populations. Additionally, it is noteworthy to mention that the “Mahres/Kerkennah” group diverged from Eastern groups in a single sub‐clade. This result was expected, the region Mahres/Kerkennah, presented a particular marine environment.Despite the strategic localization of Tunisia in the Mediterranean Sea, no phylogeographic study on sponges has been investigated along its shores. The demosponge  C. reniformis populations are subdivided into two major clades: Western and Eastern Mediterranean. This pattern seems to be associated with the well‐known discontinuous biogeographic area: the Siculo‐Tunisian Strait, which separates two water bodies circulating with different hydrological, physical, and chemical characteristics.Both haplotype Network and the biogeographic analysis showed a structured distribution according to the geographic origin.  These invertebrates are found in all oceans and at all depths , which are embedded in a complex matrix called mesohyl  or asexually. Sexual reproduction in sponges is varied, it can be gonochoric, sequential, or simultaneous hermaphrodites, it also can be through viviparity or oviparity , is a thick encrusting, smooth, and cushion‐shaped sponge. Under the effect of light intensity, its body colors vary to white, brown, black, and sometimes orange. This species was considered to have a worldwide distribution, including the Atlantic, Pacific, and Indian Oceans and Mediterranean Sea  in order to position the genus Chondrosia in the phylogenetic tree of demosponges  were collected, between January and September 2020, from eight sampling locations along the Tunisian coasts  gene was amplified using a pair of universal primers  .Hd; Nei, π; Nei, The level of DNA polymorphism, the haplotype diversity  analyses, in MrBayes v. 3.2.2  was used as outgroup.To infer the relationships of Chondrosia reniformis populations. Two supplementary AMOVA tests were carried out: for the first analysis, we tested the genetic variation between the four groups according to geographic proximity: Beja/Tabarka, Monastir/Sousse, Mahdia/Chebba and Mahres/Kerkennah. The second analysis was performed to evaluate a comparison between the group Mahres/Kerkennah and the other localities. In addition, the genetic differentiation for both eastern and western Mediterranean localities in Tunisia was tested too. All AMOVA analyses were calculated with 10,000 permutations under null distributions.The analysis of molecular variance (AMOVA)   and very low nucleotide diversity (π). The percentage of GC is from 40% to 42%, and the AT percentage is from 58% to 60%  of the genetic variation was detected between the four studied groups (Beja/Tabarka vs. Monastir/Sousse vs. Mahdia/Chebba vs. Mahres/Kerkennah). AMOVA results for the western Mediterranean and the eastern Mediterranean localities showed that more than 44% of the variation was between these two groups  were detected, indicating a remarkable differentiation among tested groups.The entire pairwise comparisons of groups based on nt Table . The FS4Chondrosia reniformis. Polymorphism results revealed very low nucleotide diversity (π). These results were congruent with previous studies, which reported low sequence variation for mtDNA in several sponge species: Crambe crambe, Astrosclera willeyana, Chondrilla nucula, Suberites diversicolor, Ianthella basta, and Xestospongia spp   gene was amplified and analyzed for 70 Mediterranean C. reniformis harbors high haplotype diversity (Hd) throughout Tunisia coasts (Hd = 0.939). Compared with the previous data, this value was higher than that detected in other marine sponges  agree with the second category proposed by these authors . Thus, we can consider that our sponge populations of C. reniformis may have undergone a bottleneck followed by rapid demographic expansion as mentioned by these authors for this category. This suggestion is strongly confirmed by the results of neutrality tests and “mismatch distribution.” However, the lower nucleotide diversity recorded could be derived from the synergy between the small sample size per population and the low polymorphism of mitochondrial region analyzed.Diversity indices nd Bowen , the valFST ranged from 0.590 to 0.788) of the current C. reniformis populations, could be maintained by the limited gene flow. In this sense, very low genetic values of mN (0.10–0.18) were detected, indicating a remarkable genetic structuring of the tested groups. It is well known that gene flow in marine invertebrates is usually expected to be related to larval dispersal capacity or marine bio‐geographic barrier. Larval dispersal capacity strongly affects the geographic distribution and genetic differentiation of habitats . Indeed, contrary to what is expected, these authors found a fairly high gene flow (mN = 1.27) between populations  to Mazara del Vallo , has been inferred to be an oceanographic and biogeographic barrier between the two major Mediterranean sub‐basins (the western and eastern) ; Data curation ; Formal analysis ; Methodology ; Writing – original draft ; Writing – review & editing . Choulak Sarra: Formal analysis (supporting); Methodology (supporting); Software (supporting); Supervision ; Writing – original draft . Rhouma‐Chatti Soumaya: Supervision ; Validation ; Visualization . Chatti Noureddine: Supervision ; Validation . Said Khaled: Methodology (lead); Supervision (lead); Validation (lead); Visualization (lead); Writing – review & editing (lead).