Insect vectors of plant diseases and insecticide resistance pose the greatest challenge to sustainable crop production in food security. In this study, we explored genetic mechanisms responsible for resistance to a common insecticide group, neonicotinoids, in a key insect vector of potato diseases—potato psyllids. These small insects with piercing-sucking mouthparts are common in potato and can transmit a bacterium that causes zebra chip disease. Zebra chip has had a devastating impact on potato producers and has contributed to a highly regimented and intense use of insecticides to suppress potato psyllids as soon as they are detected in a field, commonly using neonicotinoids. Widespread resistance to these insecticides is now evident in potato psyllid populations. Using susceptible and resistant psyllid populations, we sequenced portions of their genomes to elucidate genes involved in the evolution of resistance to neonicotinoid insecticides. We found several genes that are likely to be responsible for insecticide resistance, and these should be explored in further research. We also discovered that a method commonly used to separate potato psyllids into distinct groups based on their geographic origin does not adequately represent their genetic population structure and should be used in conjunction with other genetic techniques.Bactericera cockerelli (Hemiptera: Triozidae), a vector of Candidatus Liberibacter solanacearum, which causes zebra chip disease in potato. (2) Methods: Here, we use double-digest restriction site-associated DNA (ddRAD) to genotype eight psyllid populations (one susceptible and seven resistant to neonicotinoid insecticides). (3) Results: Association tests identified over 400 loci that were strongly segregated between susceptible and resistant populations. Several loci were located within genes involved in insecticide resistance, gene regulation, fertility, and development. Moreover, we explored the genetic structure of these eight populations and discovered that routinely utilized haplotyping was not an accurate predictor of population structure. Pairwise comparisons of the fixation index (FST) of populations of the same haplotype were not different from pairwise FST of populations that belonged to different haplotypes. (4) Conclusions: Our findings suggest that neonicotinoid insecticide resistance has a genetic basis, most likely as a result of similar selection pressure. Furthermore, our results imply that using a single maternally inherited gene marker to designate genetic lineages for potato psyllids should be re-evaluated.(1) Background: Many hemipteran insects transmit plant pathogens that cause devastating crop diseases, while pest management frequently relies primarily on insecticide applications. These intense insecticide applications lead to the development of insecticide resistance, as was the case for potato psyllid,  Many insects in the order Hemiptera, such as aphids, psyllids, leafhoppers, and whiteflies, are highly injurious to crops due to their wide host range and rapid reproduction. Their injury is more severe when these insects transmit plant pathogens—bacteria, viruses, phytoplasmas, and fungi ,2. ManagBactericera cockerelli (Šulc) (Hemiptera: Triozidae), is a vector of pathogens that are casual agents of several diseases, including “psyllid yellows” (PY) [Candidatus Liberibacter solanacearum (Lso) [Bactericera cockerelli is thought to be native to Central and North America and its current distribution ranges from southern Canada to Central America, New Zealand, and Australia [The potato psyllid, ws” (PY) , “zebra ws” (PY) , and “vews” (PY) . Among tum (Lso) ,19, and ustralia ,21. Fourustralia ,23. Notaustralia . Owing to the lack of effective control measures for any of the pathogens transmitted by the potato psyllid, management is focused on the suppression of the psyllids and relies solely on pesticides ,26. The While the incidence and severity of the potato psyllid resistance to neonicotinoid insecticides have been documented in several separate studies, the mechanisms of this resistance are not known. Generally, hypotheses about molecular mechanisms of insecticide resistance are centered around targeted site mutations, which result in target insensitivity and increased expression of detoxification genes ,33,34,35Here, we used genome-wide single nucleotide polymorphisms (SNPs) to investigate the genetic basis of resistance to neonicotinoid insecticides in resistant and susceptible potato psyllid populations collected across the potato-growing regions of the Southwestern US. We were interested in elucidating the genetic structure of the potato psyllid population and exploring the hypothesis that the selective pressure of the neonicotinoid insecticide on the potato psyllid population could result in heritable SNPs. In addition, we attempted to explore the genetic structure of the psyllid populations that are designated as Central and Western haplotypes. Given that the rapid evolution of insecticide resistance is a major challenge for sustainable agriculture, understanding the genetic resistance mechanisms is important for DNA-based resistance monitoring in field populations.Solanum lycopersicum L., variety Lance) in 60 × 60 × 60 cm insect-proof mesh cages  in the greenhouse with mean temperatures of 21 ± 3 °C (night) to 30 ± 3 °C (day) and 16:8 L:D photoperiod.Psyllid colonies were established from adult psyllids collected during 2013, 2015, and 2016, except for the Texas (TX) LRGV 2006 colony, which was originally collected in 2006 from the Lower Rio Grande Valley of Texas and was previously shown to be susceptible to neonicotinoids ,28. PotaSix biological samples (replicates) consisting of five adult psyllids pooled together were used from each of the eight populations . DNA wasp-value cutoff 0.05 for keeping an FST measurement. To obtain a putatively neutral SNP data set for genetic structure analysis, an “unlinked” data set was generated with a single SNP per locus (the first SNP per locus) to reduce the effects of linkage. We then used BayeScan v2.01 [We took a de novo approach using Stacks v2.41  because an v2.01  and usedp-value cutoff was set to 1 × 10−14.To identify loci that were strongly segregated between the insecticide susceptible (TX LRGV) and resistant populations (the remaining seven populations), we performed association analysis with the entire data set containing 12,681 variant sites using PLINK  (v1.9), −4.Gene annotations were obtained for all specific RAD loci/contigs that contained significant SNPs in the association test using the Blast2GO annotation methodology embedded in OmicsBox . Sequences were queried against the Hemiptera database under “insects” (Taxid:6960), using OmicsBox with an E-value threshold of 1 × 10We deployed three complementary approaches to model population genetic structure without a priori population assignment. First, we conducted principal component analysis (PCA) implemented in the PLINK, using neutral SNPs marker set. Secondly, we used Structure software (v 2.3.4 ) to explInsecticide resistance is a growing challenge in potato production, and our research provides a foundation for understanding the genetics of insecticide resistance in potato psyllid. In this study, the potato psyllid population that was susceptible to imidacloprid and thiamethoxam  allowed B. cockerelli, a de novo assembly was constructed using Stacks. The total ddRAD data set included 456,481 unique loci. We retained a total of 11,471 loci and 12,681 SNPs after filtering loci that did not pass sample/population constraints, and all these loci passed the HWE test . Further filtering was also used to select only one SNP at each locus (the first SNP per locus) that generated 4294 SNPs.We generated 288 million pair-end reads with an average of 5.9 M reads per sample. As no reference genome was available for −4  gene, which was reported to be one of the two key loci involved in imidacloprid resistance in populations of Drosophila melanogaster based on a large-scale genome-wide association study (GWAS) [prm encodes a protein that constitutes the structure of muscle filament, and mutation of the prm would impact the indirect flight muscle and power generation in D. melanogaster [Cephus cinctus inositol 1,4,5-trisphosphate receptor (IP3R), a family of the Ca2+ release channel protein involved in increasing the cytoplasmic free calcium concentration. Guo et al. [ip3r in a silverleaf whitefly, Bemisia tabaci, decreased the susceptibility of adult B. tabaci to cyantraniliprole, a diamide pesticide that is widely used to manage sucking insect pests. The last locus within this group, CL_51462, is homologous to the non-lysosomal glucosylceramidase-like gene. Although the function of glucosylceramidase in insects is not well characterized, it was also reported to be differentially expressed in the abdomen of insecticide-resistant strains of mosquito, Anopheles gambiae, a vector of malaria [Within insecticide resistance loci, we found the locus CL_8229 to be homologous of the Paramoysin (y (GWAS) . The prmnogaster . In addio et al.  reported malaria .Diaphorina citri  CD109 antigen, which is induced by ecdysone signaling and involved in the cellular immunity of insects [Bt-resistant and Bt-susceptible bollworm, Helicoverpa zea, several immunity genes were found to be differentially expressed between resistant and susceptible populations, including two CD109 antigen genes [Moreover, several loci that differed between the susceptible and resistant populations were related to the innate immunity of insects. For example, CL_2391 is homologous to spaetzle 4-like in peptides ,52. Anot insects . It is n insects ,55,56. FWolbachia homolog) that presumably originated from the microbiome of potato psyllids  insecticide targeted site gene mutations, which result in target insensitivity ,13,33,34Halotydeus destructor, a major pest of pastures and crops in Australia. These candidate loci were located in the genomic regions that code for transmembrane transport and signaling proteins, with a few other genes related to pyrethroid resistance. In another study in which whole genome sequencing was conducted on multiple field-collected fruit fly, D. melanogaster, two major loci appeared to be related to imidacloprid resistance [prm gene, and another locus was 60 k upstream of the coding region of the Nicotinic-Acetylcholine Receptor Alpha 3 gene. These studies illustrate the distinct genetic mechanisms of insecticide resistance in different arthropod species.Beyond these few well-known targets, different insect species might have evolved distinct genes and molecular mechanisms of insecticide resistance , and inssistance . One locprm gene. However, we did not identity SNPs that are on contigs that are homologous to previously reported genes involved in insecticide resistance, such as P450, sodium channel gene, gst, or coe. We speculate that this may be due to the fact that in ddRAD library preparation, only DNA fragments flanking the restriction enzyme cutting sites are included in the DNA library, resulting in just a portion of the genome being sequenced. Furthermore, the lack of a reference genome makes annotating these short de novo assembled tags/loci challenging. We anticipate that the annotation of SNPs will improve with the availability of the reference genome. In the future, examining the transcriptomics will be informative to test whether these SNPs impact the gene expression level of detoxification-related gene products.In our study, we found a strongly segregated SNP located on contigs that is homologous to the We have found multiple variants that are strongly segregated between insecticide susceptible and resistant populations; these variants and related genes could provide a candidate list for further testing. Insecticide resistance is recognized as a complex, polygenic, and quantitative trait, according to growing knowledge and studies on the mechanism and genetic basis of insecticide resistance ,38. Thus−4 to 2.5 × 10−4, and the highest was observed in the TX Weslaco population. To examine variance in allele frequencies among populations and their degree of differentiation, we also measured the fixation index (FST) using the data set containing 4294 independent SNPs. The mean FST for all pairwise comparisons was 0.44. The TX Panhandle was, on average, the most differentiated from all other populations (mean FST = 0.52), and Western-2 was the least differentiated population (mean FST = 0.31)  for all loci were estimated . The TX  = 0.31) . SurprisFST results, we did not find that populations from the same haplotypes clustered together, e.g., Western-1 and Western-2 did not cluster together, and TX Weslaco was separated from the other Central haplotype populations.Further, the principal component 1 (PC1), which explained over 30% of the overall variation , separatMoreover, Structure analysis indicated the most likely number of genetic clusters to be four (K = 4), based on the agreement between the ΔK method and estimators of the structure selector. At K = 2, Western-1 and TX Weslaco were placed in one cluster , and theFST, suggesting that haplotype does not appear to be a good indication of genetic structure.To further examine the genetic structure of psyllid populations, we used the 4294 neutral SNPs data set to conduct a phylogenetic analysis based on Nei’s distance. Similar to results in PCA and Structure, TX Weslaco and Western-1 were found to be closely related. The New Mexico and TX LRGV 2006 populations clustered together, and three TX Panhandle, Colorado, and TX Pearsall populations were found to be closely related . All WesFST between the two is moderate  was found to be somewhat related to the population of New Mexico; however, the moderate . We founpsyllids ,67. Specpsyllids , which ipsyllids . We sugg


Ipheion uniflorum is a bulbous geophyte known as Spring Starflower whose cultivation dates back to the first half of the 19th century. At least seven cultivars have been developed from natural stands. However, comparative analyses of wild and cultivated materials are largely missing. In the present study, we provide a morphological evaluation and analyses of the cytological and genetic variability of I. uniflorum that reveal significant levels of differentiation and evidence of artificial selection in the Spring Starflower. Distinctive phenotypic characters in cultivated materials that are rarely found or lacking in wild plants and natural populations, such as pink or violet flowers, together with its reduced heterozygosity and starting genetic differentiation support the view of early mechanisms of domestication acting upon Spring Starflower plants. The probable geographic origin of the cultivated forms is discussed together with perspectives for plant breeding.The cultivation and domestication of plants are human-driven processes that change the biology and attributes of a plant.  Domestication is the result of an evolutionary process in which wild species are exposed to new selective environments related to human cultivation and use, partly resulting in the increased fitness of the domesticated species . Thus, dIpheion uniflorum (Graham) Raf. , ornamental forms and cultivars have been developed that resulted in the acquisition of new phenotypic traits and are widely known as Spring Starflower.There are approximately 2000 semi- and fully domesticated plant species from 140 taxonomic families ,4. Most Ipheion uniflorum is a South American bulbous geophyte endemic to the Pampas in Argentina (Buenos Aires), Uruguay and South Brazil  and kept only specimens bearing geographical coordinates  [A total of 56 wild individuals of e et al. , plus 5 detected , we stru and UY) . The map and UY)  and ggpl and UY) .I. uniflorum ‘Rolf Fiedler’ (SSF2). A third individual was collected in a garden in San Isidro, Buenos Aires, Argentina (SSF3). The last two (SSF4 and SSF5) specimens were cultivated at the Botanical Garden and Botanical Museum in Berlin, Germany. All cultivated samples were compared against all 56 wild individuals from 10 collection sites  distributed across the natural range of the species , which were established from bulbs imported from England. One of these plants was identified as  species . Both thhttps://www.inaturalist.org/, accessed on 27 December 2021), 60 records from wild materials and ca. 1340 from cultivated materials . The collected data are summarized in Morphological exploration focused on identifying traits likely selected in cultivated plants and of potential interest for horticulturalists. Flower color, shape of the tepal apices and the size of the flower were studied in individuals from the natural populations. We also collected extra information about the analyzed traits from an exhaustive review of the literature ,11,12,16v/v) and stored in 70% ethanol at 4 °C. Root tips were then hydrolysed for 10 min at 60 °C in a solution of 1 m HCl, and stained for 20 min or more using Schiff’s reagents . Root meristems were macerated in a drop of 2% acetic orcein and analyzed using a Leica DM5500B microscope . Chromosome measurements were performed using three to six selected mitotic metaphases with the Leica Application Suite software . Chromosomes were classified as metacentric (r = 1.00–1.49), submetacentric (r = 1.50–2.99), acrocentric (r ≥ 3.00) or telocentric (r = ∞), where r = length of the long arm/length of the short arm.Fresh root tips were collected and treated for 6 hr at room temperature with a solution of 0.05% colchicine, fixed for 12–48 hr in ethanol/acetic acid  and by measures of genome-wide heterozygosity . Brieflyhttp://www.ebi.ac.uk/ena/data/view/PRJEB43919, accessed on 29 December 2021).DNA extraction, library preparation and sequencing were carried out as described in Sassone et al. . GBS locHO, HE, HT, FST, and FIS) were calculated to describe and compare overall and population-specific genetic diversity using the R package ‘hierfstat’ [FST) for each of the subpopulations, according to Weir and Cockerham [A discriminant analysis of principal components  was carerfstat’ , and filerfstat’ . We alsoockerham .Ipheion uniflorum plants generally have white flowers  and lilac or blueish flowers  suggested that the best grouping number following ΔK was K = 3 , and thiThe DAPC was performed with four a priori groupings  and reveals high levels of admixture with Northern populations    . Regardi, AS260) . The oth, AS260) , the DAP, AS260) a.I. uniflorum distribution   b.HO = 0.071, HE = 0.196, HT = 0.284, FST = 0.213 and FIS = 0.636 for the species, including cultivated specimens (p < 0.001). For the Spring Starflower subgroup alone, the statistics give values of HO = 0.051, HE = 0.090, FIS = 0.340. The estimates of pairwise fixation indices between regional and cultivated groups revealed the strongest differentiation for cultivated specimens with southern populations , floodplains  or at rural roadsides .I. tweedieanum (Griseb.) Traub and I. recurvifolium (C.H.Wright) Traub, at the intraspecific level, no chromosomal structural changes between the studied I. uniflorum samples and minor differences in cytological and karyological parameters were found, particularly among individuals from the South and Centre of the distribution  lower than expected (HE = 0.178), with overall levels of genetic diversity HT = 0.267. The fixation indices and inbreeding coefficients showed values of FST = 0.334 and FIS = 0.527, respectively, with the strongest differentiation decreasing from south towards north [I. uniflorum in morphological, karyological, and genetic attributes being geographically structured with a gradual latitudinal differentiation trend.The observed within (and among) population morphological variability e.g.,  is more ribution . In the ds north . OverallAs mentioned above, the domestication of ornamental plants was initiated more recently than other intensively cultivated crops and is largely dependent on aesthetically driven human selection ,8. This I. uniflorum have a recent origin dating back to the first half of the 19th century. Despite the relatively recent cultivation start, I. uniflorum’s cultivars have acquired new phenotypic traits that differentiate to a certain extent the cultivated materials from the wild ones. The most striking one relates to flower color and the shape of tepal apices. The cultivar I. uniflorum ‘Rolf Fiedler’ displays a deep periwinkle blue flower color in combination with rounded tepals apices that only bear a resemblance to individuals from two Uruguayan populations (but see comments in the next section). Similarly, the distinctive pink flower color of the cultivar ‘Jessie’, or the pink-to-violet flower color of the recently released cultivar ‘Tessa’ have not been observed among natural populations.Ornamental forms and cultivars of I. uniflorum grows naturally are among the richest worldwide [While in nature, plant sizes range from 6–20 cm height, individuals in cultivation often reach up to 25–40 cm. Phenotypic variation for quantitative genetic traits, such as plant height, is continuously distributed in natural populations and often responds slowly to artificial selection ,42. In oorldwide . So, if I. uniflorum populations show best-fit population models at K = 2 and 3, when adding the cultivated specimens, Evanno’s ΔK shifts the best-fit population model to K = 3, enhancing the differentiation between North and Centre populations  exhibit a considerable decline (HO = 0.071), but the expected heterozygosity (HE = 0.178) and the overall levels of genetic diversity (HT = 0.267) show a slight upsurge . Likewise, the relative fixation indices are reduced from FST = 0.334 [FST = 0.213, and the inbreeding coefficient changes from FIS = 0.527 [FIS = 0.636. On the one hand, the data suggest a slight reduction in the genetic diversity of the dataset after including the cultivated samples, likely due to inbreeding. On the other hand, the gain in overall genetic diversity and decay in fixation indices indicate some level of genetic differentiation, which agrees with the observed segregation of two Spring Starflower specimens from the natural I. uniflorum populations mentioned above.Genetic diversity parameters indicate that, by including five cultivated specimens, the values of heterozygosity observed among natural populations .Our genomic analysis provides little resolution on the original populations from which Spring Starflower cultivars used in the present study were first harvested. Yet, at least two cultivated individuals (SSF1 and SSF3) can be assigned to natural populations of a geographic group. One showing a genomic composition grouping with populations in the Centre and Northern regions (SSF1) and the other one with populations in the Southern region (SSF3). Most importantly, the proposed Uruguayan origin of the Fiedler’  is corroI. uniflorum individuals. The occurrence of natural variation of floral color and the natural changes in flower pigmentation during flower development, maturation and climatic conditions makes it difficult to obtain standardized measurements of flower colors, which would help scientists to manage biodiversity stands. Therefore, without a numerical classification of flower colors obtained, for example, by a colorimeter , the cup < 0.001) show low levels of heterozygosity (HO = 0.051) compared to the expected value (HE = 0.090), and relatively high inbreeding (FIS = 0.340). However, these parameters might show bias due to the small sampling size. The pairwise fixation indices showed the strongest differentiation of cultivated specimens to populations in the South (FST = 0.485), and the lowest genetic differentiation with populations in the North (FST = 0.139). This pattern parallels the genetic differentiation trend observed among I. uniflorum natural populations [I. uniflorum might well work as reservoirs of morphological and genetic variability for the further development of Spring Starflower cultivars. The continued development of new cultivars  depict both the interest of horticulturalists and the appreciation of the public . In an era of accelerated human-induced species losses, many natural populations are today threatened and may become extinct in the next few years if no conservation measures are taken. The adoption of measures to protect and conserve the remaining populations of I. uniflorum throughout the distribution of the species (perhaps considering geographic regions as separate genetic reservoirs) is of great importance for future breeding plans and the release of new cultivars.From a plant breeding perspective, the summary statistics of Spring Starflower specimens (ulations . Thus, w


Thuja sutchuenensis, a species that survive only in the Daba mountain chain, using chloroplast simple sequence repeats (cpSSR) and nuclear restriction site-associated DNA sequencing (nRAD-seq). Wild T. sutchuenensis populations were from a common ancestral population at 203 ka, indicating they reached the Daba mountain chain before the start of population contraction at the Last Interglacial . T. sutchuenensis populations showed relatively high chloroplast but low nuclear genetic diversity. The genetic differentiation of nRAD-seq in any pairwise comparisons were low, while the cpSSR genetic differentiation values varied with pairwise comparisons of populations. High gene flow and low genetic differentiation resulted in a weak isolation-by-distance effect. The genetic diversity and differentiation of T. sutchuenensis explained its survival in the Daba mountain chain, while its narrow ecological niche from the relatively isolated and unique environment in the “refugia” limited its distribution.Narrow-ranged species face challenges from natural disasters and human activities, and to address why species distributes only in a limited region is of great significance. Here we investigated the genetic diversity, gene flow, and genetic differentiation in six wild and three cultivated populations of  In the context of biodiversity loss worldwide, it is critical to conserve endangered species that are generally narrow ranged . The plaThe population demographic history is generally influenced by tectonic events and climatic change that, therefore, are key factors influencing patterns of genetic diversity . These tThuja sutchuenensis Franch, an endangered species endemic to the Daba Mountains in northwestern Chongqing Municipality and eastern Sichuan Province, China  and nuclear restriction-site associated DNA sequence (nRAD-seq). Previous studies showed inconsistent results for T. sutchuenensis populations from different methods, high genetic diversity with ISSR  and Platycladus orientalis (ZYB), as two outgroups, were sampled from the Changbai Mountains and the Institute of Botany, CAS, respectively.T. sutchuenensis was extracted from 25 mg of dry leaves using a Hi-DNA Secure Plant Kit  based on a modified version of the SDS method (http://pgrc.ipk-gatersleben.de/misa/). The motifs of trinucleotide, tetranucleotide, pentanucleotide, and hexanucleotide repeats included at least 5, 4, 4, and 4 repeats, respectively. SSR primers were designed using Primer 3.0 (https://sourceforge.net/projects/primer3/). The SSR primers were designed as follows: 18–24 bp in length, 40%–60% GC content, 55°C–65°C annealing temperature, and 100–300 bp PCR product. Furthermore, 400 SSR loci were selected and primers of 140 loci were screened in 20 individuals. Finally, 30 microsatellite repeats (SSR) were selected from the 140 loci to detect the polymorphic site of T. sutchuenensis populations . Finally, the amplicons were size selected once more into 350–500 bp fragments with the method mentioned above. The resulting ddRAD library was sent to Guangzhou Jierui Biotechnology Company, LTD.  and sequenced on the Illumina Novaseq platform using 150 nt with paired-end mode.A total of 100 ng genomic DNA per sample was digested with two restriction enzymes, EcoRI, and PstI  at 37°C for 8 h. The restriction enzymes were then inactivated by heating at 65°C for 20 min. After ligation individually with barcoded EcoRI adapter and universal PstI adapter with T4 DNA ligase for each sample at 16°C for 8 h, the reaction was stopped by heating at 65°C for 20 min. The ligation products of all samples were equally pooled and size selected into 300–500 bp fragments using the agarose gel electrophoresis. After manipulating gel purification, derived fragments were used as templates (about 30 ng) for PCR amplification process_radtags module in the Stacks-2.4  and M = 12 as the maximum distance allowed between stacks within an individual. We then used the cstacks module to build the catalogs for all samples with n = 12 as the maximum number of mismatches allowed between individuals. The Sstacks module generated alignment results for each individual against the catalog using default parameters. In the populations module, we set p = 8 and r = 0.6 to call consensus SNP among all 137 samples .We used acks-2.4  to demulp > 0.05) (Na), effective number of alleles (Ne), observed heterozygosity (Ho), expected heterozygosity (He), and inbreeding coefficient (FIS), were calculated for all samples from nine populations by GenAlEx version 6.503  . Geneticon 6.503 .FIS), nucleotide diversity (Pi), observed heterozygosity (Ho), and expected heterozygosity (He), were calculated using the populations module in Stacks  was calculated by analysis of molecular variance (AMOVA)  was estimated based on the formula FST values of nRAD-seq data were calculated with the populations module and tested based on 1,000 permutations with Arlequin v3.5  . The sigulations . FST vauin v3.5 . AMOVA wuin v3.5 , and theuin v3.5 . PCoA anuin v3.5 .FST/(1-FST) and logarithmic geographic distance . Departures from mutation–drift equilibrium were detected by a mode-shift test  distribution of FST in the absence of selection (FST distribution to 5% and 10% using the minimum expected heterozygosity of 0.10 and an error discovery rate of less than 0.05. The second approach is using latent factor mixed model (LFMM) (http://worldclim.org)  , to idenl (LFMM) . Extractlim.org) .After all quality filters, 62,375,634 reads with no rad tag and 1,000,938 low-quality reads were discarded. We retained a total of 3,509,088,912 reads from the initial 3,609,108,036 raw reads, with an average of 2.63 Mb reads per sample. After trimming and clustering, we obtained loci number ranging at 6,933–395,804 bp and mean locus coverage across all samples was 72.64X from 32.69 to 135.05X, with an average length of 135 bp per loci and with 44.92% GC content. After the “cstacks” module processing, we obtained 4,886,198 catalogs and 6,119 SNPs.Out of the 5,990 polymorphic SNPs loci, 2 (14063_15 and 3630303_6) and 1 (3630303_6) SNPs were considered as responsible for local adaptation by the OutFLANK and LFMM approaches, respectively. Because there is no existing reference genome for Cupressaceae, it is impossible to annotate these selected loci.T. sutchuenensis populations showed high chloroplast genetic diversity (cpSSR) and low nuclear genetic diversity (nRAD-seq). The Ho and He of cpSSR in T. sutchuenensis populations were 0.43–0.92 and 0.23–0.53, respectively  (pi) of T. sutchuenensis populations ranged from 0.082 to 0.092 for variant position and from 0.0053 to 0.0062 for all positions  . The nucositions .Ho and He of cpSSR in cultivated populations BQR and GMR were comparable with wild populations BQ and QS  (FST) in pairwise comparisons of BQ-QS and MZ-MY-XY was low, with high gene flow (Nm = 7.13–10.62)  and high gene flow (Nm = 5.56–22.48) (FST and low Nm from other populations (FST = 0.033–0.055) with high gene flow (Nm = 4.26–8.32)  . The gen3–10.62) . The cul6–22.48) . The cululations . However26–8.32) . This in similar , which w similar .2 = 0.029, p = 0.08)  , indicatThe structure analysis of cpSSR showed two clusters (best delta K = 2) , with onFastsimcoal results from nRAD-seq showed that the wild populations Pop 1 (BQ and XY) and Pop 3 (MY and GM) were from an ancestral population at ∼203 ka . An admiTreeMix analysis of cpSSR revealed historic migrations between QS, BQ, and GM populations , while nT. sutchuenensis using chloroplast simple sequence repeats (cpSSR) and nuclear restriction site-associated DNA sequencing (nRAD-seq) and found relatively high chloroplast genetic diversity but low nuclear genetic diversity, which provides molecular explanation in its distribution in the Daba mountains as a “refugia”.Assessing the genetic diversity and genetic structure of rare, endemic, and endangered species, i.e., narrow-ranged species, is not only the basis for developing scientific and rational conservation strategies , but alsT. sutchuenensis was widely distributed in China in the late Pliocene based on its fossil in the Shanxi province  than other narrow-ranged species such as the Camellia huana  (Pinus kesiya (He = 0.654 for cpSSR) (Ambrosia trifida (He = 0.203–0.645 for cpSSR)  and T. occidentalis (SSR He = 0.574–0.624)  on genetic diversity. An alternative explanation is that chloroplast genomic inheritance is conservative and maintain genetic material, and chloroplast genome is mainly paternally inherited in Cupressaceae  , or wider cpSSR)  and Ambrr cpSSR) . T. sutc4–0.624) . This reessaceae .pi = 0.082–0.091 for variant positions, pi = 0.0053–0.0062 for all positions) of T. sutchuenensis in this study is relatively low compared with other narrow-ranged species, such as Tetraena mongolica  based on nRAD-seq  (T. sutchuenensis in our study was higher than six single-copy nuclear loci (pi = 0.00219)  within populations than regional or widespread species using STMS (sequence-tagged microsatellite sites)-based data, while there is no difference between them using RAPD (random amplified polymorphic DNA)-based data  . Nuclear0.00219) . Qin et 0.00219) . Thus, msed data . In addised data .T. sutchuenensis populations (FST < 0.2), with high gene flow. This might result from the low population size of T. sutchuenensis . It is consistent with other narrow-ranged species, for example, the Cupressaceae species Platycladus orientalis (FST = 0.024–0.386) (Camellia huana (FST = 0.2159 for cpSSR) (Rhododendron cyanocarpum (FST = 0.0314–0.0452) (Tetraena mongolica (FST = 0.010–0.026) , and genetic and geographic distance indicating a weak effect of isolation by distance.Generally, low genetic diversity result from high genetic differentiation and low gene flow, but the genetic differentiation was low between 4–0.386) , endanger cpSSR) , threate–0.0452) , and Tet0–0.026) . The gen0–0.026) . Due to 0–0.026) . T. sutcIn fact, population structure results based on nRAD-seq of three cultivated and six wild populations failed to reveal any meaningful cluster patterns, which consisted with a previous study . HoweverT. sutchuenensis should collect plant material from populations with high genetic diversity such as the QS and BQ populations. In these populations, plant material from different age and size categories should be collected wherever possible in order to sample representative genetic diversity . The relatively high chloroplast and low nuclear genetic diversity of T. sutchuenensis populations might explain why it can and only survive in the Daba mountains as a “refuge.” After long-term (∼203 ka) adaptation, T. sutchuenensis prefers to grow on limestone cliffs, crest ridges, or steep slopes with special climate, soil (pH = 7.53), and sunshine (T. sutchuenensis and further to a low nuclear genetic diversity. These restrict its distribution only in the Daba mountains, although the seeds of T. sutchuenensis are wind-disseminated with membranous wings that facilitate population spread. Thus, enforcing the reintroduction of plant material from populations with high genetic diversity facilitates the conservation of T. sutchuenensis.In conclusion, we found the wild populations of sunshine , which l


Saccharina latissima) persisted in the Gulf of Alaska during ice‐age maxima when the western margin of the Cordilleran ice sheet covered coastal areas around the NE Pacific Ocean. We estimated genetic diversities within and phylogeographical relationships among 14 populations along 2,800 km in the NE Pacific and Bering Sea with partial sequences of mitochondrial DNA 5′‐cytochrome oxidase subunit I , chloroplast DNA ribulose‐1,5‐bisphosphate carboxylase large subunit‐3′ , and 11 microsatellite loci. Concatenated sequences of rbcL and COI showed moderate levels of within‐population genetic diversity (mean h = 0.200) but substantial differences among populations . Microsatellites showed moderate levels of heterozygosity within populations (mean HE = 0.391). Kelps in the same organellar lineage tended to cluster together, regardless of geographic origins, as indicated in a principal coordinate analysis (PCoA) of microsatellite genotypes. The PCoA also showed evidence of nuclear hybridizations between co‐occurring organellar lineages. Individual admixture plots with population clusters of K = 2, 6, and 9 showed increasing complexity with considerable historical admixture between some clusters. A time‐calibrated phylogeny placed divergences between rbcL‐COI lineages at 1.4 million years at most. The time frames of mutation in the rbcL‐COI lineages and microsatellite population clusters differed among locations. The existence of ancient lineages in the Gulf of Alaska, moderate levels of genetic diversity, and the absence of departures from neutrality are consistent with northern refugia during multiple Croll‐Milankovitch climate cycles in the Pleistocene Epoch.Many Northeast (NE) Pacific fishes and invertebrates survived Pleistocene glaciations in northern refugia, but the extent that kelps survived in northern areas is uncertain. Here, we test the hypothesis that populations of sugar kelp ( COI and rbcL sequences and 11 microsatellite loci were assayed in over 500 plants from 14 localities to test the hypothesis that populations survived Pleistocene ice ages in northern refugia. The substantial genetic diversity and chaotic distribution of genetic markers among populations indicated the likely survival in multiple northern refugia.Organellar  Several) Figure . Neverth) Figure . Abundann chromosome complement) produce large numbers of meiospores, which settle after a brief planktonic phase and sprout into haploid, filamentous gametophytes with an n chromosomal complement. Male gametophytes produce spermatozoa that fertilize oogonia on female gametophytes, producing zygotes that grow in place into large‐bladed sporophytic kelps. Even though spores can potentially be transported in coastal currents for several days  and chloroplast (cp) DNAs were used to reconstruct gene genealogies and test for genetic population structure. We also used microsatellite DNA markers to estimate recent processes influencing genetic variability  and sugar kelp Saccharina latissima  . Young individuals of H. nigripes and S. latissima generally have bullated blades and similar morphologies. However, the blades of older H. nigripes are leathery and slippery to the touch, whereas those of S. latissima are thinner and lack mucilaginous glands  and protected (S. latissima) microhabitats. A similar association between wave exposure and the occurrences of H. nigripes (as Saccharina nigripes) and S. latissima was found in the Bay of Fundy, in the NW Atlantic Ocean . Standard extraction kit protocols were followed, except dried tissues were homogenized at room temperature by crushing or chopping on weighing paper with a scalpel.A 4‐cm2.2COI‐5′ was amplified by PCR with forward GazF2 (5′CCAACCAYAAAGATATWGGTAC3′)   was amplified with the forward rbcL‐543F (5′CCWAAATTAGGTCTTTCWGGWAAAAA3′) and reverse rbcL‐1381R (5′ATATCTTTCCATARRTCTAAWGC3′) primers  were used to amplify DNA with an initial denaturation at 94°C for 3 min and 35 cycles of 94°C for 45 s, 50°C (COI), or 52°C (rbcL), for 1 min , 72°C for 1.5 min, and a final step at 72°C for 5 min.A segment of mtDNA COI and 735 bp of rbcL were used for population analyses. Quality control consisted of re‐extracting and re‐sequencing unique haplotypes from each 96‐well plate.Amplified DNAs were routinely sequenced in forward and reverse directions by GENEWIZ Inc. , or by the University of Arizona Genetics Core . Forward and reverse complement sequences were aligned and edited with MEGA 7.0.20 , 0.20 mM of each nucleotide , 0.10–0.25 µM of forward and reverse primers, 0.1 mg/ml of BSA , 0.05 U GoTaq Flexi DNA polymerase , and deionized water. Thermal cycling profiles varied with locus , nucleotide diversity, θπ (standard deviation), and number of private haplotypes in a sample, NPH, for COI and rbcL sequences. Departures from neutrality were tested with Tajima's D ] for organellar DNA or [FST/(1 − FST)] for microsatellites, and approximate shoreline distances between samples. Tests were made with and without log transformations of geographic distances and with 1,000 randomizations.We used  Tajima, . SequencrbcL‐COI haplotypes with BEAST 1.8.4  and expected (HE) heterozygosity averaged over loci, to count the number of alleles at each locus, and to estimate the inbreeding coefficient, FIS. HP‐RARE  of the data under the model. Web‐based STRUCTURE HARVESTER  loci are unlinked, (2) loci are at linkage equilibrium within population clusters, and (3) genotypes in population clusters are in Hardy–Weinberg proportions. To begin, we tested for linkage between loci overall and in the sampled populations with GENEPOP. We found that 2 locus pairs were significantly linked over all Table  and 17 o3rbcL‐COI sequences to make our major inferences about population structure.We present the results for each of the organellar markers separately to facilitate comparisons with other studies, but use the results for concatenated 3.1COI defined 11 haplotypes, but only three haplotypes were abundant. A central abundant haplotype (MT040306 in 73.1% of kelps) was connected to 9 peripheral haplotypes by one mutation  ranged from 0.0 in 8 samples to 0.145 in sample 7 and was 0.429 ± 0.024 overall (Table π) ranged from 0.0 in several samples to 0.029% in sample 9 and was 0.079% ± 0.076% in the pooled sample. Tajima's D was marginally significant at three locations and in the pooled sample . Overall, the number of observed haplotypes (NH) was 11, when only 4.39 were expected under neutrality (NEH). A total of 10 private haplotypes appeared at six locations.Haplotype diversity (ll Table . NucleotST) between populations ranged from 0.0 between pairs fixed with the same haplotype to 0.968 between populations fixed, or nearly fixed, with different haplotypes  ranged from 0.0 in 6 samples to 0.515 ± 0.027 in sample 3 and was 0.702 ± 0.013 in the pooled sample (Table π) ranged from 0.0 in 6 samples to 0.070% ± 0.067% and was 0.104% ± 0.085 in the pooled sample. Tajima's D was not significant in any of the samples, nor in the pooled sample .The overall number of le Table . NucleotST) between populations ranged from 0.0 between populations with the same haplotype to 1.0 between populations with different haplotypes (Table ST = 0.788 (p < 0.00005) among samples. AMOVA indicated that 78.8% of the total variation was due to differences among populations and 21.2% was due to differences among kelps within populations  ranged from 0.0% to 0.048% and averaged 0.016%. Tajima's D was significant (p < 0.05) in populations 2, 7, and 9, but was not significant overall (p = 0.203).Sample sizes of the concatenated sequences were slightly smaller than those of the two genes individually, because both genes were not successfully sequenced in some kelps. A total of 13 polymorphic sites defined 16 haplotypes in 447 sequences from 14 locations Figure . The numST ranged from 0.0 between populations fixed with the same haplotype to 0.971 between populations 1 and 13  to 0.096 (heterozygote deficit) per sample, except for one population for which it was 0.217, and averaged 0.038.The number of alleles among 11 loci ranged from 5 to 17 and averaged 11.3 per locus Table . AllelicFST between populations varied from 0.009 between locations Homer Spit (5) and Humpy Creek (6), both in Kachemak Bay, to 0.675 between distantly separated Nateen Bay (1) and Cordova (9) (Table p < 0.001), except between populations 5 and 6 in Kachemak Bay (p = 0.054). Overall, FST = 0.239 (p < 0.0001) among populations. AMOVA indicated that 23.9% of the variability was due to allele frequency differences among populations, on average, and 76.1% was due to differences among individuals within populations (Table FST) and geographic distance (km) was r = 0.231 (p < 0.080)  was r =0 .401 (p = 0.005)  Figure  and betw) Figure . Back asmer Spit  and HumprbcL‐COI lineages in colors corresponding to those in Figure rbcL‐COI lineage, but others included more than one lineage. Location 3 included 2 closely related haplotypes in lineage D, and widespread locations 4, 5, 6, 7, 9, and 14 contained haplotypes of lineage C. Locations 9 and 11 contained low frequencies of lineage A.A PCoA of microsatellite allele‐frequency variability among individuals resolved several population clusters. Separations defined by axes 1 and 2 accounted for only 25.4% of the variability in 13 samples Figure . Axis 3 C), 2 (B), and 11 (A) were distinguishable from other locations by both microsatellites (FST: 0.179–0.675) and rbcL‐COI DNA (ΦST: 0.356–0.994). Kelps at locations 5 and 6 (both B‐C) in Kachemak Bay were indistinguishable from each other with both rbcL‐COI (ΦST = 0.0) and microsatellites (FST = 0.0). Locations 3 and 9 shared haplotypes in the same lineage (D) but were distinguishable by both rbcL‐COI (ΦST = 0.356) and microsatellites (FST = 0.404). Locations 10, 12, and 13 were fixed with the same rbcL‐COI haplotype (A) (ΦST = 0.0) but differed significantly from each other in microsatellite allelic frequencies (FST: 0.326–0.470).Kelps at locations 1  among 10 replicate runs for test values of K did not plateau to indicate a likely value of K  and far eastern  areas of the Gulf of Alaska  was similar to the mean values for 2 populations in British Columbia  to 0.096 (heterozygote deficit) per sample, except for one population for which it was 0.217, and averaged 0.038. These relatively small inbreeding coefficients and the general lack of departures from Hardy–Weinberg proportions indicate that null alleles did not consistently occur at high frequencies at any particular locus. Furthermore, a component of the FIS values may have been due to inbreeding  and population groups, based on microsatellite allele‐frequency variability, also appeared on both sides of the North Atlantic , which included a C‐lineage haplotype among D lineage kelps that did not show convergence in the PCoA plot. This likely reflects a recent migration of C lineage kelps into this population. In general, we would expect to find strong heterozygote deficits (Wahlund's effect) at microsatellite loci at locations where rbcL‐COI lineages co‐occurred if the divergent lineages were reproductively isolated, but this was not the case . Large expanses of glaciers around the Gulf of Alaska and along the coast of British Columbia were fed by precipitation from moist air masses moving across the North Pacific Ocean from the west COHMAP, . ImportaDespite coastal glaciers, environmental reconstructions indicate that oceanic conditions remained conducive to the growth of kelps around the Gulf of Alaska and along the coast of British Columbia. Ocean temperatures in the Gulf of Alaska were relatively warm, dropping 5–6°C from present temperatures during glacial maxima  from kelps located between 48.36° and 49.84°N all consisted of the most abundant haplotype in the Gulf of Alaska (MT040306). These samples originated from coastal areas that were potentially covered by the margins of the Cordilleran ice sheet, and hence do not provide a rigorous test of the diversity–gradient hypothesis. In the absence of samples from southern latitudes, we argue from substantial levels of organellar and nuclear diversity and from emerging insights into the disjunct distributions of tidewater glaciers around the Gulf of Alaska that sugar kelp populations survived in northern refugia during glacial maxima.A robust test of the diversity gradient hypothesis with microsatellites or organellar DNA is not possible because of the absence of samples from unglaciated areas of coastal Washington, Oregon, and California. However, five rbcL‐COI haplotypes separated by only 1–5 mutations, these markers detected considerable diversity among populations  that cannot be interpreted as a postglacial expansion from a southern refuge. Eight of 14 populations were fixed, or nearly fixed, with different rbcL‐COI haplotypes. This population heterogeneity is inconsistent with dispersals from a southern refuge, but reflects isolations in several northern refugia.Several genetic signatures of sugar kelp populations in the Gulf of Alaska are consistent with northern ice‐age refugia. First, although both mtDNA and cpDNA genealogies across the Gulf of Alaska are shallow with the combined Second, the relative timings of glacial retreats along NE Pacific would have influenced patterns of genetic diversity among present‐day populations of sugar kelp. The growth and decline of coastal glaciers were out of phase between northern and southern shores. In Alaska, glaciers reached maximal extents twice between 23 and 17 ka, and most coastal areas were ice‐free by 16 ka. However, at the southern edge of the Cordilleran ice sheet, glaciers did not retreat from southwestern British Columbia and Puget Sound until 14–13 ka , two barnacles , a sea star (Patria miniata), and an intertidal blenny (Xiphister atropurpureus) were consistent with stable populations that had not been displaced to southern refugia during glaciations  shows high diversities in northern populations, also indicating persistence during glacial maxima in northern refugia    using random amplified polymorphic DNA revealed a phylogeographic break between populations between SE Alaska and south‐central and western Alaska that likely indicates secondary contact between populations isolated in northern glacial refugia ; data curation (lead); formal analysis (lead); funding acquisition (lead); methodology (supporting); resources (lead); writing‐original draft (lead); writing‐review & editing (lead). Erica Chenoweth: Conceptualization (supporting); data curation (supporting); methodology (lead); writing‐review & editing (supporting).Samples were collected under Alaska Department of Fish and Game authorization.Appendix S1Click here for additional data file.


Recent studies have showed the diverse genetic architecture of the highly consanguineous populations inhabiting the Arabian Peninsula. Consanguinity coupled with heterogeneity is complex and makes it difficult to understand the bases of population-specific genetic diseases in the region. Therefore, comprehensive genetic characterization of the populations at the finest scale is warranted. Here, we revisit the genetic structure of the Kuwait population by analyzing genome-wide single nucleotide polymorphisms data from 583 Kuwaiti individuals sorted into three subgroups. We envisage a diverse demographic genetic history among the three subgroups based on drift and allelic sharing with modern and ancient individuals. Furthermore, our comprehensive haplotype-based analyses disclose a high genetic heterogeneity among the Kuwaiti populations. We infer the major sources of ancestry within the newly defined groups; one with an obvious predominance of sub-Saharan/Western Africa mostly comprising Kuwait-B individuals, and other with West Eurasia including Kuwait-P and Kuwait-S individuals. Overall, our results recapitulate the historical population movements and reaffirm the genetic imprints of the legacy of continental trading in the region. Such deciphering of fine-scale population structure and their regional genetic heterogeneity would provide clues to the uncharted areas of disease-gene discovery and related associations in populations inhabiting the Arabian Peninsula. The Arabian Peninsula (AP) is a melting pot of human diversity and culture. Its geographical location, as a land bridge connecting Africa and Eurasia, served as a gateway for human migration from the African continent to the rest of the world . RecentlKuwait is located in the northeastern corner of the AP and is bordered by Saudi Arabia to the south and Iraq to the north. Several archeological explorations reflect Kuwait as a vital location which has been used by ancient civilizations as a military or trade station , 16. TheThe detailed materials and methods are presented in the We included DNA samples of 620 Kuwaiti individuals from the State of Kuwait who were part of a larger cohort collected for studies of metabolic disorders , 33, 34.The 620 individuals were genotyped using HumanOmniExpress arrays for 730,525 SNPs  in accordance with the manufacturers' protocol. Quality control checks and data filtering were conducted using the PLINK v1.9 . After Qhttps://evolbio.ut.ee/). The combined dataset was filtered using PLINK v1.9 [r2 value of >0.4 using a sliding window of 200 SNPs, shifted at intervals of 25 SNPs. The pruned dataset yielded 155,744 SNPs that were used for the relevant population genetics analyses, including Wright’s F-statistic (FST), principal component analysis (PCA), the ADMIXTURE for ancestry estimation, and runs of homozygosity (RoH).We merged our data with the published genome-wide SNP genotype data of global populations  implemented in the ADMIXTOOLS package [f-statistics analyses of modern individuals. We merged the dataset from the modern and ancient human genomes, which contained a combined total of 231,418 SNPs and 3697 individuals. We computed the derived allele sharing of the Kuwaiti population using outgroup f3 to measure the genetic similarity of different Kuwaiti groups. Admixture f3 was calculated to infer the plausible admixing sources in the history of the Kuwaiti population. We calculated the f4 statistic to evaluate the level of gene flow between contemporary populations of Kuwaitis and their regional neighbors and allele sharing between modern Kuwaitis and available published data of ancient individuals from surrounding regions. Further, we computed f4-ratio [We computed the  package . A datasWe used the phased dataset with 244,688 SNPs and 2139 individuals for haplotype-based analyses. ChromoPainter was used to “paint” each individual as a combination of all other sequences . We exechttps://www.r-project.org/), as described elsewhere [Starting from the copying vectors obtained with the ChromoPainter, we reconstructed the ancestry profile of each cluster or individual by applying a slight modification of the NNLS function of R v3.5.1  from Arabians, Middle Easterners, Caucasians, South Asians, and Europeans, and the Kuwait-B subgroup with Arabians, and the Kuwait-S subgroup with Arabians and Middle Easterners. The highest degree of differentiation (FST > 0.05) was observed between the Kuwait-P/Kuwait-S subgroups and Africans/East Asians. The Kuwait-B subgroup was genetically distant from East Asians, but not Africans  when one of the ancestral sources was either Bedouin or Saudi, while the second source was a different African population  in the Kuwaiti subgroups . More specifically, the mean inferred time for the Kuwait-B subgroup was 13 generations ago (5–95%: 2–27 generations ago), and similar values were observed for the Kuwait-P and Kuwait-S subgroups   Fig. . To gainThe results of the classical allele frequency-based analyses conducted in the present study are in agreement with our previous genome-wide study of the genetic substructure of the Kuwaiti population, which showed genetic heterogeneity . Additiof3 and f4 confirmed the existence of a heterogeneous genetic pattern among the Kuwaitis, signaling out a probable impact of distinct population dynamics that characterized the current genetic diversities of different Kuwaiti subgroups. The f3 admixture results , and the low level of variation in North African genetic ancestry profile for all eight Kuwaiti clusters in addition to the admixture signal with Mandenka, we infer a recent possible admixture event in the Kuwait-B subgroup with a West African (Mandenka or Yoruba) or sub-Saharan African group.Taking into account that both the West and East African populations were transported to the Middle East, Arabia, and Indian Ocean during the slave trade in the 15–19th centuries , 48, theFurthermore, the GLOBETROTTER results reinforce different major and minor ancestral sources of all three Kuwaiti subgroups  for Kuwaiti populations inhabiting the AP and remained highest when the TVD was calculated only among Kuwaiti individuals in a single cluster. Consequently, the results indicate that the heterogeneity of clusters was due to the Kuwaiti population, suggesting that Kuwaitis are one of the most genetically heterogeneous populations inhabiting the AP region. NNLS analysis also confirmed the genetic heterogeneity of the Kuwaiti population by showing the differential amount of genetic ancestries that contributed to each of these clusters by three different major donor populations of Yoruba, Bedouins, and North-East Europeans.In general, Kuwaiti populations are demographically characterized by large families and a remarkable rate of consanguinity, which is a potential threat to human health, especially in the context of rare autosomal recessive genetic diseases. As there have been historical population migrations in this region, a complex genetic diversity is expected, which was clearly reflected in this study. Our thorough investigations on the Kuwaiti population genetic structure at the finest scale highlights the precise genetic history and distinct heterogeneity of the Kuwaiti people, which could enormously aid in the systematic discovery of population- and/or family-specific diseases, especially in deciphering deleterious founder genetic variations. Overall, our study presents the fine-scale genetic structure of the distinctively heterogeneous Kuwaiti population and further highlights the recent historical population influx and gene flow from Western/sub-Saharan Africa to the AP region.Supplementary Information


Aedes albopictus is a native mosquito to Southeast Asia with a high potential for disease transmission. Understanding how Ae. albopictus populations that develop in the species’ native range is useful for planning future control strategies and for identifying the sources of invasive ranges. The present study aims to investigate the genetic diversity and population structure of Ae. albopictus across various climatic regions of Vietnam. We analyzed mitochondrial cytochrome oxidase I (COI) gene sequences from specimens collected from 16 localities, and we used distance-based redundancy analysis to evaluate the amount of variation in the genetic distance that could be explained by both geographic distance and climatic factors. High levels of genetic polymorphism were detected, and the haplotypes were similar to those sequences from both temperate and tropical regions worldwide. Of note, these haplotype groups were geographically distributed, resulting in a distinct population structure in which northeastern populations and the remaining populations were genetically differentiated. Notably, genetic variation among the Ae. albopictus populations was driven primarily by climatic factors (64.55%) and to a lesser extent was also influenced by geographic distance (33.73%). These findings fill important gaps in the current understanding of the population genetics of Ae. albopictus in Vietnam, especially with respect to providing data to track the origin of the invaded regions worldwide. Aedes albopictus  is a tree hole-dwelling zoophilic species [Ae. albopictus to survive unfavorable conditions or novel environments [Ae. albopictus populations in temperate regions, while tropical populations are active all year round [Within its native range of Southeast Asia, the Asian tiger mosquito s Skuse, 894 is a  species , facilit species ,4. Furthronments . Interesar round ,7. Severar round ,11, it rAe. albopictus, few have included samples from the species’ native range , and for this reason, little is known about the origin of invasive populations. As one of the world’s most medically important vectors, the species is capable of transmitting many arboviruses, including dengue, chikungunya, and Zika ,13,14,15Ae. albopictus dispersal [Additionally, recent studies have demonstrated that geographic distance is not the only factor that affects gene flow. Indeed, the population structure may also be affected by environmental factors, either alone or in conjunction with geographic distance . In partispersal . HoweverAe. albopictus and is suspected as the source of documented invasions into several tropical nations, including Cameroon, Brazil, and Madagascar [Aedes albopictus occurs throughout Vietnam [Ae. albopictus. In addition, Vietnam has always ranked among the countries most heavily affected by mosquito-borne diseases [Aedes aegypti  and Ae. albopictus, represents a major threat to both the public health and economy of Vietnam [Ae. albopictus populations is essential to managing the further spread of dengue fever in Vietnam. However, the genetic availability of Ae. albopictus populations in its native range in Vietnam has not been conducted yet. Vietnam is known as one of the native ranges of dagascar ,21,22. Tdagascar . Moreovedagascar . Aedes a Vietnam  and is a Vietnam . Given tdiseases ,28,29,30 Vietnam ,32. GiveCOI) gene sequences from Ae. albopictus populations in Vietnam, to investigate the population structure of the species among temperate and tropical regions, and to evaluate the role of geographic distance and climatic factors in determining genetic differentiation. The study’s findings are expected to improve the current understanding of the geographic origins of recent Ae. albopictus invasions and to provide evidence for ongoing gene flow restriction between the species’ temperate and tropical populations. The implications of these findings for mosquito control in Vietnam are also discussed.The present study aims to evaluate the variability of mitochondrial cytochrome oxidase I  were collected from a variety of habitats, including domestic containers, discarded tires and containers, bonsai tanks, flowerpots, and tree holes. Adults were collected using aspirators and larvae were collected using a small net and forceps. Larvae were either allowed to mature under laboratory conditions or preserved in absolute ethanol in the field. All specimens were initially identified morphologically using taxonomic keys  and wereCOI was amplified using the AccuPower PCR PreMix kit  and a previously published universal primer pair  [Total genomic DNA was extracted from two or three legs from each adult specimen or from muscle tissue from the abdomen (segments II–VI) of each larval (4th instar) specimen. The DNA was extracted using a DNeasy Blood & Tissue Kit , following manufacturer instructions, and was then stored at −20 °C. A 658-bp fragment of ATCA-3′) . The 20 Amplification was confirmed by electrophoresis using 1.5% agarose gels and visualization under UV light, and the validated PCR products were then purified and sequenced using an ABI Prism Sequencer 3130 . The resulting sequences of both directions (reverse and forward) were aligned and edited using MEGA v.7  and wereCOI haplotypes  and genetic diversity indices, including haplotype diversity (Hd) and nucleotide diversity (π), were estimated using DnaSP v. 6.12.03 [D [Fs [The number of  6.12.03 . Meanwhi3 [D [Fs  were cal3 [D [Fs , with po3 [D [Fs .https://inkscape.org/ accessed on 19 July 2020). To gain a broader understanding of genetic relationships between populations from Vietnam and those from other parts of the world, the sequence dataset was also compared to other available Ae. albopictus COI sequences, which were identified using the basic local alignment search tool (BLAST) from GenBank .A minimum-spanning tree was generated using ARLEQUIN and then used to construct a haplotype network (HAPSTAR v.0.7), which was optimized using InkScape v. 0.92.4 (STF) among populations was estimated using differences in haplotype frequencies, and sequential Bonferroni correction was used to adjust the significance levels in ARLEQUIN. Pairwise genetic differentiation  of STF values was performed, and GENALEX v.6.5 [STF, climatic separation [CT), among populations within regions (ΦSC), and within populations (ΦST) using permutation tests with 1000 random permutations. Moreover, spatial analysis of molecular variance (SAMOVA) was applied to further determine the genetic and geographic clusters of homogeneous populations [F-statistics  using the AMOVA approach to maximize between-group variation.To infer the geographic structure of EX v.6.5  was usedparation  and urbaparation . Statistulations . The numSTF values were used as a measure of genetic differentiation. A genetic distance matrix was used as a response variable, and geographic distance and climatic variables were used as explanatory variables. Geographic distance was transformed using third-order polynomials, and spatial variables that retained three spatial components were used for subsequent analyses. A total of 19 climatic variables related to temperature and precipitation, which are the most influential to this species’ biology and distribution [www.worldclimate.com accessed on 23 August 2020). The most explanatory factors were selected, and collinearity variables were minimized using the principal component analysis (PCA) and Pearson’s correlation. Global dbRDA was then performed between STF and selected spatial and climatic variables   was usedP-values are presented for each model.The best model was selected using a stepwise approach that optimized the model by considering the Akaike information criterion (AIC). Condition tests were conducted to detect the individual effect of either climates or geographic distance on genetic variation. Variance components of dbRDA were then partitioned by running two models: (1) a partial model in which geography explains genetic data conditioned on climatic variables and (2) a reciprocal model in which climatic variables explain genetic data conditioned on geography . The totSTF/(1 − STF) on the natural logarithm (Ln) of straight-line geographic distance [The isolation-by-distance hypothesis was also tested using Mantel’s test based on the regression of pairwise distance  . All Fu’s Fs values, except that for S13, were also significant and negative. However, the Tajima’s D values were non-significant and negative values for all but four sites  being sampled from the NE region. In addition, 45 (69.2%) of the 65 haplotypes were singletons, being represented by single specimens  was surrounded by closely related haplotypes, thus forming a ‘star-shape’ B, and a pecimens B.COI sequences obtained from GenBank revealed that the majority of the 65 haplotypes from Vietnam were novel, with only 15 being shared with sequences originating in other countries , whereas comparisons among the remaining populations yielded low and rarely significant STF values , and the greatest ΦCT value was detected when populations were clustered into two groups . The spatial structure of  regions A, which  regions A. PCoA a regions B, and sp regions C. IndeedCT = 0.2914, P < 0.001). The separation of populations into three groups that corresponded to the three haplotype regions , as was the separation of populations into seven groups that corresponded to climatic regions . Finally, the separation of populations into three groups that were randomly selected among regions based on the urbanization level of each sample site  also revealed significant geographic structuring among the 16 populations, with statistical support for almost all biogeographic hypotheses . One str regions B was alsIn comparison to the full model (Equation (1)), the best and final models were those that retained bio8 (mean temperature of wettest quarter), bio11 (mean temperature of coldest quarter), and geo3 (geographic component). Ae. albopictus populations  was the most explanatory variable for the separation of NE populations  from the other populations . In addiAe. albopictus is native to Vietnam and its impacts have been reported for several decades, the current study provides the first comprehensive investigation of the genetic variation and population structure of Ae. albopictus in Vietnam. The results reveal high genetic diversity with an admixture of temperate and tropical haplotypes, as well as an apparent population structure that is mainly determined by climatic differences. The current study’s findings are able to inform future vector control strategies in the country.Even though Ae. albopictus in Vietnam is relatively high (65 haplotypes) and is higher than almost all previous estimates for either native or invasive Ae. albopictus populations around the world , which harbored both the temperate and tropical haplotypes, followed by Region 3 , which harbored only tropical haplotypes, and then Region 1 , which harbored mainly temperate haplotypes. The results of the present study suggest a new perception of the accessing level of genetic diversity in a certain region, which is not higher in tropical or temperate regions, but in a region of contact between them, such as the several northern localities (Region 2) of Vietnam  using microsatellite loci [COI gene in northern Asia [Ae. albopictus populations in Vietnam. Particularly, the analyses in the current study clearly revealed a regional clustering of temperate and tropical haplotype groups that can be evidenced by the geographical distribution of haplotype groups  than by geographical distance  that were translocated to temperate regions or of temperate populations that were subjected to tropical conditions [Ae. albopictus population was also reported by Zhong et al. [Ae. albopictus to colonize temperate environments [Ae. albopictus populations in Vietnam. For instance, Ae. albopictus specimens from northern Vietnam (Hanoi) are able to undergo diapause [Ae. albopictus subpopulations in Vietnam are encouraged, perhaps using translocation experiments, to validate the findings of the present study.It is obvious that geographical distance must be an important barrier to dispersal preventing genetic exchange of individuals between populations. Thus, previous studies have reported the effects of geographic distance on the genetic structure of ulations ,10,55. H 11.42%; . The mea 11.42%; . The NE  11.42%;  that is nditions ,62. The g et al.  and thatronments . Extensironments ,65. It idiapause  and possdiapause . The gendiapause . HoweverAe. albopictus are more likely to extend their ranges to the north because the effects of global warming could limit the range of the temperate zone [Ae. albopictus populations in wider ranges (both native and invasive regions) is encouraged to further assess the effects of climate change to the species’ genetic variation.Moreover, the maintenance of restricted gene flow by climatic factors could promote successful invasion from other environments by the effects of climate change  since adaptation under rapid environmental change is likely unnecessary . In thisate zone . In the Ae. albopictus may improve predictions of vector-borne disease outbreaks and facilitate the development of dengue mosquito control in Vietnam. High levels of genetic diversity were observed in both the temperate and tropical Ae. albopictus populations and especially in the admixture population (Region 2; Ae. albopictus in Vietnam may allow them to resist changes in environmental conditions, pesticide resistance, or increase the likelihood of invasion success. A distinct population structure requires a specific control method for each subpopulation. Indeed, the success of biological control, in particular, requires the intensive selection of suitable biocontrol agents [Ae. albopictus populations but would not affect populations in the tropics. The present study’s robust evidence for recent population expansion in Vietnam may indicate the consequences of urbanization [Ae. albopictus [The findings of the present study regarding the population genetics of l agents . Moreovenization , which hbopictus  or may sbopictus . Ae. albopictus population in Vietnam is unknown. The present study of Ae. albopictus populations in Vietnam revealed high genetic diversity with evidence for the admixture of gene pools from temperate and tropical populations, thereby suggesting Vietnam could be an important original source for invaded areas globally. Interestingly, an uncovered population structure between these phenotypic strains of Ae. albopictus was found in a native range in Vietnam. The present study also revealed that the genetic variation observed among populations in Vietnam depended heavily on climatic factors, and to a lesser extent, geographic distance. These findings enrich the current understanding of the genetic diversity and population structure of Ae. albopictus in its native range and possibly allow for a better understanding of the worldwide invasion scenario. However, further studies conducted in larger areas and using different genetic markers are needed to validate our conclusions.Currently, the genetic variability among climatic regions of


Esox lucius) to investigate associations between offspring performance (hatching success) and parental genetic similarity in experimentally purebred and admixed families, based on neutral (microsatellites), genome-wide neutral (RADseq SNPs), and adaptive (SNPs under selection) markers.Fitness consequences of intraspecific genetic admixture can vary from positive to negative depending on the genetic composition of the populations and environmental conditions. Because admixture has potential to influence the success of management and conservation efforts, genetic similarity has been suggested to be used as a proxy to predict the outcome. Studies utilizing microsatellites  to investigate associations between genetic distance and admixture effects show conflicting results. Marker types that yield information on genome-wide and/or adaptive variation might be more useful for predicting responses to inter-population hybridization. In this study we utilized published data for three populations of pike (Estimated similarity varied among the markers, likely reflecting differences in their inherent properties, but was consistently higher in purebred than admixed families. A significant interaction between marker type and admixture treatment reflected that neutral SNPs yielded higher estimates than adaptive SNPs for admixed families whereas no difference was found for purebred families, which indicates that neutral similarity was not reflective of adaptive similarity. When all samples were pooled, no association between similarity and performance was found for any marker. For microsatellites, similarity was positively correlated with hatching success in purebred families, whereas no association was found in admixed families; however, the direction of the effect differed between the population combinations.The results strengthen the notion that, as of today, there is no proxy that can reliably predicted the outcome of admixture. This emphasizes the need of further studies to advance knowledge that can shed light on how to safeguard against negative consequences of admixture, and thereby inform management and promote conservation of biological diversity.The online version contains supplementary material available at 10.1186/s12863-021-00974-3. Intraspecific genetic admixture (henceforth ‘admixture’) occurs when separated populations starts interbreeding . It occuAdmixture affects the genetic composition of the involved populations and tends to increase the genetic variation in the receiving population. Increased genetic variation is generally considered to positively influence aspects of population performance. For example, it has been shown that genetically and phenotypically more diverse populations are better able to cope with environmental change, and to colonize novel environments , 7–10. AThe motivation for management and conservation efforts resulting in admixture is to increase productivity, viability, and adaptability of populations , 11, 14.The development of next generation sequencing techniques, such as Restriction-site associated DNA sequencing (RADseq) has enabled to genotype many markers at a low cost, even for non-model species . RADseq Esox lucius) [FST = 0.044–0.071), and some gene flow may occur [FST = 0.136–0.226) [i) variation in parental similarity estimates among pairs of experimentally mated males and females were consistent or differed among the markers; ii) parental similarity was associated with offspring hatching success; and iii) the associations between parental similarity and offspring performance were consistent or depended on whether parental similarity was estimated based on neutral (microsatellites), genome-wide neutral (RADseq SNPs) or adaptive (RADseq SNPs under selection) genetic markers.The overall aim of the present study was to evaluate the potential of neutral (microsatellites), genome-wide neutral (RADseq SNPs), and adaptive (outlier SNPs under selection) parental genetic similarity to predict admixture effects on offspring performance (hatching success) resulting from experimental matings of individuals representing genetically separated and locally adapted natural populations. To this end, we used previously published genetic and phenotypic data for three populations  of anadromous Baltic Sea pike ( lucius) , 36. Pik lucius) –40. All  lucius) , 39, 41  lucius) , but theay occur , 39, 41.6–0.226) , 39, 41.6–0.226) –45. MoreF2,178 = 36.38, P < 0.001, Fig. P = 0.014; microsatellites – neutral SNPs: P < 0.001; adaptive SNPs – neutral SNPs: P = 0.003). Estimated parental similarity was highest for neutral SNPs (mean ± SD: 0.55 ± 0.07), intermediate for adaptive SNPs (mean ± SD: 0.49 ± 0.15), and lowest for microsatellites (mean ± SD: 0.43 ± 0.15). In addition, the range of similarity estimates was smaller for neutral SNPs than for both microsatellites and adaptive SNPs  differed  , which reflected that neutral SNPs yielded higher estimates for the admixed families than did microsatellites and adaptive SNPs , whilst there was no difference between neutral SNPs and adaptive SNPs for purebred families  .When all the samples were pooled, a large variation in hatching success was evident throughout the range of parental similarity for all three datasets Fig. . ParentaF1,176 = 0.24, P = 0.62; effect of parental similarity: F1,176 = 0.045, P = 0.85; effect of interaction between parental similarity and admixture treatment: F1,175 = 0.67, P = 0.41) or adaptive SNPs  , reflecting that there was a positive relationship between similarity and hatching success for purebred families (a positive slope), whereas parental similarity was not associated with hatching success in the admixed families , to test whether associations between parental similarity and hatching success differed between purebred and admixed families, no significant interaction effect was found for either neutral SNPs , but not for the neutral SNP dataset  whereas adaptive SNPs approached marginal significance . No significant interaction effects between parental similarity and population combination were found for the admixed population combinations for any of the marker types .When the data was further separated into specific population combinations  Fig. , Studenti) estimated parental similarity differed between the marker types, and were consistently higher for purebred families compared to admixed families; ii) parental similarity was not consistently or clearly associated with hatching success for any of the marker types; and iii) the association between hatching success and parental similarity as estimated based on microsatellites was different for purebred and admixed groups, and also differed between population combinations.Increased knowledge about the effects of admixture can further the understanding about evolution of genetic structure and what shapes patterns of diversity, and also help avoid undesirable effects associated with conservation measures and management actions. In the present study, we used data for pike to evaluate whether estimates of parental genetic similarity based on three different marker types  could predict the outcome of admixture. To our knowledge, this is the first attempt to systematically evaluate and compare the utility of different markers as proxies of parental compatibility and predictors of offspring performance. The main findings were that: The findings that both values and ranges of estimated parental similarity differed between the marker types Fig.  were expThe finding that purebred families had higher parental similarity than admixed families for all datasets Fig.  was alsoWhile there was no overall association between hatching success and parental similarity for any of the marker types Fig. , the resSadly, the conclusion that parental similarity is a poor predictor of the response to admixture extends to other candidate proxies, such as geographic distance, neutral genetic differentiation, genetic diversity or environmental similarity. Empirical studies using these different proxies , 53, 54 The present study showed that parental similarity was not consistently or clearly associated with hatching success for any of the marker types . Our present study thus strengthens the conclusion that, as of today, there is no proxy that can reliably predict the outcome of admixture. There is therefore a clear need for further studies and different approaches to advance knowledge that can shed light on how to safeguard against negative consequences of admixture, and thereby inform management and promote successful conservation of biological diversity.Pike is a long-lived fish that inhabits both freshwater and brackish water systems . As one FST = 0.136–0.226, P < 0.01) [FST = 0.044–0.071, P < 0.01) [The three populations of anadromous pike included in this study reproduce in different spawning habitats in the Kalmar Sound region . Two of  < 0.01) , 39, 41  < 0.01) . Despite < 0.01) , 39, 41, < 0.01) , 41. In  < 0.01) , salinit < 0.01) , growth  < 0.01)  and vert < 0.01) , 65, and < 0.01) . Moreove < 0.01) .Nmales = 12, Nfemales = 10, Lervik: Nmales = 12, Nfemales = 9, Okne: Nmales = 10, Nfemales = 11). In short, separate batches of eggs from each female were artificially fertilized with milt from one male from each population, thus creating one purebred  and two admixed  treatments per female. Each combination was done in duplicates, and this resulted in a total of 180 units from 90 female/male pairs (30 purebred and 60 admixed). Eggs, and subsequently hatched fry were reared in a common garden environment, and three offspring performance measures: 1) hatching success (proportion of eggs that hatched), 2) fry survival (15 days following hatching), and 3) fry body length (at 15 days post hatch) were estimated . Becausails see  found thP-value cut-off of 0.01 instead of applying FDR correction for multiple testing) to not exclude potential outliers, and to increase the number of included outlier loci. This resulted in a total of 17 loci identified as putatively under selection  were used to test whether and how parental similarity varied among the three marker types , whether hatching success was associated with parental similarity, and to explore whether and how associations between hatching success and parental similarity were affected by admixture treatment (differed between purebred and admixed families). For this, we used the lme4 package v1.1–15 in RStudio with R. The different models were chosen based on the response distribution of the data: general linear mixed models for parental similarity  and generalized linear mixed models with a logit-link function for hatching success .P-values were adjusted using the FDR method [For all tests, we treated marker type as a fixed categoric factor, parental similarity as a fixed continuous factor, and family  as a random factor. Statistical significance was assessed with Type III partitioning and an α–level of 0.05, and the Satterthwaite’s method was used to approximate degrees of freedom. For tests of interactions between factors, we excluded the interaction term and rerun the analysis in case of no significant interaction effect. For tests with significant terms, we further analyzed the data with Student’s t-test with non-pooled SD or Tukey’s test to determine the nature of the interactions and evaluate pairwise differences. R method  to accouAdditional file 1: Table S1. Information on the 17 outlier loci that were used as adaptive dataset in the present study.


Puma concolor). Puma populations along California's central and southern coastal habitats have experienced rapid fragmentation from development, leading to calls for demographic and genetic management. To address urgent conservation genomic concerns, we used double‐digest restriction‐site associated DNA (ddRAD) sequencing to analyze 16,285 genome‐wide single‐nucleotide polymorphisms (SNPs) from 401 pumas sampled broadly across the state. Our analyses indicated support for 4–10 geographically nested, broad‐ to fine‐scale genetic clusters. At the broadest scale, the four genetic clusters had high genetic diversity and exhibited low linkage disequilibrium, indicating that pumas have retained genomic diversity statewide. However, multiple lines of evidence indicated substructure, including 10 finer‐scale genetic clusters, some of which exhibited fixed alleles and linkage disequilibrium. Fragmented populations along the Southern Coast and Central Coast had particularly low genetic diversity and strong linkage disequilibrium, indicating genetic drift and close inbreeding. Our results demonstrate that genetically at risk populations are typically nested within a broader‐scale group of interconnected populations that collectively retain high genetic diversity and heterogenous fixations. Thus, extant variation at the broader scale has potential to restore diversity to local populations if management actions can enhance vital gene flow and recombine locally sequestered genetic diversity. These state‐ and genome‐wide results are critically important for science‐based conservation and management practices. Our nested population genomic analysis highlights the information that can be gained from population genomic studies aiming to provide guidance for the conservation of fragmented populations.Urbanization is decreasing wildlife habitat and connectivity worldwide, including for apex predators, such as the puma ( In both the Santa Ana and Santa Monica Mountains, phenotypic evidence of inbreeding depression has been observed, similar to that of Florida panthers  from 401 individuals using a double‐digest, restriction‐site associated DNA sequencing method  from pumas collected between 2002–2015 , found dead (~2%), poached (<1%), or through depredation permits (>90%), which had never been used in any previous genetic survey. Samples were well‐distributed throughout the state, except for smaller populations in smaller mountain ranges. To bolster our sample size in the Los Angeles region of southern California, we added the only remaining DNA extracts (2.2We reduced the genome size of our samples and identified single‐nucleotide polymorphisms (SNPs) using modifications to the double‐digest restriction‐site associated DNA sequencing (ddRAD) protocols developed by Peterson et al. . We usedhttps://gc3f.uoregon.edu/) for 150 bp single‐end sequencing on an Illumina HiSeq 4000 (Illumina).We then ligated 48 uniquely barcoded P1 adaptors  and two common P2 adapter pairs  to each sample's double‐digested fragments using the protocols of Peterson et al.  to ident2.3FastQC v0.11.5  or longitudes  among libraries. However, because the southern California libraries constructed from pre‐existing extracts were from a small geographic region, there ended up being some latitudinal  and longitudinal  mean differences between those libraries and the libraries constructed from the new samples. However, as indicated below, there were no detectable biases of the southern California libraries in any analyses.In each library of 48 samples, we strategically included puma samples from across a large geographic area so libraries would have no correlation with the spatial location. For example, there was no significant difference between mean sample latitudes , we used 2.4adegenet 2.1.1  at 100,000 iterations each. We kept the most highly supported model  within each of the 20 replicates. To test for evidence of loci under selection, we identified outlier loci among populations  and a spatially explicit population structure analysis in program R , gene diversity (SH), and allelic richness (rA) using hierfstat::basic.stats  using poppr::private_alleles  using the linkage disequilibrium model, random mating, allele frequencies >0.05, and with a correction factor of 19 haploid chromosomes  based on genomic distance between SNPs. We correlated loci using binned intervals of 100,000bp from 0 to the maximum scaffold size of PumCon1.0. Meiosis should break up linkage, resulting in low R2 values. However, populations experiencing strong selection, low mutation, inbreeding, low migration, or strong genetic drift will have higher R2 values. In short, SNPs that are close together on chromosomes are expected to be correlated , but SNPs far away are expected to assort randomly during recombination. However, if sequences are too similar, which they may be in small and inbred populations, we will not be able to detect events of crossing over despite their occurrence, resulting in higher estimates of linkage disequilibrium, which is still an important indicator of genetic diversity and Ne.We used 33.1We recovered 16,285 SNPs that were randomly distributed among 125 draft‐genome scaffolds. The first three axes of the PCA accounted for 14.6% of the variance and indicated that there were four broad‐scale genetic clusters distributed across California Figure , the fouK = 2 panel of Figure K = 3 panel of Figure K = 4, and there is a major increase in variance at K = 5, but there is a steady increase in statistical support at higher K values. However, single pumas formed individual clusters at K > 10, at which point K lost biological meaning. When K was set to 4, the genetic clusters corresponded to the broad‐scale genetic groups identified by the PCA  emerged; at K = 6, the Eastern Sierra Nevada (ESN) cluster separated from the Western Sierra Nevada (WSN); at K = 7, the Santa Ana (SA) cluster separated from the Eastern Peninsular Range (EP); at K = 8, the San Gabriel–San Bernardino (SGSB) cluster emerged; at K = 9, the Klamath–Cascades (KC) cluster emerged; and at K = 10, the Central Coast South (CC‐S) cluster separated from Central Coast Central , gene diversity (HS), polymorphic loci (Poly), allelic richness (Ar), and the private alleles (Ap) indicate that the Sierra Nevada cluster had higher genetic diversity than the Southern Coast, Central Coast, and North Coast  or finer‐scale substructure. Effective population sizes were not reported for broad‐scale clusters because substructure introduced major biases  into Ne estimates.For st Table . AlthougSTF estimates which ranged from ~0.1–0.2  was lowest in the Sierra Nevada and slightly increased in the Central Coast, Southern Coast, and North Coast clusters  had an LD R2 of ~0.3 which decreased rapidly to less than 0.1 at a distance of 0.3 Mbp, then approached 0 at farther distances. Nearly, the same result was observed in the Sierra Nevada. The Central Coast also had a major reduction in LD with distance but did not fall under 0.1 until ~3 Mbp in distance. In contrast, the Southern Coast and North Coast started with an LD R2 of ~0.4 which remained above 0.1 even at distance of 8 million bp , indicating substantial gene flow throughout this region , SA was the most differentiated among the 10 finer‐scale genetic clusters . The SGSB cluster had relatively lower pairwise STF estimates with the Sierra Nevada and EP clusters, moderate STF estimates with CC‐C and CC‐S, and was more strongly differentiated from the CC‐N and NC. The EP cluster showed similar patterns of differentiation but was least differentiated from the geographically adjacent SA and SGSB clusters. Although EP exhibited LD estimates similar to the Southern Coast as a whole, SGSB and SA started with a high LD R2 of ~0.5 which decreased to just more than 0.3 at a distance of 0.3 Mbp, then remained high (more than 0.25) at farther distances  and appeared to correlate with distance , indicating 10 genetic populations at a finer scale. Generally, the 10 genetic populations identified with SNPs correspond strongly to those identified in previous studies using microsatellite markers and different samples  observed between the Klamath–Cascade and Western Sierra Nevada populations. The 10 populations varied considerably in genetic diversity estimates , effective population sizes (eN range 2.3–54.4), and genetic differentiation (STF range: 0.22–0.32) as discussed below.Although the four major genetic clusters are highly consistent among our structure analysis and PCA, there was also statistical support for substructure  and gene flow occurring only with the Central Coast Central population to the south. This pattern could also be driven by carrying capacity processes associated with habitat limitations (Dellinger et al., A major difference between this and previous studies is the observation that pumas in the Central Coast North population have genetic diversity estimates as low as those in the Santa Ana and Central Coast South populations, which are highly isolated by urbanization and transportation infrastructure and exhibit evidence of inbreeding depression (Benson et al., Ne, but had intermediate levels of genetic diversity. Occasional migrants could alter eN estimates and temporarily inflate estimates of heterozygosity (Gustafson et al., Notably, the San Gabriel–San Bernardino population had the lowest Ne, including the San Gabriel–San Bernardino, Santa Ana, and Central Coast South populations, have the smallest available amount of habitat (Dellinger et al., eN, and linkage data, the San Gabriel–San Bernardino and Central Coast North populations may be approaching levels of genetic drift and inbreeding similar to the well‐monitored and genetically depauperate Santa Ana and Central Coast South populations (Ernest et al., The three populations with the lowest Populations with intermediate genetic diversity include the North Coast, Central Coast Central, and Eastern Peninsular Range. Measures of genetic diversity were lower than expected for the North Coast population, given that there are no obvious anthropogenic barriers to gene flow with the Klamath–Cascade, Western Sierra Nevada, or pumas from Oregon (Gustafson et al., The Central Coast Central population has ample habitat for maintaining a breeding population (Dellinger et al., Ne. Dispersal in and out of the Eastern Peninsular Range is extremely limited, and the degree to which pumas disperse across the border between USA and Mexico remains unknown (Gustafson et al., Despite having less than half of the overall habitat of the Central Coast Central population (Dellinger et al., Our linkage decay analysis suggests that in the Central Coast South, San Gabriel–San Bernardino, Santa Ana, and perhaps the Central Coast North populations, pumas may have long runs of homozygosity that are identical by descent. This is consistent with the genome resequencing results of Saremi et al.  in the SPanthera leo; Miller et al., P. concolor; Ralls et al., Panthera tigris; Armstrong et al., Panthera pardus; Perez et al., Genetic restoration or rescue has been successfully demonstrated for isolated, large‐felid populations, such as the African lion (K = 4 or K = 10. Detection of outlier loci with RAD‐seq is limited by the reduced representation of the genome, yet it has often been shown to be an effective approach (Catchen et al., We tested for outlier loci using multiple methods (Narum & Hess, 5Our population genomic analyses provide decision makers a contemporary and thorough evaluation of the genetic diversity, effective population sizes, and connectivity of puma populations throughout California. These state‐ and genome‐wide results are critically important for conservation and management practices in California, especially considering the increasing demand for development and the current political climate surrounding the petition to list pumas in Southern and Central California as threatened under the California Endangered Species Act (Yap et al., The authors declare no conflicts of interest.