Sogatella furcifera (Horváth) , is a migratory pest of rice in Asia. Shandong Province, in northern China, is located on the migration pathway of WBPH between southern and northeast China. The potential sources of WBPH in northern China are poorly understood. We studied the sources of WBPH in Shandong Province by determining the population genetic structure of WBPH in 18 sites distributed in Shandong and in six regions of the Greater Mekong Subregion (GMS). We used mitochondrial gene and single-nucleotide polymorphism (SNP) markers for analysis.The white-backed planthopper (WBPH), ST values based on mtDNA ranged from − 0.061 to 0.285, while FST based on SNP data ranged from − 0.007 to 0.009. These two molecular markers revealed that 4.40% (mtDNA) and 0.19% (SNP) genetic variation could be explained by the interpopulation variation, while the rest came from intrapopulation variation. The populations in the seven geographic regions comprised four hypothetical genetic clusters (K = 4) not associated with geographic location. Eighty-four of 129 individuals distributed across the given area were designated as recent migrants or of admixed ancestry. Although the substantial migration presented, a weak but significant correlation between genetic and geographic distances was found .All of the WBPH populations studied in the seven regions had low genetic diversity. Pairwise F. These results help clarify the migration route and the source of WBPH in northern China.The Greater Mekong Subregion was the main genetic source of WBPH in Shandong, while other source populations may also exist. The genetic structure of WBPH is shaped by both migration and geographic barriers Sogatella furcifera (Horváth), often causes serious yield losses to rice in Asia [The white-backed planthopper (WBPH),  in Asia . This pe in Asia . In 1970 in Asia . The WBP in Asia , 5. Base in Asia , 7. Mole in Asia , 9.Shandong Province in China is a typical region for growing single and midseason rice. Rice growing areas in Shandong Province are important because they are on the migration path of WBPH between southern and northeast China. Trajectories analysis demonstrated that some WBPH individuals migrating into China can reach northeastern China by late June or early July. The WBPH can then move between the Shandong and Liaoning provinces (or the Korean Peninsula) in both directions , 5. The Previous studies on WBPH migration mainly depended on the trajectory analysis method , 10. RecIn this study, we used both mitochondrial DNA and genome SNP markers to analyze the genetic diversity and connectivity of WBPH populations in seven geographic regions in Shandong Province, China and the GMS, including Cambodia, Laos, Myanmar (Burma), Thailand, Vietnam, and Yunnan Province. We characterized the genetic differentiation among WBPH populations and identified the possible source populations of WBPH in Shandong Province.A total of 663 bases in the mtDNA COI genes from 133 individuals (deposited in GenBank under Accession Nos. MN718018–MN718150) were obtained. Of the 663 sites, 653 were conserved, and 10, including nine singleton polymorphic sites and one parsimonious informative site, were variable. Of the 133 samples, 11 haplotypes were identified  values ranged from 0.227 in Myanmar (MM) to 0.272 in Shandong (CN_SD) . The observed heterozygosity ranged from 0.181 in Shandong (CN_SD) to 0.227 in Yunnan (CN_YN). The unbiased expected heterozygosity  (P < 0.001), as well as between CN_YN and TH (P = 0.045)  showed that the TH was separated from the other populations  Table . The hapST values computed over SNP loci were quite low, ranging from − 0.007 to 0.009, with an average FST of 0.002. Pairwise FST value (0.009) between CN_SD and CN_YN was significant (P < 0.05) (Table Pairwise F5) Table . The PCoST, we set the group = 2, which considered CN_SD as one group and the other populations as another group. Significant variability was found among SNP between these two groups  (Table The results of the AMOVA test on mtDNA and SNP markers in different populations are shown in Table 5) Table .Table 4RWe also analyzed the population genetic structure based on SNP data using STRUCTURE software. The STRUCTURE analyses suggested that WBPH most likely forms four genetic clusters Fig. . Indeed,Based on population assignment test of the SNP data, 84 individuals were identified as migrants, there were connections among the Shandong population and other populations, all of which were expected to be a possible source of the Shandong population Table . There wST  effects were detected with the standardized pairwise FThe analysis based on the mitochondrial gene (COI) showed that all of the WBPH populations have relatively low genetic diversity. Only two dominant COI haplotypes widely exist in all populations suggesting a high-level gene flow among them. Among the 11 COI haplotypes identified, four haplotypes, including the two dominant haplotypes, were also found by previous study . The numThe genome SNP markers (2b-RAD) analysis showed that a heterozygosity deficit existed in all populations. This result may be explained by demographic expansion, and it is consistent with  who alsoThe mtDNA and SNP data revealed different patterns of genetic connectivity among the WBPH populations Table , Fig. 2.SNP data provided information about the genetic structure of WPBH populations. Genetic connectivity among the GMS populations and Shandong population was close. Most of migrants were from Yunnan population as showed by results of population assignment. Compared with the mtDNA data, the SNP data were more consistent with the IBD pattern, perhaps as a result of high information of SNPs. Besides, mtDNA is often unsuitable for detecting isolation by distance . This fiHelicoverpa spp. [In this study, the SNP markers showed that all of the populations exhibit high levels of admixture between the clusters identified with STRUCTURE. This indicates the occurrence of long distance migration events within geographical regions. Long-distance migration of WBPH allowed genetic mixing between populations from remote geographical origins . This parpa spp. . A populrpa spp.  who reviWeather conditions are thought to expedite long-distance immigration of planthoppers . In soutThis study demonstrated that WBPH populations have a low level of genetic diversity and a mixed genetic structure. We arranged the samples in chronological order, which depended on the occurrence of WBPH. Rice planting in Shandong mainly begins in May, and the WBPHs often have population outbreaks in July and August. Although the GMS were revealed as the main genetic source of WBPH in Shandong, WBPHs expand their range in a stepwise manner. Populations reproducing in other areas of China, such as Yangtze River Valley, Guangxi Zhuang Autonomous region, and Guizhou Province, may also be important sources. Future study is needed to examine more geographic populations and understand the temporal and spatial genetic structure of WBPH in China.WBPH individuals were sampled in various geographic regions (China and Southeast Asia) Table , Fig. 1.Insect DNA was extracted using the TIAMamp Micro DNA Kit  according to the manufacturer protocol. The mtDNA COI gene was amplified using primers 2195-MF(5′-CTGGTTYTTTGGTCATCCRGARGT-3′)  and a neThe 2b-RAD sequencing and genotyping were outsourced to Shanghai OE Biotech Ltd. . Libraries were constructed following the 2b-RAD protocol . BrieflyNumbers and distribution of haplotypes, composition of haplotypes in each population, numbers of unique haplotypes, within-population mean number of pairwise differences, and nucleotide diversity were assessed using DnaSP v.5.10 .The statO), unbiased expected heterozygosity (uHE), and fixation index (F), were estimated by using GenALEx 6.5 [The genotype data contained information for each locus and each individual. The primary SNP loci number was 13,565, which could genotype all 133 individuals. We used Plink version 1.07  to filteALEx 6.5 , 33. HarALEx 6.5 .ST) among populations, (ii) Principle Coordinate Analysis (PCoA) (iii) hierarchical analyses of molecular variance (AMOVA), (iv) Bayesian model-based clustering, and (v) Isolation by distance (IBD).We evaluated population genetic structure using five different approaches: (i) measuring genetic differentiation  was used to find and plot the major pattern within a genetic distance matrix dataset. The PCoA using GenALEx, performed on genetic distance, was used to display genetic divergence among the populations. To determine the proportion of genetic variation that could be attributed to differences between sampling sites, hierarchal analyses of molecular variance (AMOVAs) were performed. A hierarchical AMOVA was performed using Arlequin, with 1000 permutations. Populations were grouped corresponding to two major criteria, i.e., geographical area, and population genetic structure, to test genetic homogeneity in different hierarchies.For mtDNA data, the pairwise F.3.5.1.2  and usin.3.5.1.2 . For SNPThe Bayesian approach was used to determine genetically distinct groups (or clusters) using the program STRUCTURE v.2.3.1 –40. We sTo estimate the admixture between geographic populations, we used a Bayesian assignment method as implemented in Geneclass2 . This anAdditional file 1: Figure S1. (A) Delta k value of data across 10 replicates of STRUCTURE, where k = 4 is shown as the best fit of the data for the highest level of hierarchical genetic structure. (B) The mean lnP(D|K) and SD for each k where the model of k = 2, 3 or 4 is indicated as the best fit. Table S1. Assignment test for Sogatella furcifera individuals in the seven geographic populations. Individuals are presented in rows according to their sampling locations as individuals assigned to their own population (self) and those assigned to other putative source population. Individuals in CN_YN were randomly selected as 20 individuals participated the analysis.


Eupoecilia ambiguella (Lepidoptera: Tortricidae) is causing significant damage to grape berries, however, little is known on population genetics of this lepidopteran pest insect, hindered so far by the lack of suitable molecular markers. Here we report on the development of ten microsatellite markers of which six were used to characterise 21 E. ambiguella populations obtained from two viticultural regions in Germany and Italy. Moths were sampled during two subsequent generations (flights) in the same vineyard as well as in vineyards surrounded by different landscape types. German and Italian populations were genetically differentiated and a significant isolation by distance was evident. No significant divergence was observed among the populations from first or second flight moths, however, inbreeding was higher in first than in second flight populations. Moreover, inbreeding was influenced by habitat composition and complexity of landscape around vineyards, being positively associated with the percentage of area covered by grapevine. Population genetics of E. ambiguella could thus be affected by the presence of alternative host plants in viticultural landscapes, which is important in the light of both insecticide resistance management and sustainable pest management.The grape berry moth  Dispersal of individuals inhabiting either cultivated or natural habitats can result in gene flow between pest populations and can be important for sustainable pest management on the landscape scale. For example, a lack of natural habitats can reduce gene flow in pest populations favouring the development of insecticide resistance or can lower the pest’s exposure to parasitoids and predators. Grapevines are cultivated on 7.5 Mio ha throughout the world2, mostly in temperate regions with cool, wet winters, and hot, dry summers. Viticultural landscapes can be characterized by grapevine grown both in monoculture or in patchy smaller vineyards, located in highly structured settings with diverse natural habitats in close vicinity to crop areas3. Various fungal diseases and insect pests target grapevines throughout the season, resulting in very high levels of pesticide treatments4.Genetic diversity of populations of polyphagous insects is shaped by the availability of host resources and thus by landscape composition and heterogeneity. In agricultural landscapes, natural habitats such as hedges, forests, grasslands or other uncultivated sites may provide alternative host plants, overwintering sites or other refugee niches for herbivorous insects at several key stages of their life cycleEupoecilia ambiguella (Hübner) and Lobesia botrana (Denis & Schiffermüller) (Lepidoptera: Tortricidae), both native from the Palearctic region, are recognized as key pests of West-European vineyards since the eighteenth century5. Typically, E. ambiguella is common in the northernmost regions of Western Europe, whereas L. botrana is widespread in warmer regions reflecting their different ecological requirements6. E. ambiguella has been detected from Sicily to southern Scandinavia even outside grape-growing areas and is reported as dominant species in vineyards located in Germany, France (northern regions), Switzerland, Austria, Hungary, Czech Republic and Slovakia7. The two species coexist in transition areas and their ratio depends on climatic conditions: cooler and rainier seasons favor the dominance of E. ambiguella populations while warmer and drier seasons favor L. botrana. This is frequently observed in northern Italy8. During the last decades, L. botrana distribution shifted northwards and the pest invaded new viticultural areas across Europe, even replacing E. ambiguella9. This trend is expected to become more severe in the future under climate change scenarios11. Moreover, L. botrana is recognized as a highly invasive pest in Californian as well as in Chilean and Argentinian vineyards13, with E. ambiguella assumed to have the same invasive potential in cooler viticultural areas14.The grape berry moths, E. ambiguella, most of the investigations support that at least two generations are completed in a season5. In some European areas like France and Italy as well as in warmer years occurrence of a third generation has been reported6. Larvae of both species attack flower clusters and grapes of Vitis species, causing serious economic damage due to direct feeding activities on berries and increased risk of fungal infections of injured berries. Moreover, grape berry moths are polyphagous with E. ambiguella being able to oviposit and develop on more than 30 plant species belonging to the genera Silene, Clematis, Ribes, Prunus, Rubus, Sorbus, Rhamnus, Cornus, Lonicera, Viburnum, Euonymus etc.7. Most of these woody plant species are common in European landscapes, in particular in natural or secondary stands as hedges or shrubs or at forest edges. Population genetics of E. ambiguella in vineyards could therefore be affected by presence of alternative host plants and thus by landscape complexity, yet, the degree to which E. ambiguella existence, distribution and persistence in viticultural landscapes is influenced by alternate hosts is not well understood so far.Both grape berry moth species overwinter as pupae, usually under the bark of grapevine plants, and flight of first generation moths takes place at the time of bud burst. For 15. Here, we report on the development of the first set of SSR markers for E. ambiguella using a next generation sequencing technique. We further apply these markers to describe genetic structures and diversities in populations of this insect pest from two different regions in Europe . Moths were sampled in the same vineyards during two subsequent generations (flights) as well as in vineyards surrounded by different landscape types. Therefore, we were particularly interested in asking the following questions.E. ambiguella populations originating from two viticultural regions in Germany and Italy genetically differentiated?Are E. ambiguella individuals sampled in the same vineyard but in two subsequent generations genetically differentiated?Are Are genetic structures of populations sampled in vineyards surrounded by forests or other semi-natural habitats different compared to populations sampled in landscapes dominated by monoculture vineyards?Assessment of population structures and dispersal capacities of insect pests requires the availability of molecular markers such as microsatellites , which are known to be robust, follow a co-dominant inheritance and usually show a high degree of polymorphismEupoecilia ambiguella adults were collected in five vineyards in south-western Germany  and six vineyards in north-eastern Italy (Veneto region) (Table E. ambiguella. The categories were as follows: (A) isolated vineyards with less than 10% of the area cultivated with grapevines, fully surrounded by forests and woody elements (ca. 90% of the area) or other semi-natural habitats; (B) vineyards surrounded by other vineyards, but located at the border or nearby forests with 20–30% of the area covered by woody elements; (C) vineyards located in highly structured landscapes with orchards, other crops, small villages or houses and 10–25% of the area covered by small patches of woody elements; (D) vineyards surrounded mainly by other vineyards with less than 30% of the area covered with other crops and no or only few woody elements. As a basis for landscape analysis, satellite images provided by Google Earth were used and landscape elements were digitalized by hand in QGIS 2.18.9. For a picture of respective vineyards and surrounding landscapes within a radius of 500 m see Table n) Table . These v16 modified by the addition of an isopropanol precipitation step. DNA concentration was measured spectrophotometrically and DNA preparations were stored at − 20 °C.Total genomic DNA was extracted from adults by using a CTAB-based methodL. botrana17. For initial generation of SSR markers via 454 pyrosequencing, DNA was isolated from the head and thorax of ten adult E. ambiguella individuals obtained from a laboratory rearing at Geisenheim University as described above. Individual DNA was pooled in equimolar amounts and a total of 5 µg DNA was subjected to 454 pyrosequencing. Microsatellite identification and 454 pyrosequencing was performed commercially at Ecogenics, CH. In a first step, size-selected fragments from genomic E. ambiguella DNA were enriched for SSR content by using magnetic streptavidin beads and biotin-labeled CT and GT repeat oligonucleotides. The SSR-enriched library was then analysed on a Roche 454 platform using the GS FLX Titanium reagents. In total, 5526 reads were obtained, which had an average length of 164 base pairs. Of these, 983 contained a microsatellite insert with a tetra- or a trinucleotide of at least six repeat units or a dinucleotide of at least ten repeat units. Applying a stringent set of criteria in a tailor-made pipeline (property of Ecogenics) based on the Primer 3 core code (available from http://primer3.sourceforge.net/releases.php) design of primers flanking the microsatellite motifs was possible in 283 reads. A set of 20 oligonucleotide primer pairs having the highest probability of being functional were accordingly picked and tested for polymorphism in the present study.Microsatellite marker generation, PCR analysis as well as statistical and population genetic analysis was basically carried out according to our previous study on SSR marker identification in the European grapevine moth E. ambiguella populations, each including four individuals using the conditions described below. 10 markers failed to amplify in most of the individuals, formed extensive stutter peaks or showed an excessive background in this initial screening. From the remaining set  tail at its 5′-end (5′-TGTAAAACGACGGCCAGT-3′), 5 pmol of an unlabelled SSR-specific reverse primer, and 5 pmol of a fluorescently labelled universal M13(-21) primer, which will incorporate the fluorescent dye into the PCR product18. For multiplexing of the reactions for capillary electrophoresis this M13(-21) primer was either labelled with BMN5 , DY-681  or DY-751 . For markers Ea_01, Ea_02, Ea_05 and Ea_15 cycling conditions were according to the following PCR program using the Phire Hot-Start II DNA Polymerase (Thermo Scientific): initial denaturation and hot-start step at 98 °C for 3 min, followed by 30 cycles of 98 °C for 5 s, 63 °C for 5 s and 72 °C for 10 s, additional eight cycles of 98 °C for 5 s, 53 °C for 5 s and 72 °C for 10 s and a final extension at 72 °C for 10 min. Marker Ea_06 and Ea_11 were amplified using the DreamTaq DNA Polymerase (Thermo Scientific) and the following program: initial denaturation at 94 °C for 5 min, followed by 30 cycles of 94 °C for 30 s, 56 °C for 45 s and 72 °C for 45 s, additional 8 cycles of 94 °C for 30 s, 53 °C for 45 s and 72 °C for 45 s and a final extension at 72 °C for 10 min.The 20 obtained microsatellite markers were initially classified in polymerase chain reactions (PCR) according to their performance and degree of polymorphism in five different PCR products were analysed for SSR allele size via capillary electrophoresis on a GenomeLab GeXP DNA Genetic Analysis System (Beckman). Reactions were loaded as a multiplex analysis and included PCR products of three different fluorescently labelled primers . A subset of respective samples was genotyped twice to assess genotyping and allele-scoring error. Reactions that failed in the first run were also repeated at least twice. Allele sizes were determined using GenomeLab GeXP Version 10.2 (Beckman) software.O) and expected heterozygosities (HE) as well as deviations from Hardy–Weinberg equilibrium (HWE) and null allele frequency (F0) at each locus were calculated using Cervus version 3.0.319. Within population summary statistics such as number of alleles, HO and HE was computed with Arlequin version 3.520. In addition, the program Genepop version 4.7.521 was used to estimate the inbreeding coefficient (FIS) and to test for the presence and frequency of null alleles in the populations. To investigate population differentiation, a global estimate of FST as well as population pairwise measures of FST were estimated and tested for significance using FSTAT version 2.9.3.222, with a Bonferroni correction for multiple comparisons23 applied to all P values from FST estimates. A correction for a positive bias induced by presence of null alleles in the data set on FST estimates was performed using the program FreeNA24. To test for an isolation by distance a Mantel test was performed based on the relationships between genetic differentiation (represented by the linearized FST transformation FST/(1 − FST)) and the logarithm transformation of the geographic distances (in km) using the ISOLDE option in Genepop version 4.2. An analyses of molecular variance  was performed to assess hierarchical genetic population structures among populations from (1) two different geographical origins , (2) two different generations and (3) the four different landscape types surrounding respective vineyards.Descriptive statistics such as average number of alleles per locus obtained with the respective microsatellite marker or observed  iterations25. Subsequently, the method described by Evanno et al.27 implemented in the program Structure Harvester28 was used to infer the most likely value of K. Simulations in the program Structure were then run again for the most likely K with a burn-in period of 50,000 steps and 100,000 MCMC iterations.The genetic structure of the FIS) and habitat composition by fitting a general linear mixed model using the PROC MIXED of SAS ver. 9.4. In the model we considered the inbreeding coefficient (FIS) as dependent variable and the percentage of area covered by vineyards in a 500-m radius scale as continuous predictor; effect was tested with an F test (α = 0.05). The model accounted for country of origin and generation as random term. Degree of freedoms were estimated using Kenward–Roger correction29. Prior to the analysis data were checked for normal distribution and untransformed data were used.As inbreeding results from mating of related individuals and can be influenced by the availability of host plants in the immediate vicinity, one can assume that inbreeding is higher in landscapes dominated by monoculture vineyards. We assessed the relationship between the inbreeding coefficient , plum fruit moth Cydia funebrana (Treitschke) and European grapevine moth L. botrana. Two adult individuals per species obtained from laboratory cultures of the respective insect species were used for DNA extraction, PCR amplification and marker screening as described above.Amplification of the 6 SSR markers isolated from E. ambiguella populations analysed and data were thus not included in the subsequent analysis.Of the 20 markers analysed in an initial marker screening, ten showed stable and reproducible amplification patterns with distinct peaks present in capillary electrophoresis . Averaged over all 21 populations, null allele frequencies varied between 0.07% (marker Ea_2) and 47% (marker Ea_11) (Table Six polymorphic microsatellite markers with a stable amplification profile were further assessed for their suitability and were used to characterise 21 C. pomonella) individuals tested (data not shown). For none of the other E. ambiguella SSR markers developed in this study an amplification product was detected in individuals of the three other tortricid species tested, indicating that respective primer sequences and repeat motifs are not conserved in members of this family.SSR marker Ea_3 successfully amplified a fragment of 210 bp size in one of the two codling moth  excluded zero  implying a significant amount of genetic differentiation. If null alleles were excluded from the analysis according to the method described by Chapuis and Estoup24 and using program FreeNA the global FST value was similar  suggesting that the presence of null alleles in the dataset had no impact on the accurate estimation of FST.The overall FST values calculated among the 21 populations, 129 had a 95% CI excluding zero, suggesting a significant genetic differentiation between the respective populations . Except for two population pairs, all German populations significantly differed from all Italian ones with a moderate to pronounced level of differentiation. Genetic differentiation between Italian populations was often not evident and, in most cases, not significant with overall very low FST values).Of the 210 pairwise ns Table . PairwisP < 0.0001, Fig. P = 0.004, Fig. P = 0.004, Fig. We found significant relationships between genetic and geographic distances when all populations from both countries and both generations were included in the analysis  was influenced by habitat composition being positively associated with the percentage of area covered by vineyards , flights (generation) of moths or landscape type surrounding vineyards. AMOVA indicated weak but significant variance among E. ambiguella populations from the two different geographic regions , however almost 89% of the genetic variation was explained by variability within populations (model (1), Table FCT = 0.004, P = 0.19 for German and FCT = − 0.003, P = 0.76 for Italian populations) (model (2), Table E. ambiguella populations from the four different landscape types , yet in this model 92% of the genetic variation occurred within populations (model (3), Table To estimate hierarchical genetic structure of E. ambiguella populations. Results showed a moderate to high degree of diversity between E. ambiguella populations sampled in two viticultural regions in Germany and Italy. Moreover, a clear geographic structure was observed in our samples, evident through a significant isolation by distance and through results from Structure analysis. In the later, E. ambiguella populations were grouped according to their German or Italian origin in two clearly separated clusters. This indicates a limited genetic exchange between both geographic regions and thus a limited active or passive dispersal of moths from both countries. Active dispersal via flight of moths is unlikely to occur as adult grape berry moths have a relatively low active dispersal pattern and do not migrate over substantial distances32, however, human-aided dispersal resulting for example from the global trade of plant material could be one way for displacement of e.g. pupae overwintering on the grapevine plant.By using a new set of microsatellite markers this study is the first one to analyse genetic structures in European grape berry moth E. ambiguella populations included in this study had positive FIS values indicating a deficiency of heterozygotes. This might be due either to the presence of nonamplifying (or null) alleles which is frequently observed in Lepidoptera including the closely related species L. botrana17. Alternatively, a sampling bias might have caused the apparent heterozygote deficiency, producing a Wahlund effect33. Sampling via pheromone-trapping of adults might have actually sampled a pool of individuals from the respective vineyard and/or from subpopulations established on alternative host plants in close proximity. Thus we might in fact have sampled a mix of two or more genetically different subpopulations. A Wahlund effect and null alleles both increase FIS values and produce apparent heterozygote deficits.All E. ambiguella, and this can be one of the explanations for the differences observed here. A different dispersal capacity between the two E. ambiguella flights has implications on its management. First generation larvae infest flowers, while larvae of the second generation damage berries, causing weight loss and reduced berry quality. Accordingly, economic injury levels adopted for the second generation are much lower than those for the first one, with control intervention being unnecessary in most of the cases34; however, one can argue that intervention against the first generation brood can have consequences on the extent of second generation infestation. Data on inbreeding coefficients provided here suggest that grape berry moth populations of the first (derived from the first flight adults) and second (derived from the second flight adults) generation are not strictly related, with the first likely derived from adults which developed in the same vineyards, while the second originated from individuals coming also from surrounding areas. Consequently, the control of the first generation infestation cannot be justified as a preventive measure for the control of the second generation.The comparison between adults belonging to the first and second flights facilitated additional insights on factors affecting genetic structures of grape berry moth populations. In both German and Italian populations, inbreeding was higher in the first than in the second flight. We can assume that adults caught in pheromone traps in spring originated from overwintered pupae, thus from larvae which developed in these sites in the previous summer and fall. Adults collected during the second flight could include individuals coming from different vineyards and host plants occurring in the surrounded areas. Based on these results, we can speculate that dispersal among different vineyards and alternative host plants might be influenced by seasonal parameters and is apparently higher in the second flight than in the first one. An increase in population densities as well as higher temperatures during summer can promote the flight activity of E. ambiguella populations. Flight distances for grape berry moths are usually less than 100 m32 and adult moths tend to mate with individuals nearby in the vineyard right after emergence from pupae. Thus, a certain degree of isolation, with segregation into metapopulations in different vineyards, is likely to occur in diverse and fragmented landscapes which offer other host plants beside grapevine. This is supported by results of hierarchical genetic structure analysis of E. ambiguella populations. AMOVA indicated a weak but significant genetic variation among E. ambiguella populations sampled in vineyards surrounded by landscape types of different complexity and with different percentages of areas covered by grapevine plants. Insights in the effects of landscape features on genetic composition of pest insect populations is however rare so far. Codling moth (C. pomonella) populations in Chile showed a significant but weak genetic differentiation between adults collected in managed and unmanaged orchards, indicating active adult movement between both habitat types35. For melon aphids (Aphis gossypii Glover) it has been shown that genetic diversity of aphid populations on watermelons cultivated in greenhouses was significantly associated with certain landscape elements in two counties in China36. In contrast, a limited gene flow between grain aphid (Sitobion avenae Fabricius) populations colonizing cereal crops as well as uncultivated hosts growing in field margins was detected in an area in Western France37. Overall, research on interactions between landscape features and evolutionary processes such as gene flow and selection, a research topic referred to as landscape genetics38, is becoming increasingly important for understanding dispersal capability of pest insects as well as their natural enemies. In this regard, a recent review by Tscharntke et al.39 point to the fact that natural habitats surrounding agricultural fields can be a major source of pests if they provide an environment that is of greater benefit for pest insects than for natural enemies. Understanding the relevance of landscape composition on genetic structures of pest populations is also important in the light of insecticide resistance management and for the deployment of sustainable pest management strategies taking ecosystem services of landscape elements into account. The availability of molecular markers, like the SSR markers developed in this study, are a prerequisite for future studies on insect landscape genetics.A lower level of inbreeding was found in Italian compared to German populations, which might again be due to a Wahlund effect and/or the presence of null alleles as discussed above. Moreover, most of the German populations were collected in vineyards mainly surrounded by other vineyards, thus in intensively cultivated areas, while Italian populations were obtained from vineyards located in landscapes with a higher complexity and percentage of non-vineyard habitats . Habitat composition at 500 m’ radius scale, as considered in the present study, thus seems to affect the population structure of Supplementary Information.


Rhinichthys osculus) endemic to two basins (Owens and Amargosa) in the Death Valley ecosystem. These fish persist in the Mojave Desert as isolated Plio‐Pleistocene relicts and are of conservation concern, but lack formal taxonomic descriptions/designations. Double digest RAD (ddRAD) methods identified 14,355 SNP loci across 10 populations (N = 140). Species delimitation analyses [multispecies coalescent (MSC) and unsupervised machine learning (UML)] delineated four putative ESUs. FST outlier loci (N = 106) were juxtaposed to uncover the potential for localized adaptations. We detected one hybrid population that resulted from upstream reconnection of habitat following contemporary pluvial periods, whereas remaining populations represent relics of ancient tectonism within geographically isolated springs and groundwater‐fed streams. Our study offers three salient conclusions: a blueprint for a multifaceted delimitation of conservation units; a proposed mechanism by which criteria for intraspecific biodiversity can be potentially standardized; and a strong argument for the proactive management of critically endangered Death Valley ecosystem fishes.The tips in the tree of life serve as foci for conservation and management, yet clear delimitations are masked by inherent variance at the species–population interface. Analyses using thousands of nuclear loci can potentially sort inconsistencies, yet standard categories applied to this parsing are themselves potentially conflicting and/or subjective . One potential solution for consistent categorization is to create a comparative framework by accumulating statistical results from independent studies and evaluating congruence among data sets. Our study illustrates this approach in speckled dace (Leuciscidae:  Rhinichthys osculus), an endemic to two basins (Owens and Amargosa) in the Death Valley ecosystem (DVE). Our study offers three salient conclusions: a blueprint for a multi‐faceted delimitation of conservation units; a proposed mechanism by which criteria for intraspecific biodiversity can be potentially standardized; and a strong argument for the proactive management of critically endangered DVE fishes.The tips in the tree of life serve as foci for conservation and management, yet clear conservation unit delimitations are masked by inherent variance at the species‐population interface, and modern genomic applications can be difficult to parse for this purpose. Here, we present a study that provides a framework for parsing these data using speckled dace (Cyprinidae:  Yet, they often lack those distinct demarcations expected of their categorization and represent instead mere waypoints along evolutionary pathways  define the questions to be addressed, (b) detail different analytical approaches that quantify inter‐ and intraspecific diversity using NGS data, (c) place detected patterns within a spatial and temporal framework so as to understand underlying evolutionary and ecological drivers, and (d) combine our data with those available from prior publications and gray literature reports to evaluate diversity within the context of adaptive potential and ecological boundaries , within a region well understood with regard to biogeography and paleohydrology. We use our framework to develop a strong argument for the proactive management of critically endangered fishes in one of the world's most unique environments, the Death Valley ecosystem of arid southwestern North America.1.2The issues introduced above globally transcend regions, ecosystems, and organisms. We focus in this study on a desert ecosystem (Mojave Desert) with aquatic organisms largely restricted to freshwater springs  from the Death Valley ecosystem of southwestern Nevada and eastern California (Appendix R. o. nevadensis) is formally described , their hydrology differs. The high desert of the Owens Basin exists in the rain shadow of the eastern Sierra Nevada and accumulates surface water through snowmelt runoff from the surrounding mountains. However, available habitat for speckled dace has been systematically depleted by aqueducts constructed in 1913 and 1970 to supply the metropolis of Los Angeles. This has forced considerable reliance upon local groundwater resources Danskin,  which poThis study provides a comprehensive, population‐level evaluation of speckled dace as a case study for conservation unit delineation in a system with confused intraspecific variability. We sequenced thousands of nuclear loci (SNPs) so as to: (a) gauge gene flow within and among populations; (b) identify nuclear loci under selection as a proxy for local adaptation; (c) generate a phylogeny using species tree methods; and (d) employ MSC and machine learning algorithms to delimit intraspecific diversity. As a means to validate conservation units and underscore management, we then (e) interpreted the genetic data in the context of morphological and ecological descriptions available from published sources. This comparative framework also allowed us to interpret the evolutionary drivers that shaped this unique and endemic biodiversity.22.1N = 50 samples; Amargosa: 5 localities, N = 80 samples). Populations were evaluated from nine locations , and Amargosa Canyon). Sampling spanned 1989–2017, with Long Valley sampled twice  and Oasis Valley three times . Two closely related taxa served as outgroups: eastern blacknose dace  and Lahontan speckled dace , but excluding a population from a private pond (RUP) because of its uncertain origin. Samples representing the same locality but collected decades apart were grouped together for analyses presented here, but were treated separately in preliminary analysis (data not shown), and did not influence the final results. To account for incomplete lineage sorting, the reversible polymorphism‐aware phylogenetic model (pomo) was applied in iq‐tree v1.6.9  methods and unsupervised machine learning (UML) algorithms. Machine learning algorithms have been proposed as alternatives to MSC methods, which seemingly oversplit taxa under certain conditions . Samples were selected from the most recent temporal collection of each genetic cluster identified by admixture in order to provide the best representation of extant diversity. Results were ultimately congruent with prior analysis that involved greater numbers of samples (N = 60) and SNPs (N = 601), but included fewer populations  negatively impacted the final results.A MSC‐based method  between R. osculus and its sister taxon R. cataractae (Longnose Dace). This value was set as the mean of a gamma‐distributed prior. The lineage birth rate (λ) of the Yule model was fixed using pyule (https://github.com/joaks1/pyule). A λ‐value of 181.49 assumed tree height as one‐half of the maximum observed pairwise sequence divergence. Path sampling was set to 48 steps of 500,000 MCMC generations, with 100,000 discarded as burn‐in. Bayes factors (BF) were calculated from normalized marginal likelihoods  in BFD* using the mean pairwise sequence divergence (7.99 × 10K) was determined by partitioning around medoids (PAM) with gap statistic using k‐means clustering , using the 200 SNPs previously sampled for BFD*  and R. atratulus samples were excluded).Five UML algorithms were also applied  present in at least 33% of the ingroup samples. Mean sequencing depth per locus was 62.08× (σ = 21.17×). After filtering by bcftools, 14,355 loci were retained. Missing data  ranged from 8.42% to 70.96%  within individual samples. We attribute extraneous amounts of missing data (>50%) in a minority of speckled dace samples (N = 10) to be a consequence of either degraded or low‐yield DNA. No single population or collection event exhibited noticeably greater amounts of missing data.bayescan recovered 210 loci under positive selection, whereas lositan found 632. We cross‐referenced them to acquire a consensus of 106 loci (the “SNP‐select” dataset) representing a subsample of the 14,355 locus “SNP‐all” data.3.2Genetic diversity within the Owens and Amargosa basins was best represented by seven genetic clusters representing SNP‐all Figure  and SNP‐FST = 0.50; SNP‐select FST = 0.96; p < .001). Genetic divergence among admixture‐defined clusters was also high , but with variability among localities reduced within clusters . The proportion of genetic variance distributed among admixture‐identified clusters was greatest for SNP‐select  whereas for SNP‐all it was within sampling localities . The proportion of variance distributed among localities within clusters was very low for both  indicating that stochastic temporal sampling variance is unlikely the cause of the observed strong genetic differences among localities.AMOVA results for both datasets revealed high genetic divergence among localities . The second greatest comparison  also involved an Owens Basin population, the upstream location in Benton Valley (HAR). Similar trends were observed for SNP‐select, with the most notable exception being a lack of significant pairwise FST values when comparing temporal sampling events within populations, which strongly suggests that stochastic temporal sampling is not driving the differences among sites. Pairwise FST values were greater overall, with two Owens Basin locations again reflecting elevated divergence .Pairwise ts Table . All pai3.3newhybrids demonstrated a similar trend using the 390‐locus dataset filtered in genepopedit .A hybrid index was calculated for crosses between Ash Meadows (ASH) and the two Oasis Valley populations (AMA and RFO). Data represented fixed differences between the two populations  when compared with Oasis Valley speckled dace at Beatty (AMA). Only Ash Meadows samples from Bradford Spring (ASH) yielded a significant result with moderate bootstrap support when compared against Oasis Valley samples from Roberts Field (RFO).Results of de Table  were con3.4pomo and svdquartets results converged on the same tree topology. While the five Amargosa Basin sites formed a clade, Long Valley speckled dace (LVD) forced a paraphyletic Owens Basin group . The other uncertainty concerned the relationship between the downstream Owens River (ORB) and the upstream Benton Valley (HAR) populations, which together formed a moderately supported clade in both methods .p Figure . Most no3.5admixture . The next two most highly ranked models collapsed subspecies within the Amargosa Basin: the first collapsed Oasis Valley populations into a single entity (BF = 397.58), while the second grouped Ash Meadows with Amargosa Canyon (BF = 228.43).The BFD* analysis Figure  decisive3.6N = 13, x¯=25.67%, σ = 17.94%, min = 0%, max = 53.5%). All other individuals exhibited a much lower proportion of missing data . Clusters of individuals defined by missing data were typically small in size .The machine learning methods recovered a minimum of three clusters and a maximum of 10 Figure . Random An unnecessary division of populations occurred in two other instances. The t‐SNE PAM algorithm recovered two clusters within Oasis Valley yet neither was clearly defined by a geographic locality . A second instance occurred for Owens Basin localities (HAR and ORB) when evaluated with the RF isoMDS Hierarchical algorithm. Each cluster again contained a mixture of individuals from both localities, so both instances were therefore interpreted as “oversplitting.” Final UML classifications were “corrected” by removing spurious clusters, yielding a minimum of 3 clusters and a maximum of 9.Results of two algorithms (VAE and DAPC) matched BFD* results. Random Forest and t‐SNE methods varied in the number of clusters recovered, with most  finding fewer clusters than recovered through BFD*. The RF cMDS PAM algorithm was the lone exception. It produced results similar to BFD*, the exception being that nine total clusters were recovered [the ninth a result of Ash Meadows being split according to the two springs sampled in that refuge (ASH and ASR)].N = 3), corresponding to Amargosa Basin , Long Valley (LVD), and Owens Valley/ Lahontan speckled dace . The remaining RF and t‐SNE methods recovered 5–7 clusters, with Lahontan speckled dace (PRC), Long Valley (LVD), Ash Meadows (ASH), and Oasis Valley (RFO and AMA) consistently recognized. Discrepancies corresponded to splitting or lumping of Owens Valley populations , and the treatment of Amargosa Canyon (AMC) .All other RF and t‐SNE methods recovered fewer clusters. The RF isoMDS PAM method recovered the fewest , as a mechanism by which new questions relating to intraspecific genetic diversity could be addressed.One potential mechanism to interpret results of molecular studies that evaluate intraspecific diversity, and to translate them into management solutions would be to place them within a comparative framework with other available data. Here, the difficulty is to (a) know that such data exists, and (b) locate and access it for integrated analyses. In the present situation, this would provide the platform from which results of individual studies could be compiled in a consistent format, compared, and potentially categorized. A recent example is a platform that accommodates a trait‐based approach to classification by delineating taxa from which ancestral morphologies and their functions are reconstructed  Associate patterns of population‐level diversity with the consensus of species delimitation methods; (b) Address complications resulting from the hybrid status of Amargosa Canyon speckled dace; (c) Draw comparisons between our results and previous morphological work, as support of intraspecific divisions; and (d) Evaluate genetic variation in the context of previously recognized subspecific diversity. This allowed us to address the functionality of genomic data as a mechanism for parsing intraspecific biodiversity within both a systematic and conservation context. Finally, we discuss the specific conservation implications regarding the proposed speckled dace conservation units in the Death Valley ecosystem.4.2admixture‐defined populations in producing seven Death Valley lineages.MSC‐based species delimitation methods have been successful in delineating taxonomic units within problematic groups Hedin, . HoweverR. o. robustus).UML analyses have emerged as an alternative classifier to delineate groups. In comparison to our species delimitation results, UML analyses detected fewer clusters in all instances except two, with a mean of six following adjustments for spurious results due to missing data and oversplitting. The search for a consensus indicated a minimum of four unique conservation units: Long Valley, Owens Valley, Ash Meadows, and Oasis Valley Table . We consadmixture indicated differentiated populations within each. Species delimitation methods similarly concurred, with a minority  splitting Roberts Field (RFO) from the rest of Oasis Valley, whereas a majority  separated Benton Valley (HAR) from Owens River. The latter is the most intriguing candidate for designation as a separate conservation unit, due largely to its elevated divergence from other local populations, as measured via FST. However, a previous phylogenetic reconstruction based on restriction‐site mapping of mtDNA failed to distinguish it from Owens River  with R. o. nevadensis (Ash Meadows: ASH) reflects a spread from historical to approximately F2‐hybrid status. The hyde results indicate AMA as being the more probable Oasis Valley parental population than RFO, in that comparisons between the latter and R. o. nevadensis were not as well supported. The Amargosa Canyon samples were collected 10‐months following a substantial flood that temporarily reconnected this area with upstream populations . Interestingly, nonadmixed parentals and F1 offspring were not detected, and 10 months is inadequate time for speckled dace to yield F2 offspring  recognized it as discrete, while those that clustered it with other lineages did so inconsistently. The two RF isoMDS methods clustered Amargosa Canyon with Oasis Valley, while the t‐SNE Hierarchical method classified it with Owens River. Neither is consistent with the placement of Amargosa Canyon as sister to 4.4Next, we applied a comparative framework to solidify support for our conservation units. While speckled dace lineages were statistically significant on a genetic basis, they should also be validated morphologically, ecologically, and with additional life history data. This process acts to confirm them as distinct biological entities warranting conservation unit status. A morphological overview of putative subspecies in the Death Valley ecosystem focused on broad, regional trends, but it also found “... highly significant differences among all populations for all meristic and mensural characters” , and subspecies do seemingly segregate morphologically, albeit without statistical tests as confirmation. In contrast, multiple lines of genetic data provide clear signals of distinct entities, and inferences from modern genomic techniques also reinforce observed gaps in ecological data  as a distinct taxon and conservation unit. These taxa meet all three criteria for ESU designation: geographic isolation, genetic differentiation, and local adaptation (as detected via FST outlier SNPs).Modern genomic tools offer an in‐depth view of population histories and allow unique genetic lineages to be discriminated. In our study, they phylogenetically validate a rare situation where the majority (80%) of anecdotal subspecific designations (our OTUs) are recognized as genetically discrete units. We deem this as a response to the extreme geographic isolation imposed upon each lineage. Until formal descriptions can occur , we thus recognize Long Valley, Owens Valley, and Oasis Valley speckled dace as ESUs Moritz, , and subEndemicity resulting from habitat isolation provides a unique challenge for conservation. While isolated habitats are often prone to human disturbance, they are readily identified and monitored as well . Hydrological changes underlying these patterns occurred on considerably different timescales in the two basins. Loss of surface water in the Owens Basin is directly tied to anthropogenic activities during the past century, with available habitat being severely restricted . The issue is further compounded by a lack of policy explicitly addressing hybrids , deemed a hybrid between endangered gray wolf (Canis lupus) and coyote (Canis latrans) . Our results demonstrate the necessity of using multiple approaches in a comparative framework to diagnose conservation units , validate three lineages  as distinct ESUs, and argue that Amargosa Canyon speckled dace is eligible for protection as a DPS under existing environmental laws.Distinct speckled dace lineages within the Death Valley ecosystem represent Plio‐Pleistocene hydrological connections among basins. They are narrowly endemic and relictual components of more pluvial Plio‐Pleistocene ecosystems that now persist as small pockets within desert oases. The majority represent entities previously identified anecdotally, yet without formal description. Despite their academic recognition, legal protection is absent and their existence remains manifestly precarious, save for one described entity ; data curation ; formal analysis ; investigation ; methodology ; project administration ; writing – original draft (lead); writing – review and editing . Marlis R. Douglas: Conceptualization ; investigation ; methodology ; project administration ; writing – original draft (supporting); writing – review and editing . David D. Oakey: Conceptualization ; investigation ; writing – review and editing . Michael E. Douglas: Conceptualization ; investigation ; methodology ; project administration ; writing – original draft (supporting); writing – review and editing .Appendix S1Click here for additional data file.


Sceloporus arenicolus). This species is found exclusively in dune blowouts and patches of open sand within the shinnery oak-sand dune ecosystem of southeastern New Mexico and adjacent Texas. We find evidence of phylogeographic structure consistent with breaks and constrictions in suitable habitat at the range-wide scale. In addition, we find support for a dynamic and variable evolutionary history across the range of S. arenicolus. Populations in the Monahans Sandhills have deeply divergent lineages consistent with long-term demographic stability. In contrast, populations in the Mescalero Sands are not highly differentiated, though we do find evidence of demographic expansion in some regions and relative demographic stability in others. Phylogeographic history and population genetic differentiation in this species has been shaped by the configuration of habitat patches within a geologically complex and historically dynamic landscape. Our findings identify regions as genetically distinctive conservation units as well as underscore the genetic and demographic history of different lineages of S. arenicolus.Phylogeographic divergence and population genetic diversity within species reflect the impacts of habitat connectivity, demographics, and landscape level processes in both the recent and distant past. Characterizing patterns of differentiation across the geographic range of a species provides insight on the roles of organismal and environmental traits in evolutionary divergence and future population persistence. This is particularly true of habitat specialists where habitat availability and resource dependence may result in pronounced genetic structure as well as increased population vulnerability. We use DNA sequence data as well as microsatellite genotypes to estimate range-wide phylogeographic divergence, historical population connectivity, and historical demographics in an endemic habitat specialist, the dunes sagebrush lizard ( Patterns of population genetic diversity within species are shaped by both evolutionary and contemporary history . Though Stenopelmatus species  in southwestern North America, for example, revealed limited dispersal among populations, and identified a recent response to anthropogenic change )c change . A meta-c change . Alternac change ,9) even c change ). IdentiEcological specialists can have greater population genetic and phylogeographic structure than generalists because individuals and populations may be restricted to spatially isolated patches of suitable habitat –13. EcolLocal processes are often linked to patterns observed across long-term, evolutionary time scales and at broader spatial scales . ThuSceloporus arenicolus, is endemic to the Mescalero and Monahans Sandhills ecosystem of southeastern New Mexico and adjacent Texas [Sceloporus graciosus clade [S. arenicolus is a habitat specialist. Within this ecosystem, it only uses shinnery-oak sand dune formations with interconnected dune blowouts (sandy depressions created by wind) and in some cases shinnery hummocks in dunes with steep slopes [Quercus havardii) which stabilizes the dunes [S. arenicolus lizards demonstrate a nested hierarchy of habitat selection [The dunes sagebrush lizard, nt Texas ,22 . Int. IntS. as , the Claremont Colleges (protocol number not assigned), Texas A&M University , and Pacific University (Protocol: R-0026). Animals were captured and released, or euthanized with injection by MS222. Field research was approved by New Mexico Department of Game and Fish  and Texas Parks and Wildlife Department .Sceloporus arenicolus throughout their range  and two protein coding nuclear loci  used previously published primers [scar298anl  and scar875anl . All PCR were conducted in 10 μl total volume. Annealing temperature for the new nuclear loci was 54°C.We targeted two mitochondrial and four nuclear loci for DNA sequencing. PCR amplification of two mitochondrial loci . Heterozygous sites in nuclear sequences were called with the appropriate ambiguity code. Sequences at each locus were aligned using the MAFFT [We used 0.4 μl ExoSAP-IT (USB/Affymetrix) and 1.6 μl water to clean 5 μl of PCR product. One μl of clean PCR template was used in each cyclo-sequencing reaction using the same locus-specific primers used in amplification. Sequencing reactions were cleaned and run on an ABI 3730xl at the Duke Sequencing Facility. Chromatograms were verified and cleaned in Geneious R9 (he MAFFT  plug-in b) into a single alignment. Each of the four nuclear loci were treated independently. All sequences at each locus were aligned in Geneious and alleles at nuclear loci were determined using the program PHASE [Because the mitochondrion is inherited as a single unit without recombination, we concatenated the two loci (ND1 and Cyt-am PHASE  and the am PHASE .http://dx.doi.org/10.5061/dryad.sxksn0316)Nuclear microsatellite loci were developed from a 454-library enriched for microsatellite motifs developed at Cornell University Evolutionary Genetics Core Facility. After initial screening of loci, we used the Qiagen Type-It microsatellite PCR kit to genotype individuals at these loci plus seven previously published loci  in five Summary statistics. We used PAUP [sed PAUP  to detersed PAUP  to calcuHaplotype networks. We constructed parsimony networks in TCS [S. arenicolus. Because the results generated by network methods can be strongly influenced by missing data [s in TCS  for comping data , we firsPhylogenetic analysis. For the mitochondrial DNA, we estimated the phylogenetic relationships among S. arenicolus under both maximum likelihood and Bayesian frameworks. Urosaurus ornatus, Uta stansburiana, Phrynosoma coronatum, Sceloporus jarrovii, S. merriami, S. occidentalis, and nine individuals of S. graciosus were used as outgroups (following [sequence_cleaner.py [http://dx.doi.org/10.5061/dryad.sxksn0316). We estimated the best-fit model of sequence evolution at each codon position of each gene in DT-ModSel [ND1 and Cyt-b respectively. We estimated the phylogeny under a Bayesian framework in MrBayes v3.2.6 [ollowing ). Concateaner.py  . These and finer-scale delineations of phylogeographic regions were based on geographic sampling and clustering of mtDNA haplotypes in network and phylogenetic analyses. We used multilocus sequence data to construct extended Bayesian skyline plots in BEAST 2.5.0 [-8 substitutions/site/year and SD of 0.27 [ST 2.5.0 –59. EachST 2.5.0   assuming that the Monahans Sandhills populations were ancestral and of constant population size [-8 substitutions per site per year [ion size  S1 Fig)Hypothesiion size . Analyseper year  and nuclSample sizes, alignment lengths, the number of unique haplotypes, number of segregating sites, average nucleotide differences, and nucleotide diversity are reported in Mitochondrial haplotype networks revealed geographically associated haplotype groups for mtDNA that largely correspond to regions of grossly contiguous habitat ; S2 Fig.S. arenicolus populations, but do not form a single haplotype group. There is high sequence divergence among haplotypes from the Monahans Sandhills despite occurring in a relatively restricted geographic area and they are distantly related to Mescalero Sands haplotypes. The EA and EC areas each have unique haplotypes without a single, most common haplotype. The EB haplotypes fall out into two main groups, one that is equally distant to northern and southern Mescalero Sands haplotypes and one that is distantly related to all other recovered haplotypes . Monahans Sandhills populations were paraphyletic with respect to Mescalero Sands populations with the southern-most Monahans Sandhills individuals forming a weakly supported clade (PP = 0.866) sister to all other S. arenicolus. Among the remaining individuals, there is strong support for a Northern Mescalero Sands clade including individuals north of the skinny zone (PP = 0.976) and moderate support for a Southern Mescalero Sands–Monahans Sandhills clade that includes individuals south of the skinny zone and the northern and central Monahans Sandhills (PP = 0.935). Within the Northern Mescalero Sands clade, we recover support for some clusters of individuals, but do not find well-supported clades corresponding to distinct geographic regions. Individuals from region A, at the northern end of the range, form a basal polytomy relative to smaller, but otherwise well-supported clades containing most individuals from the remaining regions north of the skinny Zone and one individual at the northern end of region DA  was constrained in individual ABC simulations to occur after the divergence between the Northern Mescalero Sands and the other regions (Tanc), the median estimate for the former Texp1 precedes the median divergence time, Tanc (We recover strongest support (PP = 0.9991) for a divergence scenario that involved colonization of the Northern Mescalero Sands from Monahans Sandhills populations around 34.8 Kya (CI 17.7–108 Kya) followed by colonization of the Southern Mescalero Sands from Monahans Sandhills populations more recently, around 16.3 Kya  in the central Mescalero Sands. This narrow zone of habitat for S. arenicolus is indicative of a long-standing barrier to dispersal and is now a point of secondary contact between divergent Northern and Southern Mescalero Sands populations.ulations ,30. Shinenicolus ,23,31. WWe find shallow divergence, but distinct genetic diversity among the Northern Mescalero Sands regions indicating that habitat suitability also impacts population genetic connectivity at these finer spatial scales. Populations in some of these regions, like AB and BB, have diverged in isolation, suggesting a founder effect in line with the major direction of sand dune movement . We addiS. arenicolus have emphasized the importance of a network of suitable habitat at multiple spatial scales to support metapopulation dynamics and population persistence [S. arenicolus was a driver of this pattern. As such, because the fine-scale distribution of suitable habitat is critical for local presence of S. arenicolus, we suggest the composition and configuration of the landscape with respect to unsuitable habitat types determines patterns of genetic connectivity across the range. The divergences we detect reinforce that extensive habitat may be necessary to support gene flow among populations and that habitat quality and habitat configuration at finer scales may be of critical importance to identifying potential corridors. Importantly, it is clear that the shinnery oak-sand dune ecosystem is a dynamic landscape where the configuration of habitat patches can change over decades and millennia. We know from phylogeographic studies that specialists in changing environments undergo repeated episodes of isolation and divergence [The historical demography and patterns of divergence are reflected in the microsatellite data as well as the more slowly evolving mtDNA sequence data indicating that population structure is the result of longstanding habitat dynamics and restrictions to gene flow at multiple spatial scales, not just more recent anthropogenic change. Demographic studies of sistence . Landscasistence ,26. The vergence . While ivergence ). Sourcevergence ,33, and vergence ,64,66 invergence . The SouS. arenicolus are not surprising of a habitat specialist inhabiting a dynamic landscape. Based on demographic studies [S. arenicolus [S. arenicolus mirrors the hierarchical nature of their habitat preference: individuals require suitable blowouts within a matrix of shinnery oak, and populations are supported by a network of connected shinnery oak-sand dune complexes. While the genetic consequences of metapopulation dynamics have typically been explored at fine spatial and temporal scales, our phylogeographic study shows that these metapopulation dynamics may also leave their signature at broader spatial scales, in this case, across the range of this endemic lizard.The patterns of divergence and gene flow that we see in  studies ,33 and oenicolus ,69–71, iS. arenicolus [S. arenicolus requires contiguous shinnery oak-sand dune complexes for population persistence and that individuals do not disperse across areas that are not shinnery oak-sand dune. There is most certainly restricted gene flow and genetically independent populations within each phylogeographic regions that is not detectable in our dataset because of limitations to the temporal and spatial resolution of mitochondrial DNA sequences and microsatellite markers at fine-scales. Our findings highlight regions to be considered as genetically distinctive conservation units and underscore the unique genetic and demographic history of different regions within the range of S. arenicolus. Continued fragmentation of the shinnery oak-sand dune ecosystem increases the likelihood that ancestral diversity and unique evolutionary lineages will be lost.The shinnery oak-sand dune habitats of Mescalero Sands and Monahans Sandhills have experienced severe habitat degradation and fragmentation, particularly in the southern portions of the range of enicolus ,19. Receenicolus ,19,30,32enicolus ,30,32) mS1 TableCharacteristics of microsatellite loci, primers used, and multiplex conditions for 31 loci originally screened. Annealing temperatures and including/exclusion of Q-solution refers to the Type-It Multiplex Kit (Qiagen). Twenty-seven of these loci were included in final analyses.(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S1 Fig(PDF)Click here for additional data file.S2 FigAlternative versions for individuals with color vision deficiencies. Circles represent unique haplotypes with the size of the circle corresponding to the relative abundance and the color/pattern referring to the region of origin of individuals with that haplotype (see boxes in upper left representing geographic approximations of each region). Lines connecting haplotypes represent one mutational step. Small white circles represent unsampled haplotypes.(PDF)Click here for additional data file.S3 FigSeparate networks for each of the four nuclear genes sequenced. Circles represent unique alleles with the size of the circle corresponding to the relative abundance and the color referring to the region of origin of individuals with that haplotype. Lines connecting haplotypes represent one mutational step. Small white circles represent unsampled haplotypes.(PDF)Click here for additional data file.S4 FigAlternate version of nuclear haplotype networks for individuals with color vision deficiencies. Separate networks for each of the four nuclear genes sequenced. Circles represent unique alleles with the size of the circle corresponding to the relative abundance and the color referring to the region of origin of individuals with that haplotype. Lines connecting haplotypes represent one mutational step. Small white circles represent unsampled haplotypes.(PDF)Click here for additional data file.S5 FigAnalyses conducted in BEAST for each of the five major regions  utilizing mitochondrial and nuclear sequence data.(PDF)Click here for additional data file.


Picea rubens Sarg.), an endemic and emblematic tree species of eastern North America high‐elevation forests that are facing the combined threat of global warming and increasing human activities. We sampled a total of 340 individuals, including populations from the current core of the range in northeastern USA and southeastern Canada and from the southern portions of its range along the Appalachian Mountains, where populations occur as highly fragmented mountaintop “sky islands.” Exome capture baits were designed from the closely relative white spruce (P. glauca Voss) transcriptome, and sequencing successfully captured most regions on or near our target genes, resulting in the generation of a new and expansive genomic resource for studying standing genetic variation in red spruce applicable to its conservation. Our results, based on over 2 million exome‐derived variants, indicate that red spruce is structured into three distinct ancestry groups that occupy different geographic regions of its highly fragmented range. Moreover, these groups show small eN, with a temporal history of sustained population decline that has been ongoing for thousands (or even hundreds of thousands) of years. These results demonstrate the broad potential of genomic studies for revealing details of the demographic history that can inform management and conservation efforts of nonmodel species with active restoration programs, such as red spruce.Understanding the factors influencing the current distribution of genetic diversity across a species range is one of the main questions of evolutionary biology, especially given the increasing threat to biodiversity posed by climate change. Historical demographic processes such as population expansion or bottlenecks and decline are known to exert a predominant influence on past and current levels of genetic diversity, and revealing this demo‐genetic history can have immediate conservation implications. We used a whole‐exome capture sequencing approach to analyze polymorphism across the gene space of red spruce ( Current populations of red spruce are mainly distributed in mountainous locations across the northeastern USA and the cool maritime climates of eastern Canada. However, this species also occurs in fragmented patches at high elevations along the Appalachian Mountains in the Mid‐Atlantic and southeastern USA , its closest relative. Further, while there is some indication that red spruce populations show differences in genetic ancestry corresponding to different regions within its highly fragmented range  to determine the effectiveness of whole‐exome capture sequencing at low coverage to investigate genome‐wide patterns of population genetic diversity; (b) to investigate signatures left by past demographic events on the current genetic structure across red spruce's range; and (c) to estimate regional differences in 22.1We sampled 65 populations and 340 individuals of red spruce across the entirety of its range in eastern North America Table . Samples2.2Bait design—Our aim in the bait design was to include exomic regions from as many genes as possible, together with upstream and downstream regulatory regions and intergenic sequences between nearby genes. To design the capture baits, we used two reference transcriptomes of white spruce (Picea glauca), a closely related species of red spruce  to an average size of 400 bp followed by end‐repair reaction, ligation of an adenine residue to the 3'‐end of the blunt‐end fragments to allow the ligation of barcoded adapters, and PCR amplification of the library. SureSelect probes (Agilent Technologies) were used for solution‐based targeted enrichment of pools of 16 libraries, following the SureSelectxt Target Enrichment System for Illumina Paired‐End Multiplexed Sequencing Library protocol. Libraries were sequenced on an Illumina HiSeq X to generate paired‐end 150‐bp reads.Capture2.3Read mapping—Libraries were demultiplexed, clipped of Illumina adapter sequences, and trimmed of low‐quality flanking sequence (Q < 20) using a sliding window of 6 bp using Trimmomatic , we compared the performance of three commonly used aligners: BWA  greatly increases the probability that only one of the two chromosomes of a diploid individual gets sampled during sequencing, leading to uncertainty in genotype calling , setting a minimum MapQ score to keep a read to 20 (“‐minMapQ 20”), a min nucleotide Q score to consider a site to 20 (“‐minQ 20”), a minimum number of 2 individuals with coverage to keep a site (“‐minInd 2”), a maximum of 17 reads to estimate genotype likelihood for one individual (“‐setMaxDepthInd 17”), a minimum number of 15 reads across the complete sampling to estimate genotype likelihoods for a site (“‐setMinDepth 15”), keeping only biallelic sites , and performing the base alignment quality  genome, available from congenie.org . To perform the PCA, we used the ‐doCov option of ANGSD to produce a matrix of genetic covariance among all 339 individuals, which we used to find the eigenvalues of the matrix and plot the eigenvectors based on individual loadings. We also estimated pairwise population fixation index (FST) values between each pair of the three regional subpopulations . Regions were designated a posteriori to investigate potential differences in regional genomic diversity and effective population size dynamics based on clear clustering of individuals in the PCA .We assessed the genetic structure of red spruce through principal component analysis (PCA) from the genotype likelihoods produced with ANGSD and keeping only sites at least 500 bp apart from each other to avoid linkage disequilibrium (see Results section: θw) , pairwise diversity (π), and Tajima's D were calculated using the program ANGSD and its function “thetaStat”  as a function of physical distance (bp) based on the equation initially proposed by Hill and Weir  across the evolutionary history of our sample. We converted from coalescent units to absolute units of population size and time assuming a generation time of 29 years , the timing of Core divergence (TCore), the ancestral population size (Nanc), the current population sizes , and the migration rate between each pair of regions . The parameters were estimated running 30 independent maximizations of the likelihood based on the observed 3‐dimensional joint SFS derived from the three regional SFS, and retaining the estimate with the highest likelihood. We then performed 100 parametric bootstraps to obtain the 95% confidence interval for each parameter estimate. To avoid making the models overly complex, we modeled changes to eN as discrete shifts that occurred simultaneously with the timing of population divergence.As a complement to the STAIRWAY PLOT method, we investigated the history of L), we did not convert coalescent parameter values into absolute estimates of eN or time. Instead, we compared θ per site over time.Finally, to evaluate the potential impact of non‐neutral sites on the demographic inferences, we reperformed the STAIRWAY PLOT analysis using only the synonymous and intronic sites identified from the variants' annotation. We followed the same procedures and used the same parameters as above. However, because we used only a subsample of sites from the full dataset, and it was not trivial to accurately estimate the total length of the covered genome consisting of just synonymous and intronic sites  and 1,372,627 polymorphic sites. For 1,363,244 of these variants (99.3%), we found a reliable correspondence in the Norway spruce genome and were able to annotate them Table , we foun3.3The PCA performed on the 339 individuals pruned dataset  identified three well‐differentiated genetic groups, corresponding to three different genetically and geographically defined regions across the species range: the main northern part of the range (“Core”), a transitional or marginal region in Pennsylvania (“Margin”), and a low latitude trailing edge (“Edge”) region in the southern Appalachians Figure . The firFST values <0.03. The highest estimates were found between Margin and Core (FST = 0.0275) and between Margin and Edge (FST = 0.0273), while the FST value between Core and Edge populations was 0.0229.Population fixation indices were low among the three regions with all pairwise 3.4θw, pairwise nucleotide diversity (π), and Tajima's D parameters. The SFS used to infer θw and Tajima's D were thus estimated from a genetic dataset considering only sites present in all three regional populations, resulting in a dataset of 42,328,740 covered bases  and 1,372,627 polymorphic sites.Considering the signal of genetic structure observed in the PCA, we analyzed the three different genetic regions  independently for the pattern of LD decay, Watterson's 2r was .043 (SD = 0.093) and LD decayed rapidly, with 2r dropping below .1 within less than 100 bp. The rate of LD decay when estimated independently for the three regions was strongly concordant for Core, Margin, and Edge datasets. The distance at which 2r was half of the initial value was 21, 17, and 18 nucleotides for Core, Margin, and Edge regions, respectively. The per‐region distribution of Tajima's D estimates showed value close to 0 for the Core region  and positive values for the Margin , and the Edge  regions  and pairwise divergence (π) were also very similar among the three regions , while Margin and Edge populations returned a mean per‐site value of 0.0046 (SD = 0.0043) and 0.0044 (SD = 0.0049), respectively. Similarly, π was 0.005 for Core (SD = 0.0063), 0.0054 for Margin (SD = 0.0061), and 0.0054 for Edge (SD = 0.0061).A very low level of linkage disequilibrium was observed among loci Figure . The meas Figure , suggests Figure . The Cor3.5θw and Tajima's D. STAIRWAY PLOT analyses revealed that red spruce has experienced a strong decline in Ne for hundreds of thousand years  with an acceleration of the decline visible between 600 and 400 kya  for the Core, 4,655 individuals  for the Margin, and 3,015 individuals  for the Edge regions. It is important to note that inferred timing and eN estimates are directly dependent on the generation time and the mutation rate used for the analyses (respectively 29 years and 2 × 10–9 mutation per base and per generation). Hence, eN estimates would be larger  if the true mutation rate was slower (or faster) than assumed.We used the same SFS to infer demographic history as we used to infer s Figure . The thrs Figure . The decNe estimates returned by Fastsimcoal2 were 39,143  for Core, 6,919  for Margin, and 11,812  for Edge, consistent with and within the confidence limits of the STAIRWAY PLOT results. The two events of divergence between the three regions occurred close together in time, both around 9,000 years ago, with an initial divergence of Core population 8,874 years ago  and a split between Edge and Margin 8,845 years ago  (Table NANC) of 1,036,314 individuals . This value was consistent with the values of Ne observed in the STAIRWAY PLOT 300 thousand years ago  and the Margin region Figure , the lattheta per site over time with either the full sampling or only the synonymous/intronic sites , and both agreed that red spruce has undergone a precipitous population decline to its current eN below 20,000 individuals. The first major demographic event recorded by the STAIRWAY PLOT analyses was a strong bottleneck occurring ~400 kya and ending with a loss of 65% of Ne . The timing of this bottleneck in the mid‐Pleistocene is congruent with the timing proposed in the literature for the initial divergence of red spruce from black spruce during the Pleistocene  found evidence of a more recent bottleneck in Ne from 7,209 to 535 occurring in the early Holocene, approximately 11,000 years ago. We suggest that these varied estimates, which all indicate a bottleneck in Ne but with different timing and magnitude, are consistent with the continuous nature of the demographic decline shown by the STAIRWAY PLOT analysis  and generation time (29 years) that represent our best available information for red spruce. As a result, while the general trends appear quite robust, the exact timing and magnitude of changes in Ne should be interpreted cautiously. For example, if the true generation time is longer than we assume, then the timing of demographic events we report in this study would be pushed back; likewise, a higher mutation rate would both decrease the value of Ne and move the timing of events forward.It is important to note that the approaches we used for demographic inference produce scaled parameter estimates of population diversity and time that can be converted to absolute units of Ne in red spruce, the exome data also suggested genetic subdivision across the range. PCA performed using genotype likelihoods showed a clear genetic structure, with three main clusters that aligned closely with the north–south axis of range fragmentation  species, have received considerable attention with published whole‐exome sequencing datasets for Norway spruce (P. abies)     in this study, four of the six most common spruce species present in North America now have whole‐exome polymorphism data available, opening the door to comparative analyses in this ecologically and economically important genus.Relatively few genetic studies have been conducted in red spruce, and most are based on a small set of marker loci that have revealed limited genetic diversity, especially compared to its sister taxon black spruce  even though the probes were designed based on expressed sequences  sampled from across the species' range at a cost of approximately $110 USD/exome. Given the expected low per‐site coverage, a key component of our strategy was to make use of population genomic pipelines that avoided hard‐calling genotypes and instead used genotype likelihoods to keep the uncertainty associated with each site while maximizing use of all of the data  and showed a rapid decay with physical distance . Rapid decay of LD suggests a historically large mating population, and is consistent with levels of LD found in other coniferous tree species (Jaramillo‐Correa, Verdú, & González‐Martínez, Picea.The level of LD found for red spruce was very low (mean The presence of low LD has implications for the efficacy of genome scans for genes under selection (Kim et al., 5eN values found in this region, these restoration programs are probably key to preserving red spruce populations in the south of its range, at least at short‐ to medium‐term temporal scales.Conserving biodiversity requires a good understanding of the factors impacting species genetic variability and evolutionary potential. For red spruce, it seems clear that the recent population dynamics have been strongly influenced by human activities. During the two last centuries, acid rain, logging, and fires have tremendously impacted the survival capacity of the species throughout its range (Mathias & Thomas, Ne, even during or in between the different interglacial periods of the late Pleistocene. This sustained decrease in Ne probably dates back to the very beginning of red spruce as an incipient species. Red spruce is also a relatively young species (Lockwood et al., However, when looking at a longer timescale, the global trend of the species' demographic history lacks any sign of stabilization or expansion in None declared.Figure S1Click here for additional data file.Figure S2Click here for additional data file.Figure S3Click here for additional data file.Figure S4Click here for additional data file.Figure S5Click here for additional data file.Table S1Click here for additional data file.


Short tandem repeats (STRs) are essential genetic markers for forensic applications and population estimations; thus the population genetics of STR loci have been extensively studied and discussed.In the present study, we detected 21 autosomal noncombined DNA index system (non‐CODIS) STR loci in a Chinese Han population from Shanghai, calculated their forensic parameters and analyzed their genetic relationships with reported reference populations in mainland China.A total of 173 alleles were observed, with corresponding allele frequencies from 0.0020 to 0.5512. The cumulative power of discrimination (CPD) and the cumulative probability of exclusion (CPE) values of the 21 STR loci were 0.999999999999999999997337058271 and 0.99999953732495, respectively. The results of interpopulation differentiation, phylogenetic, multidimensional scaling, and structure analyses indicated a closer genetic relationship of the studied population with Han populations from other regions of China than with other populations.The 21 STR loci exhibited high genetic polymorphism in the studied Shanghai_Han population and could be used for forensic applications and population genetic studies. Twenty‐one autosomal noncombined DNA index system (non‐CODIS) STR loci were detected in a Chinese Han population from Shanghai. Forensic potential was estimated by calculating forensic parameters, such as CPD and CPE. Genetic relationships of the studied population and other reported reference populations in Chinese mainland were analyzed. With a history of more than 2,000 years, Shanghai was a small fishing village until it began to serve as a major trading port during the Tang Dynasty. Before the liberation, Shanghai received a large Han population who migrated from the central plains due to the war. Since the founding of the People's Republic of China, Shanghai has attracted a large number of migrants from diverse regions of China due to its rapid economic development. Mandarin is widely used as the official language in Shanghai, and the Shanghai dialect, a fusion of dialects brought by immigrants from the surrounding areas, is also widely used in Shanghai. Gene flow and exchange lead to continuous genetic admixture in Shanghai with the frequent inflow of populations. It is thus worthwhile to obtain an overview of the features of genetic polymorphism in the Han population in Shanghai.Shanghai, located on the eastern coast, was listed as the largest city in China by population according to the 2010 population census. There are more than 26 million citizens in Shanghai as of 2019, and approximately 98.8% of the residents are of Han Chinese ethnicity. Approximately 9 million Shanghai residents are long‐term migrants from Anhui (29.0%), Jiangsu (16.8%), and Henan (8.7%), among other regions (−3)   are DNA segments that consist of 2–100 nucleotides repeated in tandem. These repetitive elements make up approximately 3% of the human genome, and most of them are located in noncoding regions. STRs have relatively high estimated mutation rates  that form the backbone of the U.S. national DNA database. Many of these same STR loci are used by other countries around the world. Population data on CODIS STRs in the Shanghai_Han population have been reported and updated for routine forensic applications, such as personal identification and paternity testing. However, extra STRs are needed, since joint analysis of non‐CODIS STRs and CODIS STRs has been reported to be significantly informative in identifying problematic kinship cases when a definite conclusion could not be obtained with only the profile of CODIS STRs  includes 21 autosomal STR loci  and amelogenin. Amplified PCR products were separated by capillary electrophoresis in an ABI PRISM 3130xL Genetic Analyzer (Applied Biosystems). Alleles were determined according to the provided allelic ladders by GeneMapper® ID software v3.2 (Applied Biosystems). All experimental procedures were performed according to laboratory internal control standards.The extracted DNA was amplified with the GeneAmp PCR System 9700 (Applied Biosystems) according to the manufacturers’ recommendations. The AGCU2.32O (AGCU ScienTech Corporation) was used as a negative control for each batch of amplification and genotyping. Moreover, the laboratory has been accredited in accordance with ISO/IEC 17025:2005 and the China National Accreditation Service for Conformity Assessment (CNAS) (Registration No. CNAS L4476).We strictly followed the recommendations of the Chinese National Standards and Scientific Working Group on DNA Analysis Methods SWGDAM,  and the 2.4FST values and corresponding p‐values were calculated by analysis of molecular variance (AMOVA) using Arlequin v3.5.2 software between the Shanghai_Han population and 23 other populations in mainland China for which data were available, including the Liaoning_Han , and the plot of optimum K determined by STRUCTURE HARVESTER v. 0.6.94 . Nei's genetic distances, Cavalli‐Sforza genetic distances, and Reynolds genetic distances were computed separately with the PHYLIP 3.695 software , expected heterozygosity (HE), typical paternity index (PI), matching probability (MP), power of discrimination (PD), polymorphism information content (PIC), probability of paternity exclusion (PE), cumulative power of discrimination (CPD), cumulative probability of exclusion (CPE) and Hardy–Weinberg equilibrium (HWE) tests of the 21 STR loci were calculated by Modified‐PowerStat software (version 1.2) . Therefore, the 21 tested non‐CODIS autosomal STR loci could be treated as independent loci in the following analyses. The lowest values of HE, HO, PE, and PIC were observed at locus D1S1627, equaling 0.6093, 0.6063, 0.2958, and 0.5539, respectively, while the highest values of these parameters were 0.7958, 0.8627, 0.7200, and 0.7681, respectively, at locus D19S433. The MP values ranged from 0.0061 at locus D5S2500 to 0.2469 at locus D1GATA113. In addition, the PD values ranged from 0.7531 at locus D1GATA113 to 0.9939 at locus D5S2500. The CPD and CPE values of these 21 STR loci were 0.999999999999999999997337058271 and 0.99999953732495, respectively, which indicates that the 21 non‐CODIS autosomal STR loci were highly polymorphic and appropriate for individual identification and paternity testing in the studied population. On the other hand, the CPD and CPE values of the 13 CODIS STRs in the studied population were 0.999999999999992 and 0.999988192673092, respectively, revealing that the utilization of non‐CODIS STRs could significantly improve the discrimination efficiency of the testing system, thus providing solutions for intricate forensic cases. Raw genotyping data for the 21 tested non‐CODIS STR loci are available upon request to daisydaisysun@163.com.The allele frequencies and forensic parameters of the 21 non‐CODIS autosomal STR loci are listed in Table 3.2FST values and corresponding p‐values are given in Table FST value (0.00268) was observed between the Fujian_She and Gansu_Tibetan group, whereas the smallest FST value (0.00000) was found three times: between the studied population and Henan_Han group, between the Xinjiang_Xibe and Chengdu_Han group, and between Mongolian and Gansu_Yugu group. In general, the FST values increased with greater geographic distances. Based on the maximum value of corresponding FST, the interpopulation differentiation does not affect the effectiveness of this set of markers for forensic application in mainland China. The result of multidimensional scaling analysis is shown in Figure The pairwise K = 2–6 with five runs was carried out; then the optimum K was selected by STRUCTURE HARVESTER v.0.6.94 and the results are shown in Figure K = 3 was the most appropriate configuration. In the bar plot, one color represents each ancestry origin, and one bar represents each individual. One bar with several colors indicates an individual with admixed ancestry. From the bar plot, different patterns of ancestry components distribution were detected among the analyzed populations. Significant difference was reflected between the Fujian_She and the Xingjiang_Kyrgyz, which is in line with their geographic distance. Consistent with the phylogenetic and multidimensional scaling analyses, the results of the structure analyses showed that the tested Shanghai_Han population shares more similarities with Han populations from other regions of China than with other populations. The genetic components of the analyzed minorities differed from those of the Han populations. No additional subtle stratification was observed by further increasing the K value.The population structure analyses were performed on the basis of the genotypes for the same 21 STR loci among the studied Shanghai_Han and other previously published populations by the STRUCTURE program. Each of 3.3Nei's genetic distances, Cavalli‐Sforza genetic distances, and Reynolds genetic distances were all analyzed based on 21 autosomal STR loci. The phylogenetic trees constructed based on the genetic distances are shown in Figure N, where N is the effective population size. Thus if population size doubles, genetic drift will take place more slowly, and the genetic distance will be expected to increase only half as fast with respect to time. Reported simulation studies showed that the Cavalli‐Sforza distance is more sensitive in distinguishing genetically similar populations and that the Reynolds genetic distance provides the highest sensitivity for highly divergent populations. It is also suggested that using the Cavalli‐Sforza distance may provide less power for studies concerning human migration history (Libiger, Nievergelt, & Schork, The three employed measures all assume that differences between populations arise from genetic drift. However, there are somewhat different assumptions. Nei's distance is formulated for the “infinite iso‐alleles” model of mutation, in which each mutant forms a new allele. It is assumed that all loci have the same rate of neutral mutation and that the genetic variability in the population is initially at equilibrium between mutation and genetic drift, with the effective population size of each population remaining constant. Therefore, Nei's genetic distance is expected to increase linearly with time. However, the other two measures assume that frequency changes are results of genetic drift alone. The genetic distances under this assumption are expected to increase linearly with the sum over time of 1/4In conclusion, the results suggested that the 21 non‐CODIS STR loci were highly polymorphic in the tested Han population from Shanghai and hence could be utilized in forensic individual identification and parentage testing. These population data of the STR loci could be useful to enrich genetic information resources and provide reference data for population genetic studies in the future. Moreover, the interpopulation comparisons revealed population differentiation and assimilation of the Shanghai_Han and 23 other populations.The authors declare that they have no conflict of interest.K.S. and J.X. were involved in study design. C.S., and Y.L. were involved in experimental work. H.X. provide support. Z. Zhou, H.X., Y.Z., and Z.P. were involved in data collection and analysis. Z. Zhou. and K.S. were involved in writing of the initial manuscript. Z. Zhao and Q.T. were involved in revision. All authors reviewed the manuscript.Samples were collected upon approval of Ethics Committee at Fudan University, P. R. China. This study was approved by the Ethics Committee of Fudan University, P. R. China. Click here for additional data file. Click here for additional data file. Click here for additional data file.