Drosophila simulans founder populations, which were adapting to the same novel hot environment, uncovered very different architectures—either many selection targets with large heterogeneity among replicates or fewer selection targets with a consistent response among replicates.Understanding the genetic architecture of temperature adaptation is key for characterizing and predicting the effect of climate change on natural populations. One particularly promising approach is Evolve and Resequence, which combines advantages of experimental evolution such as time series, replicate populations, and controlled environmental conditions, with whole genome sequencing. Recent analysis of replicate populations from two different Here, we expose the founder population from Portugal to a cold temperature regime. Although almost no selection targets are shared between the hot and cold selection regime, the adaptive architecture was similar. We identify a moderate number of targets under strong selection  and parallel responses in the cold evolved replicates. This similarity across different environments indicates that the adaptive architecture depends more on the ancestry of the founder population than the specific selection regime.These observations will have broad implications for the correct interpretation of the genomic responses to a changing climate in natural populations.The online version contains supplementary material available at 10.1186/s13059-021-02425-9. Adaptation of natural populations to environmental change may either occur from standing genetic variation or by the acquisition of new mutations. The relative importance of these two paths crucially depends on the underlying adaptive architecture  of the fSelection targets Table  are idenThe key concept of polygenic adaptation is that multiple loci are contributing to the phenotype, and rather than focusing on particular loci, the collective effect of all loci needs to be considered to estimate the phenotypic value of a given trait. The infinitesimal model –20 is thEven when these conditions are relaxed and a distribution of effect sizes with some large effects is considered, no pronounced allele frequency changes are expected when the populations are large and in mutation selection equilibrium: targets with large effects are segregating at low frequencies only and do not contribute much to the phenotypic variance of the population upon which selection is operating , 25. TheThe consequence of this genetic redundancy is that the contribution to the phenotype can be highly heterogeneous for individual loci in differentiated populations if they vary in frequency. This expectation nicely conforms with empirical data, mostly from QTL studies, which find heterogeneous sets of contributing loci among different populations –36. In tSelection signatures not shared among natural populations are difficult to interpret, as the distinction between population-specific selection targets and false positive/negative signals can be challenging given the high impact of a largely obscure demography on selection signatures –42. ExpeTwo previous experimental evolution studies conducted in the same novel hot laboratory environment showed parallel fitness increase among replicates . Five replicate populations originating from the Portuguese founder population described by Mallard F, Nolte V, Tobler R, Kapun M, and Schlötterer C )D. melanogaster experiment, where replicate populations were exposed to the same hot and cold temperature regimes [This implies that different loci are contributing to adaptation in hot and cold environments - irrespective of whether a highly or moderately polygenic architecture is assumed. A very similar lack of shared candidates was also noticed in a  regimes . This ex regimes , 93.D. melanogaster populations [The observation of different selection targets in hot and cold environments is particularly interesting, because seasonal changes were found to be associated with cycling allele frequencies in natural ulations , which sD. subobscura populations with different genetic backgrounds also observed very little overlap in the genomic regions responding to a common selection regime [Various studies, mainly using QTL mapping and GWAS, have identified different loci contributing to the same trait in diverged populations –36, 95. n regime . Hence, Although more experiments are needed to nail down why the genetic architecture of temperature adaptation is highly dependent on the founder population and not on the selection regime, our results have important implications for all studies attempting to characterize the response of natural populations to climate change. The analysis of a single population cannot be sufficient to understand the genetic basis of adaptive traits. Thus, multiple diverged populations need to be studied to reach conclusions that can be generalized beyond a limited number of focal populations.Unless stated otherwise, analysis was conducted using R v3.6.1 .Drosophila simulans population in Northern Portugal in the summer of 2008 and used to establish 250 isofemale lines. These lines were kept for 10 generations in the laboratory before starting the experiment. Mated females of all lines were used to create the starting populations. Ten replicates were created by combining an equal number of flies from each line. Five of the replicates were then kept in a 12 h:12 h day and night cycle with temperatures of 20 °C during the day and 10 °C during the night (cold regime). The population size was kept constant at 1250 per replicate in non-overlapping generations.The set-up of the evolution experiment is described in detail elsewhere . In briehot regime).The other five replicates were used to start the evolution experiment described by Mallard F, Nolte V, Tobler R, Kapun M, and Schlötterer C , which wFor the founder population, we used the sequences described by Mallard F, Nolte V, Tobler R, Kapun M, and Schlötterer C , but addSequencing data from the time points F11, F21, F31, F41, and F51 in the cold regime were newly generated from pooled females and males. Paired-end libraries were generated with different protocols and sequenced on different Illumina platforms (see Supplementary Table S1).D. simulans genome sequence created by Palmieri N, Nolte V, Chen J and Schlötterer C [Sequencing reads were trimmed using Readtools TrimFastq version 0.2 . with thtterer C  was usedtterer C , three dtterer C  with partterer C  with deftterer C  with parThe intermediate time points  used for the detection of selected haplotype blocks were mapped with novoalign only, as this mapper is known to estimate allele frequencies most accurately .Mapped reads were filtered for mapping quality ≥ 20 and proper pairs using SAMtools view version 1.3.1 . Duplicaflybase.org (FB 2018_06), and search engine NCBI/RMBLAST v 2.2.27+ were used for repeats > 500 bp. All sync files were masked for these repetitive regions and for known Y chromosome translocations [Single nucleotide polymorphisms (SNPs) were called from the founder population by creating sync files from BAM files as described above but filtering for polymorphic sites that had a mapping quality of at least 30 and a minimal count of at least 5 and were detected by the three mapping algorithms in all founder replicates. Filtering resulted in 3.8 million SNPs that were used for further analysis. Indels were detected using the PoPoolation2 identify-indel-regions.pl script, and transposable elements were detected with repeatmasker v 1.332 . Custom ocations  using th2 tests were conducted to compare the results of the different mappers per replicate and time point. After correcting for multiple testing using the Benjamini-Hochberg procedure [To correct for false positive outlier SNPs created by libraries with different insert sizes , mappingrocedure , only SN2 tests which are adapted for genetic drift and pool sequencing noise as implemented in the R package ACER version 1.0 [10 kb, method P.planI, pool size and census size 1250) and used for CMH and χ2 tests. The CMH test was performed using all population replicates per time point whereas χ2 tests were performed for each replicate separately. All results were corrected for multiple testing (Benjamini-Hochberg). Finally, candidate SNPs detected by either test (p.adjust < 0.05) were combined to include consistent responses across replicates (CMH test) and replicate-specific responses (χ2 test).Candidate outlier SNPs were detected in the filtered sync files created from novoalign mapping results after correcting for false positive outliers as described above. Allele frequency changes between the founder and the most evolved population were analyzed using CMH and χsion 1.0 . SNPs wision 1.0  with theSelected haplotype blocks were reconstructed from candidate SNP allele frequency data of all time points and replicates using the R package haplovalidate with MNCS of 0.01 . Haploty2 test result and using the poolSeq v0.3.5 function estimateSH (method LLS) [p-value < 0.05 were used to calculate the median selection coefficient for each selected allele. Relationship of selection coefficient, starting allele frequency and block size were tested using a linear model with log10 transformation of selection coefficients. To test the robustness of our definition for selected alleles, we repeated the estimation of selection coefficients using (a) the top 20% SNPs or (b) all SNPs that had an allele frequency change > 0.1  . Only seD. simulans population evolving under a hot temperature regime [N = 10,000), and finally applied a 95 % cut-off.We used data from the same e regime  to contrD. simulans population from Florida [In addition, a different published hot-evolved  Florida  was used Florida  with MNCemmeans).We fitted a linear model with log10 transformed selection coefficients as response and main effects of population as fixed categorical effect with three levels  and a linear and quadratic covariate for starting allele frequency, to account for their non-linear relationship with the response, as explanatory variables. Residuals from this model were normally distributed and displayed variance homogeneity. The model with linear and quadratic covariate for starting allele frequency fit significantly better than a model with only a linear term. Contrasts between populations were compared based on estimated marginal means . Following Barghi N, Tobler R, Nolte V, Jaksic AM, Mallard F, Otte KA, Dolezal M, Taus T, Kofler R, and Schlötterer C [We compared the similarity of replicates calculating the Jaccard indices for the Portugal and the Florida population using the R package lentropy . We creatterer C , we creaNucleotide diversity (π) of each autosome in the ancestral populations was calculated from the allele frequency data using the formula of Tajima F . For mai2 for loci  within 10,000 kb distance. As different sets of 34 Florida haplotypes resulted in very consistent mean estimates  to the genotypic value. Fitness was finally estimated based on the phenotypic value and the corresponding fitness function. We used the following Gaussian fitness function as previously described  on polygenic adaptation after a shift in trait optimum with forward simulations based on Wright-Fisher populations using MimicrEE2 v208 . Using ted (e.g. ): N.2 in these linkage equilibrium simulations ranged from 0.03 to 0.04 (see Fig. We generated two different sets of founder populations, one with strong LD and one with linkage equilibrium. Both sets of founder populations contained loci with 200 chromosomes carrying an allele contributing to the quantitative trait towards the new trait optimum and 800 chromosomes with alleles of opposite effect. For high LD, we generated a defined haplotype structure based on four different sets of selected haplotypes. The number of contributing loci was randomly distributed between the four sets of selected haplotypes. Hence, 50 haplotypes shared the same contributing loci, but due to stochastic sampling the number of contributing loci differed among the four sets of 50 haplotypes. To generate starting populations in linkage equilibrium, we randomly distributed the contributing loci across 200 haplotypes until each of the loci had a final frequency of 0.05. Average rAfter 50 generations, we generated “Pool-seq data” with 50× coverage and added sequencing noise by binomial sampling based on the allele frequencies. We recorded the phenotypic and mean frequency change across loci and replicates as well as the coefficient of variation in the mean allele frequency change across the five replicates as an indicator for the degree of parallel response.Additional file 1: Figure S1. Female fecundity as proxy for fitness in the ancestral population and for each evolved replicate in the Florida and Portugal populations. Figure S2. The inverse relationship between starting allele frequency and selection coefficient is robust to the definition of the selected allele. Figure S3. Histogram of the number of genes per reconstructed haplotype block in the cold-evolved Portugal population. Figure S4. Selected haplotype blocks from replicated D. simulans populations from Portugal evolving in a novel hot environment. Figure S5. Allele frequency changes between the founder and the evolved populations. Figure S6. Selected haplotype blocks from replicated D. simulans populations from Florida evolving in a novel hot environment. Figure S7. The adaptive architecture is population-specific but does not depend on the temperature regime. Figure S8. Replicate populations derived from Portugal founders show a more similar selection response than those with Florida ancestry. Figure S9. Linkage disequilibrium in the ancestral Florida population.Additional file 2: Table S1. Details of DNA extraction, Illumina library preparation and sequencing for all samples used in this study.Additional file 3. Review history.


Ancestry informative markers (AIMs) are useful to infer individual biogeographical ancestry and to estimate admixture proportions of admixed populations or individuals. Although a growing number of AIM panels for forensic ancestry origin analyses were developed, they may not efficiently infer the ancestry origins of most populations in China. In this study, a set of 52 ancestry informative deletion/insertion polymorphisms (AIDIPs) were selected with the aim of effectively differentiate continental and partial Chinese populations. All of the selected markers were successfully incorporated into a single multiplex PCR panel, which could be conveniently and efficiently detected on capillary electrophoresis platforms. Genetic distributions of the same 50 AIDIPs in different continental populations revealed that most loci showed high genetic differentiations between East Asian populations and other continental populations. Population genetic analyses of different continental populations indicated that these 50 AIDIPs could clearly discriminate East Asian, European, and African populations. In addition, the 52 AIDIPs also exhibited relatively high cumulative discrimination power in the Eastern Han population, which could be used as a supplementary tool for forensic investigation. Furthermore, the Eastern Han population showed close genetic relationships with East Asian populations and high ancestral components from East Asian populations. In the future, we need to investigate genetic distributions of these 52 AIDIPs in Chinese Han populations in different regions and other ethnic groups, and further evaluate the power of these loci to differentiate different Chinese populations. Ancestry informative markers (AIMs) refer to genetic variations that exhibit high allelic frequency divergences between different ancestral populations . AIMs arDeletion/insertion polymorphisms (DIPs) are one type of genetic variations that arise from random deletion or insertion of DNA fragments . This kiIn this study, we firstly selected 52 AIDIPs for ancestry origin predictions of different continental populations based on the 1,000 Genome Project  and prevhttp://www.ncbi.nlm.nih.gov/SNP/). The primer design and multiplex assay construction of 52 AIDIP loci proceeded based on the workflows described by Pereira et al. , respectively, and all of the primers were synthesized by Sangon Biotech .We aim to construct a multiplex PCR assay of 52 AIDIPs based on the capillary electrophoresis platform for forensic individual biogeographic ancestry inference and population genetic structure and background analyses. A batch of 52 AIDIPs located on autosomal chromosome were selected; they showed high allele frequency divergences among European, East Asian, and African populations, which were confirmed by previous studies . AIDIPs a et al. ; 2012b. 2O. The PCR cycling conditions were as follows: 95°C for 5 min; 30 cycles of 94°C for 15 s, 60°C for 50 s, and 62°C for 55 s; and a final extension at 70°C for 20 min.The PCR reaction of the 52 AIDIPs was finally optimized to amplify in a single tube with the 25-μl reaction volume. The reaction system composed of 5 μl of primers, 10 μl of reaction mix , 1 μl of template DNA, 1 μl of polymerase , and 8 μl of sdH® formamide  and 0.5 μl of AGCU SIZ-500 internal size standard (AGCU Biotech). Detection and separation for 52 AIDIPs were performed on 3500xL Genetic Analyzers (Thermo Fisher Scientific) under default injection conditions. The raw data were genotyped with GeneMapper® ID-X v1.5 software (Thermo Fisher Scientific).For capillary electrophoresis, 1 μl of amplification products were added to 12.5 μl of loading mixtures, which consisted of 12 μl of deionized Hi-Di® 100 resin-based method , respectively. Genomic DNA was diluted to 1 ng per microliter with Tris-EDTA buffer and stored at −20°C for later use.Buccal samples were obtained from the volunteers with written informed consents for the above-mentioned research purposes, approved by the Ethics Committee of Xi’an Jiaotong University, China (No. XJTULAC 2013). Buccal samples stored on FTA™ cards  were collected from 345 unrelated healthy Han individuals who lived in eastern China for more than three generations, including 200 individuals living in Wuxi city, Jiangsu Province and 145 individuals living in Hangzhou city, Zhejiang Province, China. The genomic DNA was extracted and quantified using Chelexd method  and the R software v4.1.0. Pairwise fixation index (FST) and informativeness (In) values of 50 AIDIPs among continental populations were calculated by Arlequin software v3.5.1.2  of 50 AIDIPs in each continental population were estimated by a previous report (K = 2–7 (Snipper online tool v2.5 (http://mathgene.usc.es/snipper/) based on 50 AIDIPs.Firstly, we assessed allelic frequency distributions of the same 50 AIDIPs in different continental populations. A heatmap of deletion allelic frequencies of 50 AIDIPs in African, American, East Asian, European, and South Asian populations was plotted by the pheatmap package v1.0.12 of v3.5.1.2  and Infov3.5.1.2 , respectv1.09861  and ggpls report . Next, ts report , and the, green (HEX), yellow (TAMRA), and red (ROX) dye channels according to dye color and expected amplicon size . The sizAlthough 52 AIDIP loci were selected and successfully incorporated into the novel assay for ancestry origin inference, the population data of rs3033053 and rs1305047 loci were not available in the 1,000 Genome Project. Thus, genetic data of the same 50 AIDIPs were assessed in five different continental populations. To visually display the analytical results, the distributions of deletion allele frequencies of the 50 AIDIPs are shown by a heatmap. As shown in FST values were also calculated, as shown in FST values between East Asian and other continental populations, especially between East Asians and Europeans, whereas most loci showed low FST values between American and European/South Asian populations. In is commonly used to evaluate the ancestral information of genetic markers in different populations . Linkage disequilibrium (LD) analyses of pairwise AIDIPs in the Eastern Han population are listed in p = 0.05/1,326 = 0.000037). LD between the two loci in the Eastern Han population may be caused by genetic linkage because both of them are located on chromosome one and just 292,573 bp apart from each other. The locus rs140864 exhibited a little more excellent characteristic of forensic statistical parameters, therefore, it was preferentially selected for further data analysis in Eastern Han population. Furthermore, to better understand the associations among loci in different population groups, further evaluations containing more populations and lager sample sizes need to be investigated.HWE tests of 52 AIDIPs in the Eastern Han population are given in Allelic frequencies of 52 AIDIPs in the Eastern Han population are presented in Snipper. The obtained results revealed that all Eastern Han individuals could be categorized into East Asian population, implying that these AIDIPs could perform ancestry origin analyses of Eastern Han population well. Besides, these results also reflected that the studied Eastern Han population had intimate genetic relationships with East Asian populations.Based on the raw data of 50 AIDIPs, PCA of Eastern Han and continental populations was conducted. We found that the studied Eastern Han individuals were predominately superimposed on the East Asian individual cluster located on the right part of the plot . AncestrLang et al. assessed genetic structure of Eastern Han population by 27 Y-STRs and 143 Y-SNPs and found that the Han populations showed closer genetic affinities with East Asian populations than South Asian populations. Furthermore, they also pointed out that genetic differentiations between Southern Han and Northern Han populations were observed . Lu et aIn summary, we developed a multiplex PCR panel for ancestry origin predictions of different continental populations that contained 52 AIDIP loci. Most loci out of these 52 AIDIPs showed high genetic divergences between East Asian and non-East Asian populations. We also demonstrated that this AIDIP panel could be employed for inferring biogeographical origins of continental populations, specifically for East Asian, African, and European populations. In addition, these 52 AIDIP loci also showed relatively high application values for forensic identity testing in the Eastern Han population. For ancestral component analysis of the Eastern Han population, the novel panel could accurately estimate its close genetic affinities and high ancestral components with East Asian populations. In the future, we need to assess genetic distributions of the 52 AIDIPs in other populations from different regions to unveil genetic portraits of these populations. Only in this way could the performance of the developed panel to infer sub-populations and estimate inter-ethnic admixture proportions be completely understood.


Barbus barbus. Four populations of native Barbus species (B. plebejus and B. tyberinus) were studied: two purebred and two completely introgressed with alien B. barbus. Across the four populations, differences in their biological traits  and trophic ecology  were tested. While all populations had similar body condition and were dominated by fish up to 2 years of age, the introgressed fish had substantially greater lengths at the same age, with maximum lengths 410–460 mm in hybrids versus 340–360 mm in native purebred barbel. The population characterized by the highest number of introgressed B. barbus alleles (81 %) had the largest trophic niche and a substantially lower trophic position than the other populations through its exploitation of a wider range of resources . These results attest that the genetic introgression of an invasive congener with native species can result in substantial ecological consequences, including the potential for cascading effects.Genetic introgression with native species is recognized as a detrimental impact resulting from biological invasions involving taxonomically similar invaders. Whilst the underlying genetic mechanisms are increasingly understood, the ecological consequences of introgression are relatively less studied, despite their utility for increasing knowledge on how invasion impacts can manifest. Here, the ecological consequences of genetic introgression from an invasive congener were tested using the endemic barbel populations of central Italy, where the invader was the European barbel The online version contains supplementary material available at 10.1007/s10530-021-02577-6. Interspecific hybridization is a widespread process in animal communities that has been suggested to negatively affect species through depressing the fitness of hybrids (i.e. outbreeding depression)  somatic growth rate, body condition and population demographic structure ; and (2) diet composition and trophic ecology (e.g. trophic niche size and trophic position), enabling the assessment of their functional roles  population (located in the upstream section), and one invaded and introgressed (“i”) population (in the lowland section). Pure vs. hybrid status of populations have already been tested using mitochondrial (D-loop) and nuclear  DNA markers , while at the analysed nuclear marker, a different proportion of B. barbus alleles was detected between the two invaded populations, resulting in a higher number of B. barbus alleles in TLi (81 %) than PVi (68 %)  and the Montacchione Stream (TLp)  and the Bosso Stream (PVp)  Fig. ; Table 1Lp) Fig. . Sites iVp) Fig. .i and PVi) were characterised by a wider riverbed than the upstream sites, which results in major vegetation cover of the latter that provides shading even during summer droughts. Substrates at all sites were characterised by coarse material, primarily boulders and cobbles in varying percentages, which were mainly mesolithal (6–20 cm) and macrolithal (20–40 cm). Mesohabitats at all sites were mainly characterised by riffle-run hydro-morphologies, except for TLp that was dominated by a step-pool hydro-morphology , measured for total length (to 1 mm) and weight (to 0.1 g).Quantitative sampling of the fish communities was completed in July 2019 by electrofishing using DC electric current (2500 W). To estimate fish density, a two-pass electrofishing approach was implemented Moran , involvi2 area, 500 μm mesh). Once collected, BMI samples were preserved in formalin (4 %) and, then, in the laboratory, were sorted into families whose density  was determined by counting individuals. For each sampling site, the fish and BMI assemblages were characterised through the calculation of three common metrics: total density , richness  and diversity . Two operators carried out ageing of scales from the images independently, discarding unreadable or dubious scales. Length at age relationships of each population were then fitted to the von Bertalanffy growth model . ANCOVA was used to test for differences in LWR across the populations, with differences from isometric growth (i.e. b = 3) tested for each population using t-tests. LWR models obtained for each population were then used to back calculate fish weight, and residuals (differences between observed and predicted weight) were tested for significant differences (one way ANOVA) in fish body condition between the populations  in each population was also estimated using the following linear regression model:A subsample of barbel  were selected for gut content analysis (GCA). These fish were euthanized , placed on ice and brought to the laboratory. After their defrosting, the fish were dissected, and their guts preserved in ethanol (70 %) prior to analysis. As barbels do not have a differentiated stomach, the entire digestive tract (‘gut’) was weighed and all the prey items examined using microscopy (x 5 to x 50 magnification). Prey were initially identified to the lowest possible taxonomic level before being grouped into 15 categories according to their taxonomic affinities. Food items with a low frequency, low specific abundances (< 5 %) and/or that occurred in only one population, were grouped into broader categories . As the actions of the pharyngeal teeth of barbels makes the separation of their ingested prey difficult, this prevented the effective use of gravimetric or numeric methods and, consequently, the relative-fullness method was selected Hyslop . This mei in their guts against the total number of fish with contents in their guts (Fi % = Ni/ N x 100). Prey-specific abundances were calculated as the volume occupied by prey item i (Si) in all the guts against the total gut volume comprising prey i  was used to test for dietary differences between the four populations. SIMPER analysis was then applied to detect the contribution of each food item to the dissimilarities. The Shannon-Wiener diversity index (H) was also calculated within the same package (i.e. vegan), and ANOVA and Tukey pairwise test available in R tested for differences in H between the four populations.The GCA data were then analysed for diet composition and niche width area per population. Data were arcsine square root transformed and non-metric multidimensional scaling (nMDS) was performed with 40 % standard ellipses representing the core population trophic niche (Gutmann Roberts and Britton 13C and δ15N). All individuals analysed were of age between 1+ and 4+ years old and of lengths between 98 and 244 mm. Scales were used as the tissue of choice for SIA as they represent non-lethal alternatives to muscles , the fish used differed to those used in the GCA but were collected concomitantly. This was partly due to logistical reasons related to both sample collection and to the Covid-19 lockdown that prevented the fish from the GCA being analysed in a timely manner for the purposes of this study. Consequently, 10 barbel per site were analysed for SIA (δp), Hydropsychidae, Leuctridae (except TLp), Simuliidae (except TLi)) that were mostly present at all sampling sites were selected  and three replicates of each family  were processed. These families mainly comprised a mix of collector gatherers, grazers and scrapers, but also included shredders, filter feeders and predators  were randomly collected by hand at each sampling site and combined to form three replicates.To provide SIA data for the baselines and putative prey, BMI, biofilm (periphyton), benthic algae and fine and coarse particular organic matter (FPOM and CPOM respectively) were sampled on the same date of the fish. A dedicated BMI sample was collected at each site with the same method used to characterize the BMI communities, but this was put on ice and frozen upon arrival in the laboratory. After defrosting, six families  and nitrogen  of the fish, putative prey and baseline samples were then analysed at the Cornell Isotope Laboratory, New York (USA). Across the four sites, 142 samples were analysed: 40 fish, 57 BMI (five families per site) and 45 primary producers . Samples were ground to powder, weighed (to the nearest 1000 µg) and put in tin capsules, before being analysed on a Thermo delta V isotope mass spectrometer (IRMS) coupled with a NC2500 elemental analyser. Data accuracy and precision were tested every 10 samples reporting an overall standard deviation for internal animal standard (deer) of 0.08 for δ15N and 0.03 for δ13C. The C:N ratios of all animal samples were below 3.5 and so did not require lipid correction  in each population through linear regressions as implemented in R. A Bayesian approach available in the R package ‘tRophicPosition’  was 4.2 ‰ ± 0.2 ‰, with this specific to scales for B. barbus, derived experimentally from individuals that had fed on an invertebrate-based diet . Although it is recommended to estimate consumer TPs through the use of baseline taxa that are long-lived   utilised the δ13C data of the BMI using the following equation  of the same macroinvertebrates  Post , there wB). SEAB was tested for significant differences in niche width between populations and obtained through Markov Chain Monte Carlo simulations (104 iterations per group), with differences calculated as the probability that the posterior distribution relative to each population niche was larger or smaller than others (α = 0.05). Maximum likelihood estimate of SEAs were used to plot the niches in the isotopic space, where they represent the population ‘core’ niche (40 %), and enabled identification of the extent of isotopic niche overlap between the different barbel populations  and Cottus gobio Linnaeus 1758; at PVi, Gobio gobio . These taxa are all native, except for G. gobio at PVi. At PVi, three of eight fish species present were alien and at TLi, five of eight were alien (Table S2). All fish species at PVp and TLp were native, except for two salmonid species . At TLi there was the highest density of fishes followed by PVp, with both sites having a relatively lower diversity than the other two sites  to 0.96 (TLp vs. PVi). The TLp community differed the most from the other communities (Bray-Curtis index > 0.87) and was dominated by the gastropods Lymnaeidae and Planorobidae (Table S3). At PVi, there was the highest BMI density while the lowest was in TLp , with values of the Bray-Curtis index ranging from 0.54 (PVLp Table . At PVp es Table .p and PVp), with fish in the introgressed populations reaching a maximum age of 3+ (TLi) and 4+ (PVi) years  was selected as the best-fitting model, indicating that the introgressed barbel (both at TLi and PVi) had significantly larger maximum theoretical lengths than purebreds , with hybrids having greater mean lengths at ages equal and/or greater than 2 years old  were present at fish lengths of 38 to 286 mm. Fish of 5+ and 6+ years were only present in the purebred barbel populations  and within each population, LWR models were highly significant  was detected in all populations except for TLp (b = 3.0). Conversely, body condition indices were all around zero .Length-weight relations (LWRs) varied significantly across the populations . The proportion of fish with empty guts  ranged between 0 % (TLp) to 21 % (TLi) . Prey accumulation curves were approaching the asymptote in all populations, suggesting the subsample of gut analysed was sufficient to represent the diet variability of barbel populations . The most frequent food items in barbel diets across all sites were aquatic insect larvae, particularly Chironomidae . Feeding strategy plots  indicated generalized feeding behaviour in all populations, with all barbel frequently consuming certain prey items (e.g. Chironomidae and Simuliidae), but with some differences in the contributions of others . However, an exception was in TLi and PVi, where there was some dietary specialization through some individuals feeding on fish . This resulted in considerable differences in diet composition among sites, with significant differences in the population trophic niches . The widest trophic niche was in TLi and then PVp , with the highest overall average dissimilarity in the diet of TLi barbels . The diet of hybrids in TLi lacked the items that were frequent and abundant in the diets of the other populations , while consuming food categories (e.g. fish bones and plants) that were absent or infrequent in the other populations . The diet of barbel at TLi was also the most distant from the macroinvertebrate community present at the site of capture as shown by the nMDS analysis , pairwise comparisons indicated these were only significant between TLi and the other sites   and 6.1 ‰ (PVp) . Values of δ15N were more similar for both BMI and primary producers between TLp and PVi , except for fish in PVp where δ13C decreased as fish length increased (Table S5). There was no significant difference in the length of the fish analysed between rivers .Across the four sites, δi compared to TP in the other populations  and length was subsequently removed due to its non-significant effect (p > 0.05). The isotopic niche size of the barbel was significantly larger in TLi and smaller in TLp (as indicated by posterior distributions of the core isotopic niche as SEAB), with the niches being similarly sized in PVp and PVi  was detected for barbel in TLB. tyberinus and B. plebejus following B. barbus invasion revealed substantial changes in some biological traits of introgressed populations and an altered trophic ecology in one of the hybrid population. These results reveal important insight into the ecology of the hybridized progenies and highlight the potential impacts of hybridization at wider ecosystem scales.The testing of the ecological and biological consequences of introgressive hybridisation of the endemic B. barbus × B. tyberinus and B. barbus × B. plebejus), including their maximum theoretical lengths and lengths at age, were higher in the invaded populations than in the purebred populations, as per the prediction. These results are also similar to those from previous studies on B. barbus hybrid populations of central  and Rutilus rutilus , and, conversely, only minor differences between the PVi hybrid population and its reference purebred population (PVp). The introgressed barbel of TLi differed to the other three populations studied in their relatively high proportions of small fishes and plants in their diet, which resulted in a relatively low trophic position. The diets of the other populations were all dominated by benthic macroinvertebrate prey. These differences were then reflected in their trophic niche size, with the hybrids in TLi having the broadest isotopic and trophic niches.The second prediction concerned the differences in trophic ecology between hybrids and purebred barbel populations and were tested using a combination of gut contents (GCA) and stable isotope (SIA) analyses. These techniques are considered to be largely complementary  of all populations, but as these fish had both plant and fish material present then they actually had the widest trophic niche. Moreover, TLi site had the highest fish density and a lower macroinvertebrate density than PVi site, so these data suggest that the hybrids of TLi preyed upon smaller fish through the combination of their high availability and relatively lower availability of macroinvertebrate resources, a pattern that was not evident elsewhere . We can thus speculate that the hybrids in TLi were primarily consuming common food resources in the site rather than preferentially selecting small fishes as dietary items. However, it highlights both their diet plasticity and a shift in their functional feeding guild  and downstream (PVi and TLi) sites, could have influenced the trophic strategy shift and niche broadening of fish in TLi , an invertivore  has been detected in hybrids between native Japanese Cyprinus carpio lineages and non-native strains


Is the modified newborn genetic and hearing screening feasible in China?In this population-based cohort study including 32 512 infants, incorporating the limited and expanded genetic screening into physiological screening was associated with identifying 31 newborns with hearing loss missed by the conventional hearing screening, providing etiologic information to 1299 participants, and targeting 517 children at risk of late-onset hearing loss to improve prevention.Large observational studies are needed to evaluate the cost-effectiveness and long-term benefits of integrated genetic and hearing screening programs. This cohort study investigates the feasibility and utility of a modified genetic and hearing screening program among newborn infants in Nantong, China. Early identification and intervention for newborns with hearing loss (HL) may lead to improved physiological and social-emotional outcomes. The current newborn hearing screening is generally beneficial but improvements can be made.To assess feasibility and evaluate utility of a modified genetic and hearing screening program for newborn infants.This population-based cohort study used a 4-stage genetic and hearing screening program at 6 local hospitals in Nantong city, China. Participants were newborn infants born between January 2016 and June 2020 from the Han population. Statistical analysis was performed from April 1 to May 1, 2021.Limited genetic screening for 15 variants in 4 common HL-associated genes and newborn hearing screening (NHS) were offered concurrently to all newborns. Hearing rescreening and/or diagnostic tests were provided for infants with evidence of HL on NHS or genetic variants on screening. Expanded genetic tests for a broader range of genes were targeted to infants with HL with negative results of limited genetic tests.The detection capability for infants with hearing impairment who passed conventional hearing screening, as well as infants with normal hearing at risk of late-onset HL due to genetic susceptibility.SLC26A4 variation and 92 infants with MT-RNR1 variation were at risk for enlarged vestibular aqueduct and aminoglycoside-induced ototoxicity respectively, indicating early aversive or preventive management.Among a total of 35 930 infants, 32 512 infants completed the follow-up and were included for analysis. Among the infants included in the analysis, all were from the Han population in China and 52.3% (16 988) were male. The modified genetic and hearing screening program revealed 142 cases of HL and 1299 cases of genetic variation. The limited genetic screening helped identify 31 infants who passed newborn hearing screening, reducing time for diagnosis and intervention; 425 infants with normal hearing with pathogenic This study found that performing modified genetic and hearing screening in newborns was feasible and provides evidence that the program could identify additional subgroups of infants who need early intervention. These findings suggest an advantage for universal adoption of such a practice. Moreover, variants in SLC26A4 and MT-RNR1 are of particular interest, because they are responsible for HL associated with enlarged vestibular aqueduct (EVA) and aminoglycoside-induced ototoxicity, respectively. Identification of genetic carrier status provides an opportunity for reproductive counseling. Avoidance of potential event triggers  in individuals with such variants would dramatically reduce the incidence of HL.13Newborns with PCHL have benefitted from the universal newborn hearing screening (UNHS) program.GJB2, SLC26A4, and MT-RNR1) to improve the detection of late-onset prelingual HL was first proposed in 2006.2 Afterwards, concurrent limited genetic screening and hearing screening programs have been evaluated in several cities and provinces.18 The results show that limited genetic screening can identify newborns who otherwise might be missed by hearing screening alone and may identify individuals at risk for late-onset HL. Meanwhile, with the advent of next-generation sequencing technology, there has been an expansion in the number of deafness-related genes that can be screened, indicating the need for further evaluation.19Limited genetic screening of a small number of HL-associated genes  reporting guideline for cohort studies. This study was approved by the ethics committees of Nantong municipal Health Commission and all hospitals involved. Written informed consent was obtained from the infant’s parents.This study used a concurrent newborn genetic and hearing screening program in Nantong city. From January 2016 to December 2020, the population-based cohort study was conducted at 6 hospitals: (1) Maternal and Child Health Hospital affiliated to Nantong University, (2) The Second Affiliated Hospital of Nantong University, (3) Nantong Second People’s Hospital, (4) Nantong Third People’s Hospital, (5) Nantong Sixth People’s Hospital, and (6) Nantong Rich Hospital. Race or ethnicity was collected by self-report at enrollment. All of the recruited infants received both newborn hearing screening and genetic screening for free, funded in part by the municipal government and research project foundations. This study followed the Strengthening the Reporting of Observational Studies in Epidemiology (Inclusion criteria were as follows: (1) the infants were born between January 2016 and June 2020 from the Han population in China; (2) the infants’ health condition was good enough to tolerate the screening procedures; (3) the parents were urban residents of Nantong city; and (4) the parents agreed to have their babies participating in the modified genetic and hearing screening program. Exclusion criteria were as follows: (1) the infants’ samples were unqualified for the genetic tests according to criteria of the National Health Commission of China’s technical specification for neonatal screening of congenital diseases; or (2) the infants were lost to hearing or genetic follow-up.The population-based longitudinal databank for all children with congenital HL in Nantong city commenced in January 2016 and maintained indefinite recruitment and ongoing follow-up.The flowchart of the modified genetic and hearing screening program is illustrated in the 20Newborn hearing screening was conducted by otoacoustic emission (OAE). Hearing rescreening consisted of a repeat OAE and automated auditory brainstem response. Hearing diagnosis was made with auditory brainstem response and auditory steady state response. The severity of hearing loss was graded as mild (26-40 dB), moderate (41-60 dB), severe (61-80 dB), and profound (≥81 dB).GJB2 gene); c.1174A>T, c.1226G>A, c.1229C>T, c.1975G>C, c.2027T>A, c.2168A>G, c.IVS7-2A>G, c.IVS15 + 5G>A (SLC26A4 gene); m.1494C>T, m.1555A>G (MT-RNR1 gene); c.538C>T (GJB3 gene). The results were categorized as (1) negative, (2) carrier , and (3) refer .12 The expanded genetic screening was conducted with next generation sequencing (NGS) panel  to identify 228 deafness-related genes by BioelectronSeq 4000 high throughput sequencing instrument   and tested using a deafness gene variant detection array kit  with LuxScan 10K-B Microarray Scanner . A 2-step variation screening procedure, consisting of 4 genes , then 228 genes (expanded genetic screening for those who tested negative at the first step and confirmed HL) was performed. The limited genetic screening entailed genotyping 15 variants in 4 genes: c.35delG, c.176_191del16, c.235delC, c.299_300delAT (SLC26A4 and MT-RNR1 variant carriers), and routine hearing checkups  from April 1 to May 1, 2021. χBetween January 2016 and June 2020, a total of 35 930 infants born in Nantong were initially screened for the program. The health condition of 1426 infants did not allow newborn hearing screening tests within 3 days. The parents of 724 infants declined the genetic screening. For the 33 780 infants who underwent the modified genetic and hearing screening program, 986 blood samples were unqualified for genetic tests, 195, 81 and 6 infants lost to follow-up of newborn hearing screening, hearing rescreening and hearing diagnosis respectively. Overall, 32 512 infants who completed follow-up were included for analysis, of whom 52.3% (n = 16 988) were male, 47.7% (n = 15 524) were female, and all were from the Han population in China. The neonatal birth weight ranged from 2150 to 5100 g . There were 1273 premature newborn infants (<37 weeks), accounting for 3.9%. Results of stages 1, 2, 3, and 4 of the concurrent genetic and hearing screening program were presented in eTable 2, eTable 3, eTable 4, and eTable 5 in the GJB2 and SLC26A4 were most enriched in the infants with HL with positive genotypes, accounting for 77.6% (52 of 67) of cases . However, none of the GJB3 variations was diagnosed with HL.Of 1260 participants in the carrier group, 31  were reported to have HL, significantly higher than the population prevalence . Besides, both subgroups had predominantly severe to profound HL. Among 92 infants with HL passing limited genetic screening, expanded genetic screening further detected 17 infants (18.5%) with a variation  and sHL genes .Among 22 participants in the refer group, 19  were reported to have HL. The rates of HL in the ariation , includiOf the 142 infants confirmed with HL at 3 months of age, 111 infants were diagnosed following the protocol of the conventional NHS program, whereas 31 infants  with variable degrees of HL passed NHS and were identified by the limited genetic screening, indicating early intervention . Among tSLC26A4 variation who were at risk for EVA-associated HL  that needs referral for special care and early aversive or preventive management.The genetic and hearing screening program identified 425 of 32 512 infants  with iated HL . For theGJB2 variation (3 cases), SLC26A4 variation (1 case), and multiple genes heterozygous variation (5 cases) were involved in late-onset genetic HL in the population.Except for the 142 infants with congenital HL, the follow-up system identified 11 cases of HL  during the follow-up of past 5 years . The medEarly identification and intervention for newborns with hearing loss was associated with improved physiological and social-emotional outcomes. The current newborn hearing screening is generally successful but improvements could be made. In the past decade, genetic testing has emerged as an important etiological diagnostic test for childhood HL. Some variants have been associated with environmental factors and thus regarded as preventable. Combined genetic and physiological screening may result in multiple benefits, including (1) identifying newborns with HL missed by the current physiological screen, (2) providing etiologic information, and (3) decreasing the number of children who would otherwise have late-onset HL. To our knowledge, we present a novel framework for integrating limited and expanded genetic testing into the current NHS and the performance assessment of the modified genetic and hearing program.22 OAE is considered to have low accuracy and lead to some false-negative errors.23 Besides, delayed onset of sensorineural HL limits our ability to achieve early identification of a substantial number of deaf infants.24 Of note, the median age at confirmation of permanent HL following a negative result of NHS was 25.7 months and 17.6 months for well babies and NICU graduates, respectively.22 The modified screening program identified not only babies born with HL but also those at risk for childhood-onset HL. Specialty counseling service to the parents would improve the hearing and language development of the HL children and protect those at risk.15Among the 142 infants confirmed with HL at 3 months, we found that 21.8%  passed the conventional newborn hearing screening. This is consistent with an earlier study that found the sensitivity of otoacoustic emission screening ranged from 79% for well babies and 96% for neonatal intensive care (NICU) graduates.25 High prevalence of variation, especially of the 4 most common HL-associated genes, has been uncovered among the general population.26 Besides, an estimated 30% of genetic HL is syndromic. There are more than 100 genes associated with syndromic HL and more than 400 genetic syndromes that include HL as a feature, such as Pendred , Usher (retinitis pigmentosa), and Waardenburg  syndromes.1 Varying syndromic forms of HL may raise concerns about comorbidities that need specialty referral. Our study helps to understand the limits of limited genetic testing. When the result of limited genetic testing is negative, we have to be aware that HL would still have genetic etiology, because the variant associated with disease risk factors may be located in the region that has not been tested. The advantage to applying expanded genetic screening to this particular group is that targeted screening exhibits a high detection rate , favorably impacting clinical utility and cost-effectiveness.The genetic study of HL has greatly broadened our knowledge of normal auditory function and the pathophysiological processes that can disrupt it. HL-associated genes encoding different proteins involved in the development and function of the ear can affect any component of the hearing pathway.29 However, there are still many safety concerns and translational hurdles to overcome before gene-editing technology can be used to treat HL.30 In our study, all the 153 HL infants have been referred to the specific hospitals and speech therapy service as well. Moreover, there has been no case of drug-induced HL during the follow-up period.If the newborn genetic and hearing screening is to be a priority, services for timely intervention must be established as well. Otherwise, early identification of HL provides no advantage. For congenital HL, hearing aids and cochlear implants are the primary treatments for the patients to improve hearing. Gene therapy, as a biological treatment, has the potential to restore hearing.31We should keep in mind that genetic screening has social and ethical implications. The 2 major concerns are (1) the privacy and confidentiality of genetic testing results and (2) the social and medical implications of any positive findings. Legislation and relevant policies should be available to protect individuals from discrimination by health insurers or employers based on genetic results.32 General population-based longitudinal studies are needed in the future to highlight the cost-effectiveness of this 4-stage screening strategy. Second, congenital cytomegalovirus (CMV) infection, the leading cause of nongenetic HL, was not included in this study. Although our previous decision model-based study suggested that screening for CMV may improve detection of newborns at risk for HL,33 we have not implemented the CMV screening project until early this year. Third, this study reported a surprisingly low incidence of childhood-onset HL during follow-up, considering difficulty of communication with families. The communication difficulties increased when the children grew older. Finally, this study was conducted in a relatively homogeneous Chinese population. It has been documented that etiologies  of congenital HL might differ across different populations.35This study has several limitations. First, due to budget constraints, only an urban resident population was selected in the pilot study and observed for limited duration. Internal migrants  were not included as the dropout rates among this subpopulation may be high.Today, approximately 30 Nantong infants are identified each year with congenital hearing loss within 3 months of birth, such that early intervention can be initiated to improve outcomes. Despite this, awareness and precaution of nearly 300 HL-associated gene variation carriers annually may bring further potential benefits. The results of our pilot study are expected to contribute to the knowledge concerning yield of the UNHS program. This study’s findings suggest that universal screening of a limited number of hotspot variants plus targeted screening for the hearing-impaired subpopulation would offer inexpensive and timely clinical benefits. The performance of Nantong's modified screening program highlights the need for universal adoption of such a practice. Our results call for rigorous large-scale observation to evaluate the cost-effectiveness and long-term benefits of integrated genetic and hearing screening programs.


Clk on the neo-Z chromosome potentially related to the endemicity of a refugial population. In the colonized populations, genes in pathways of energy metabolism and wing development showed signatures of selection. These different genomic signatures of refugial and colonized populations point to multiple impacts of Quaternary climates on adaptation in an extant species.The Quaternary climatic oscillations are expected to have had strong impacts on the evolution of species. Although legacies of the Quaternary climates on population processes have been widely identified in diverse groups of species, adaptive genetic changes shaped during the Quaternary have been harder to decipher. Here, we assembled a chromosome-level genome of the oriental fruit moth and compared genomic variation among refugial and colonized populations of this species that diverged in the Pleistocene. High genomic diversity was maintained in refugial populations. Demographic analysis showed that the effective population size of refugial populations declined during the penultimate glacial maximum (PGM) but remained stable during the last glacial maximum (LGM), indicating a strong impact of the PGM rather than the LGM on this pest species. Genome scans identified one chromosomal inversion and a mutation of the circadian gene  The oriental fruit moth is a pest species native to East Asia with refugial and colonized populations throughout the region. Here, a chromosome-level assembly for the species is reported and used to identify genomic signatures related to Quaternary climate change. These climatic oscillations would have had extensive impacts on the evolution of species3. Understanding the genetic legacy of species contractions and expansions during such periods of extreme climate changes is a fundamental goal of molecular evolutionary studies5.In the Quaternary, species are expected to have contracted to refugial areas during periods of glaciation and then expanded their distribution during warm interglacial periods6 would have had a strong impact on species, given that many species were confined to refugial area in the LGM and colonized their current distribution range after this period8, and with genetic legacies of Quaternary climates expected to depend on this period9. In contrast, climate oscillations have been mild in East Asia during the LGM. Many species spread out from their refugia early before the penultimate glacial maximum 11. Consequently, species in East Asia are expected to have experienced different climatic changes in the Pleistocene with different genetic legacies. However, studies on genetic legacies in species from this area lag those in Europe and North America12.The effects of Quaternary climate changes on species evolution are expected to have varied with the geographical range of species and period of glaciation. In Europe and North America, the last glacial maximum 14. During the glacial and the postglacial dispersal periods, populations would have been isolated with gene flow restricted—genetic differentiation at neutral loci would have developed due to genetic drift in small refugial populations15. Neutral genetic variation has been widely investigated across populations to build an ecological and evolutionary understanding of species during periods of change19. In addition, changing climates pose selection pressures on populations that can respond to local climates by adaptive evolution21. While adaptive evolution to climate changes has been identified across many species14, populations used for genome scans of climate change adaptation have tended to focus on more recent historical periods of climate change in Europe, North America, and Australia25. We still lack an understanding of the adaptive responses of species to climatic changes since the early Pleistocene. Nevertheless, insights into the adaptive evolution of species to past environments may improve our understanding of species’ responses to ongoing and future climate changes27.Under climate changes, neutral and adaptive variations can be reshaped in different populations28. Moths of the family Tortricidae tend to distribute in temperate and tropical upland regions28. However, some species have expanded their distribution range to become global invasive species30. The oriental fruit moth, Grapholita molesta, representing one such species, has become a cosmopolitan pest of stone and pome fruits31. This species is native to East Asia and molecular data suggest two refugial regions around eastern Qinghai-Tibet Plateau boundary regions (Yunnan and Sichuan Basin)32. This moth has dispersed to temperate and tropical upland regions of all continents within the last 100 years30. Genetic lineages from one of the refugial areas (Yunnan) appear to have expanded post-glacially to the east and then to the north of China, while genetic lineages from another refugial area (Sichuan Basin) are considered endemic32. The native populations of the oriental fruit moth provide ideal models to understand adaptive genomic variation related to climate changes of the Quaternary in East Asia.The Tortricidae represent one of the largest families of Lepidoptera (Hexapoda: Insecta) with over 5000 described species, including many economically important pests in agriculture and forestryIn this study, we assembled a high-quality chromosome-level genome for the oriental fruit moth and examined genomic signatures potentially related to the Quaternary climate changes in the refugial and colonized populations. We found that refugial populations of the oriental fruit moth were mainly impacted by the PGM but not the LGM. Higher differentiation on the neo-Z chromosome was detected in the endemic refugial populations. Genomic scans identified a circadian gene that was related to the endemicity of the refugial populations, and wing development genes that were related to the colonized populations. These results indicated that both the extreme climate during the glaciation and the dispersal event in the postglacial periods might shape the genomic signatures of the oriental fruit moth in East Asia. As far as we know, this is the first attempt to identify adaptive genomic variations among populations that diverged in the early Pleistocene period of the Quaternary.We generated 53.7 Gb NanoPore long-reads, 48.7 Gb Illumina short-reads, and 120.8 Gb Hi-C short-reads for the oriental fruit moth genome assembly , which was not assembled in the oriental fruit moth genome. In the Tortricidae, the number of chromosomes ranges from 27 to 3133. The subfamily Tortricinae has 30 chromosomes, while the Olethreutinae  has 28 chromosomes28. Three fusion events were identified in the tortricid moths. A neo-Z chromosome was formed by a fusion event between the sex chromosome Z and an autosome35. The second fusion involved chromosome 7 and chromosome 8 of S. litura fused into chromosome 2 of the oriental fruit moth. The third one involved the fusion of chromosome 5 and chromosome 22 of S. litura into chromosome 4 of the oriental fruit moth  genes and compared these gene families among 13 representative genomes of Lepidoptera, as described in Supplementary Table C. pomonella, the oriental fruit moth has more genes in five annotated gene families  did not overlap. The neo-Z chromosome had the highest π and FST values compared with the autosomes when calculated for all population pairs. The average π value for the neo-Z chromosome (0.00327 ± 0.00006) was ~1.56 fold that of the autosomes (ranging from 0.00110 ± 0.00004 to 0.00279 ± 0.00009 with an average of 0.02094 ± 0.00006). Average FST values of the neo-Z chromosome  were 1.82, 1.74, and 1.40 fold higher than for the autosomes  for population pairs of SCCD vs. BJPG, SCCD vs. YNHH, and BJPG vs. YNHH, respectively , 0.1480 ± 0.0008 (BJPG vs. YNHH), and 0.1655 ± 0.0009 (SCCD vs. YNHH), indicating that the SCCD population is particularly strongly differentiated from the YNHH population.To test for expected differences in demographic changes in the three populations of the oriental fruit moth, we compared their genetic diversity and inferred demographic history based on genome resequencing data. The SCCD had a larger estimated genome size, heterozygosity, and duplication rate than the recently colonized BJPG population  and an ABC transmembrane transporter, both involved in target-site based resistance of many pesticides34. Two SNPs related to the endemicity of the SCCD population were genotyped across 12 populations  is predicted to be 0.7548. The πZ/πA ratio is usually used to predict evolutionary history based on their deviation from the expectation of 0.75, involving processes such as population size fluctuations, sex breeding system and selection, speciation, and introgression, as documented in the human, fruit fly, and silk moth51. In the oriental fruit moth genome, we identified a neo-Z chromosome, which originated from the fusion of ancestral Z and autosomes, as in other tortricid moths34. The gene content and arrangement of the ancestral Z part of the oriental fruit moth was highly concordant with the Z chromosome of S. litura , reflecting a substantial departure from a 0.75 expectation. This high genetic diversity may increase the adaptive potential of the oriental fruit moth to different environments during its dispersal and invasion53. We found particularly high differentiation on the neo-Z chromosome in the SCCD population, and a high proportion of outlier genes related to SCCD endemicity detected on the neo-Z chromosome, suggesting that this chromosome plays an important role in adaptation of the SCCD population.Several theoretical and empirical studies suggest that evolution may occur more rapidly on sex chromosomes29. Consistent with this expectation, we detected a dramatic bottleneck reducing variation in a derived northern population of the oriental fruit moth, a moderate bottleneck in a refugial population from Yunnan, and a more stable population size in another refugial population from Sichuan Basin during the Quaternary climatic oscillations. The high genomic differentiation among the two refugial populations and the lower differentiation between the YNHH and BJPG populations are consistent with the general pattern that the refugial populations usually persist with higher genetic diversity56.Lower genetic diversity might be expected in populations that are recently established due to bottlenecks associated with founder events and perhaps selective processes that favor strong dispersal/fast reproduction and lead to selective sweeps32. Larvae of the oriental fruit moth bore for long periods into fruit and tree shoots; they often disperse via the transport of fruit or nursery stock. During the last century, the oriental fruit moth has spread to almost every continent (reaching a cosmopolitan distribution) and shows high invasiveness36. Population genetic analyses showed that the globally dispersed populations are likely from other recently colonized populations rather than from the refugial area of Sichuan30. Thus, the SCCD population is likely to have remained relatively isolated. Molecular dating and demographic analysis has also shown that northerly populations of oriental fruit moth are derived from the refugial area of Yunnan in the Middle Pleistocene (about 54.0 kya) rather than Sichuan, whereas the two refugial populations of YNHH and SCCD diverged in the early Pleistocene (about 286 kya)36. Hence, strong local adaptation in the SCCD population was most likely shaped before the Middle Pleistocene.Even though the oriental fruit moth populations in the Sichuan Basin have higher genetic diversity, they have been restricted to their refugial areas, while lineages from the Yunnan region have expanded northwardClk) and two target-sites of pesticides, as candidate genes associated with endemicity of the SCCD population. The Clk gene has been associated with breeding and migration in some birds60. According to our genome scanning methods, these genes have higher genetic differentiation between the SCCD and the other two populations, and less genetic diversity in the SCCD population. These results indicate strong selection of these genes in the SCCD population. A previous study found that low genetic polymorphism at the Clk gene may limit adaptation to changing environments as suggested in other species38. It is well known that the oriental fruit moth overwinters by diapause of the last instar larvae61. Diapause induction of insects is associated with the circadian clock62. Perhaps range expansion of Sichuan populations is restricted by strong local adaptation of diapause induction. Further work is required to understand whether the Clk gene of the oriental fruit moth associates with diapause as well as with breeding and dispersal timing, and whether these relationships are sex-dependent, given that the Clk gene locates on the neo-Z chromosome.A genome scan identified 42 genes on the neo-Z chromosome, including a circadian clock gene  for whole-genome resequencing.  for assembly and annotation of the oriental fruit moth genome. The raw reads generated from the Illumina platform were filtered by Trimmomatic v0.3875 with default parameters. The assembled contigs were polished based on Illumina short reads using Pilon v1.2276. To remove the possible secondary alleles, the assembled contigs were filtered using the pipeline Purge Haplotigs77, resulting in a contig-level genome. The Illumina short reads sequenced from the Hi-C library were used to assemble these contigs into a chromosome-level genome using the Juicer v1.578 and 3D de novo assembly (3D-DNA) pipelines79. The completeness of each assembled version of the genome was assessed using a Benchmarking Universal Single-Copy Orthologs (BUSCO) v3.0.280 analysis, based on the insecta_odb9 database (1658 genes). We conducted a synteny analysis between the oriental fruit moth and the codling moth C. pomonella (Lepidoptera: Tortricidae) (Assembly accession: GCA_003425675.2)29 and Spodoptera litura (Lepidoptera: Noctuidae) (Assembly accession: GCF_002706865.1)81 using MCSCAN82.Long reads generated from the NanoPore platform were corrected and assembled using CANU version v1.883 against the Insecta repeats within RepBase Update (http://www.girinst.org) and Dfam database (20170127), with RMBlast v2.10.0 as a search engine. tRNAs were annotated by tRNAscan-SE84 with default parameters; rRNAs were annotated by RNAmmer prediction85. The protein-coding gene in the oriental fruit moth genome was annotated using ab initio, RNA-seq-based, and homolog-based methods in the MAKER version 3.01.03 genome annotation pipeline86. For the RNA-seq-based method, the RNA-seq reads were first mapped to the genome of the oriental fruit moth with Hisat v2.2.0. The transcripts were assembled using StringTie v2.1.2. For ab initio methods, parameters of SNAP v2013-02-1687 and Augustus v3.2.388 were estimated or trained before using them to predict genes in MAKER. The SNAP parameters were estimated from high-quality transcripts obtained by improvement and filtering using PASA v2.4.189. The gene model of Augustus was directly obtained from the above BUSCO analysis of the genome assembly. For the homolog-based method, protein-coding genes of Drosophila melanogaster and Bombyx mori were used. Then, fragments per kilobase per million (FPKM) values of each gene predicted by the MAKER pipeline were calculated using cufflinks version 2.2.1;90 the gene set was filtered by keeping those with an FPKM value > 0 in any RNA-seq library. Finally, PASA was used to update the annotation based on transcripts; all predictions were further filtered using GffRead v0.11.7 implemented in Cufflinks v2.2.190 to remove genes having in-frame stop codons. Functions of the protein-coding genes were annotated using eggNOG-Mapper v1.0.391 against the database EggNOG v5.092. Genes that can be functionally annotated by EGGNOG analysis were retained in gene structure annotation and used for further analysis.Repeats and transposable element families in the oriental fruit moth genome were detected by RepeatMasker pipeline v4.0.7Three representative populations were genotyped by genome resequencing, and 12 populations were genotyped using KASP  calling was performed using the Genome Analysis Toolkit (GATK) v3.595. The criteria used to filter the raw SNPs were “QD < 2.0, FS > 60, SOR > 4.0, MQ < 40”. SNPs were further filtered using the R package vcfR96 and VCFtools v0.1.1697 with the following criteria: SNPs with a sequencing depth lower than four and higher than 500 were removed; SNPs with a missing rate higher than 10% were removed. All the SNPs were annotated with SnpEff v4.398.For genome resequencing of three representative populations, individual DNA libraries with an insert size of 400 bp were constructed and sequenced on the Illumina HiSeq X Ten platform to obtain 2 × 150 bp paired-end reads. A sequence depth of ~36-fold was obtained for each sample. After filtering out raw sequencing reads containing adapters and reads of low quality, the remaining clean reads were mapped to the reference assembly using BWA v0.7.17 with default parametershttps://github.com/pinbo/SNP_Primer_Pipeline2). The KASP reactions were conducted in 1,536 microplates with 1 μl final reaction volumes containing 1–5 ng genomic DNA, 0.5 μl of KASP 2x Master Mix , 0.5 μl of ultrapure water, 0.168 pmol of each allele-specific forward primer and 0.42 pmol reverse primer. The amplifications were conducted on an LGC Hydrocycler high‐throughput thermal cycler  using the following program: 95 °C for 15 min; 10 cycles of 94 °C for 20 s and touchdown starting at 61 °C for 60 s until to 55 °C, then 26–42 cycles of 94 °C for 20 s and 55 °C for 60 s. The fluorescence was scanned by Pherastar , and the genotyping results were visualized and output files generated using Kraken software . Due to unavailable KASP amplification of the Clk gene on the neo-Z chromosome, a pair of PCR primers  was designed for Sanger sequencing of the target region. PCR amplification and sequencing followed mitochondrial genes32.For genotyping the other 12 populations of the oriental fruit moth, we used the KASP, a fluorescence-based method. This method is suitable for detecting flexible numbers of SNPs with low cost and high accuracy. We selected 22 SNPs on nine outlier genes for genotyping (see below for outlier selection). Primers for each SNP were designed based on the genome sequence using SNP_Primer_Pipeline2 (74. Pairwise genetic differentiation (FST) and nucleotide diversity (π) of the populations were calculated using VCFtools v0.1.1697 with a window size of 50 kb and widow step of 10 kb. The decay of LD against physical distance for the different populations was calculated using PopLDdecay99 with default parameters.Genetic diversity and differentiation were estimated across chromosomes and among populations Fig. . To char100 with the default Markov chain Monte Carlo (MCMC) parameters. We used SMC++ v1.15.4101 to analyze the historical effective population size spanning 4 × 106 generations based on SNPs of autosomes. We assumed a mutation rate of 2.9 × 10−9 per site per generation as estimated for Heliconius (Lepidoptera: Hesperiidae)102, a generation time of 0.25 years104.Demography history were investigated in two perspectives Fig. . The div105. Lostruct divides the genome into non-overlapping windows and calculates a PCA for each window. It then compares the PCAs derived from each window and calculates a similarity score. The matrix of similarity scores is then visualized using multidimensional scaling (MDS) transformation. Lostruct was run with 100 SNP-wide windows and independently for each chromosome. Each MDS axis was then visualized by plotting the MDS score against each window in the chromosome. We identified recombination suppression regions following106. Briefly, we manually selected the potential regions based on an MDS axis and minimum or maximum value that included windows within the region but excluded the rest of the chromosome. All SNPs within the regions defined by MDS scores were used to calculate PCAs using R to test whether they could divide samples into three groups representing 0/0, 0/1, and 1/1 genotypes. Average heterozygosity and LD within the regions were also calculated to test whether they were higher than the other chromosomes.The program lostruct  was used to detect chromosomal inversions from genome resequencing dataST values and the top 5% of π ratios between population pairs were considered selective sweep regions. Since π and FST values varied among chromosomes (see “Results”), each chromosome was analyzed independently. To detect outliers related to the northward dispersal of the oriental fruit moth, we used pairs of refuge/north populations, i.e., SCCD/BJPG and YNHH/BJPG. The intersection of these two combinations was considered as regions involving spatial sorting during northward dispersal. Missense SNPs with maf > 0.05 in these regions were extracted based on annotation results of SnpEff v4.3 analysis. These missense SNPs with higher FST values between BJPG and the other two refuge populations than between YNHH and SCCD populations were retained as outlier SNPs. To detect outliers related to the endemicity of the SCCD population, we used π ratios and FST values of BJPG/SCCD and YNHH/SCCD and filtered outlier regions SNPs and genes as above.We conducted genome scans to identify outliers potentially related to the refugial population and the northward dispersal Fig. . The comhttps://bedtools.readthedocs.io/en/latest/) to identify genes near candidate SNPs (±1 bp). We performed functional enrichment analyses using the clusterProfiler toolkit107, where the significance level was set at 0.05, and the P-value was corrected using the Benjamini-Hochberg FDR.We used Bedtools v2.2.80 (Further information on research design is available in the Peer Review FileSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Reporting Summary


Quercus franchetii complex is widespread in the southeast Himalaya fringe and northern Indo-China with a long evolutionary history. It provides a unique proxy for studying the diversity pattern of evergreen woody lineages in this region since the Oligocene. In this study, we combined chloroplast (cpDNA) sequences, nuclear microsatellite loci (nSSRs), and species distribution modeling (SDM) to investigate the impacts of geological events on genetic diversity of the Q. franchetii complex. The results showed that the initial cpDNA haplotype divergence was estimated to occur during the middle Oligocene (30.7 Ma), which might have been raised by the tectonic activity at this episode to the Miocene. The nSSR results revealed two major groups of populations, the central Yunnan-Guizhou plateau (YGP) group and the peripheral distribution group when K = 2, in responding to the rapid YGP uplift during the late Miocene, which restricted gene flow between the populations in core and marginal areas. SDM analysis indicated that the distribution ranges of the Q. franchetii complex expanded northwards after the last glacial maximum, but the core distribution range in YGP was stable. Our results showed that the divergence of Q. franchetii complex is rooted in the mid-Oligocene. The early geological events during the Oligocene, and the late Miocene may play key roles to restrict seed-mediated gene flow among regions, but the pollen-mediated gene flow was less impacted. The uplifts of the YGP and the climate since LGM subsequently boosted the divergence of the populations in core and marginal areas.The Oligocene and Miocene are key periods in the formation of the modern topography and flora of East Asian and Indo-China. However, it is unclear how geological and climatic factors contributed to the high endemism and species richness of this region. The  The late Paleogene (36∼23.3 Ma) is a key period in the formation of the modern topography and flora of Asia . During One prevailing view addressed by numerous scholars is that the rapid uplifts of the Tibetan Plateau-Himalayas (TP) since the Miocene has created new habitats and niches, which have in turn promoted sympatric speciation . MeanwhiSalix brachista (cushion willow) showed that the TP uplift induced sky island habitats, which increased population differentiation in combination with the Quaternary climate fluctuations, thus boosting in situ speciation  (Taxus (Taxaceae) (Picea (Pinaceae) (A recent study of Chinese angiosperms by eciation . Other paxaceae) , Taxus (axaceae) , and Picinaceae) .Ginkgo . However, after adding RAD-seq data from more individuals from both species and reconstructing the phylogenetic tree, neither of the two species form a monophyletic clade (unpublished data). Likewise, morphometric measurements showed no difference between Q. franchetii and Q. lanata, both at the species level as well as among different geographical regions (Quercus franchetii complex”). Data beyond the molecular dating results concur that the Q. franchetii complex has a long evolutionary history. Fossils resembling the extant Q. franchetii complex have been widely reported along the Tethys/Paratethys Seaway dating to the late Eocene to Pliocene, and they were commonly used as a proxy indicating warm and semi-humid climates , we aimed to (1) illustrate the spatial genetic structure of the Q. franchetii complex, (2) identify the key environmental factors restricting the distribution and diversity pattern of the species complex, and (3) explore the key factors that drove the divergence of the Q. franchetii complex. This study provides deep insights into the distribution and evolutionary dynamics of this subtropical woody lineage in the context of global environment change, informing efforts to safeguard this unique forestry ecosystem in the Southeast Himalaya biodiversity hotspot.In this study, we comprehensively sampled populations of the on range . We usedSampling of oaks were granted and supported by National Forestry Bureau of China, Local National Nature Reserves, and Ministry of Environmental Conservation and Forestry, Myanmar.Q. franchetii complex populations were sampled from Yunnan and Sichuan, China and Mount Victoria in Chin State, Myanmar. In total, 303 individuals from 33 populations were sampled for this study, covering the major known distribution range of the Q. franchetii complex .Total genomic DNA was extracted using a modified cetyltrimethyl ammonium bromide (CTAB) protocol . Three psbA-trnH , trnT-trrnT-trnL , and atptpI-atpH , and eigcpDNA sequences were assembled and checked using Sequencher 4.01 . The ClustalW implementation in MEGA X , within-population diversity (Hs), and coefficients of differentiation (GST and NST) for cpDNA loci were estimated using PERMUT 2.0 (h) and nucleotide diversity (π) for cpDNA loci were obtained with ARLEQUIN 3.5 (cpDNA haplotype network was constructed using the Median-Joining model in NETWORK 5.0.0.1 (He), observed heterozygosity (Ho), number of alleles (NA), and effective number of alleles (NE). Allelic richness (Ar) of nSSRs was determined using HP-RARE (Haplotypes and polymorphism statistics for naSP 6.0 . The hapRMUT 2.0 . HaplotyQUIN 3.5 . The cpD 5.0.0.1 . GenAlEx 5.0.0.1  was used HP-RARE .FST) based on cpDNA and nSSR data were estimated using ARLEQUIN 3.5, respectively (Ar) for cpDNA loci and genetic divergence (FST) based on cpDNA and nSSR data according to inverse distance weighted interpolation. For cpDNA and nSSR data, analysis of molecular variance (AMOVA) was performed to assess the genetic variation among populations and within populations. The Mantel test was also performed based on the genetic structure and geographic distance matrix with 1,000 random permutations to evaluate their relationship and test isolation by distance (IBD). For nSSR data, Principal coordinate analysis (PcoA) based on genetic distance was performed using GenAlex 6.5  was varied from 1 to 10, with 10 replicates at each value. Each run consisted of a burn-in length of 25,000 iterations with a run length of 500,000 MCMC (Markov chain Monte Carlo) iterations. The optimal K (number of clusters) was determined using the △K method  was chosen as the outgroup to root the tree. An uncorrelated lognormal relaxed clock was applied with the K81uf + I substitution model, which was selected based on the Akaike information criterion (AIC) in Modeltest 3.7  . The MCMC chains were run for 100 million generations with a sampling frequency of once every 10,000 generations. Convergence was assessed using Tracer v1.72 (3 to generate the maximum clade credibility (MCC) tree after discarding the first 20% of the trees as burn-in. The results were visualized using FigTree v1.4.3.4The divergence time dating for AST V2.4 . Quercustest 3.7 . The ear. 34 Ma)  was set er v1.72 , and thecpDNA loci, D and Fs of neutrality tests were performed to detect possible demographic expansions of the Quercus franchetii complex using ARLEQUIN 3.5  analysis as implemented in RASP  and DEC Q. franchetii complex (including Q. franchetii and Q. lanata) under the past, current, and projected future climate scenarios based on the maximum entropy model ,6 and the field collection records of our research team. Each voucher specimen in the distribution record was inspected and checked carefully. Nineteen bioclimatic variables with a 2.5-arc-min resolution for the present (1950–2000), the last glacial maximum (LGM) (CCSM) period, and the future  were downloaded from WorldClim 2.7 The nine environmental factors  were selected after eliminating climatic variables such that none that were include were highly correlated  using the Dismo package in R.8 We also chose CHELSA climate data,9 which had a higher prediction power in mountain regions , to simulate the present distribution  was used to evaluate the model’s accuracy. AUC values ranged from 0.5 to 1, where the higher AUC value indicated a better prediction. The maximum training sensitivity plus specificity thresholds for the presence or absence of species was used to draw a species distribution map in ArcGIS 10.5. In addition, we classified three categories equally between this threshold and 1, corresponding to low, medium, and high fitness areas, respectively. To compare the changes in species distribution across different periods, three indicators were calculated: locality habitat stability (NStab), habitat distribution area ratio (Na), and habitat expansion extent (Ne). These values were calculated using the following formulas: NStab = 1- | NPre - NLGM |, where NPre and NLGM are the habitat suitability of the present and LGM distribution area; Na = (present distribution areas)/(LGM or future distribution areas): a value close to 1 indicates a stable distribution of the species, while a value much higher or lower than 1 indicates that the distribution area of the species has expanded or contracted from the LGM to the present; Ne = [1 - (Distribution area overlapping between the LGM and present or the future and present/present distributions area)] × 100% represents the percentage of the distribution that has expanded from the LGM to the present.In order to improve the prediction accuracy of the model, it was necessary to optimize the model in MaxEnt 3.4  by settiQ. franchetii complex in the past and present periods were inferred based on the least-cost path analysis method using SDM toolbox 2.0  and genetic structure  with habitat and geographic factors. Five variables—population longitude and latitude, habitat suitability for the present (NPre) and the LGM (NLGM), and habitat stability (NStab) were used as explanatory covariates.The linear model in R 3.5psbA-trnH, atpI-atpH, and trnT-trnL sequence alignments were 482–520, 899–1030, and 796–838 bp in length, respectively. The combined length of the three aligned chloroplast fragments was 2,319 bp, with 94 polymorphisms across a total of 33 haplotypes among the 303 individuals analyzed. The cpDNA haplotype diversity was h = 0.956 and nucleotide diversity was π = 0.00536. The nucleotide diversity and haplotype diversity within the populations were 0–0.04184 and 0–0.644, respectively (HT = 0.982) was much higher than the average within-population diversity (HS = 0.123). The genetic diversity map showed that the NPR and HDM populations had high genetic diversity (NST (0.959) was significantly greater than GST (0.874) (P < 0.05), which indicated that a clear phylogeographic structure existed among the Q. franchetii complex populations. The network analysis resolved four distinct cpDNA haplotype clades in the Q. franchetii complex, which was consistent with the haplotype structure determined by BEAST. The haplotypes in Clade IV exhibited a star-like structure. The most common haplotype, H9, had the widest distribution, mainly within the central YGP , 2.578 (SE = 0.066), 0.537 (SE = 0.013), and 0.536 (SE = 0.016), respectively, while allele richness (Ar) was 3.18–4.34.Among the 303 individuals genotyped, a total of 115 alleles were identified using eight pairs of microsatellite primers. All eight microsatellite loci were highly polymorphic, with allele numbers varying from 8 to 18 per locus. The genetic diversity indexes cpDNA sequence data revealed greater genetic variation among populations  than within populations (13%). In contrast, the nSSRs showed substantial genetic differences within populations  and microsatellite data  both revealed significant correlations between genetic and geographic distances, but the correlations among chloroplast markers were weaker , and southwestern Yunnan  groups. Cluster b was composed of 12 populations peripheral to those of cluster a. When K = 4, the YGP group was further divided into subgroups, and the populations from RR  separated from the rest of cluster b to form a new subgroup , and Clade I (NPR) was the earliest derived. The haplotypes of the southwestern lineage (Clade II) began to diversify around 25.7 Ma (95% HPD = 11.9–37.2 Ma). The divergence of Clades III (HDM region) and IV (YGP region) was dated to ca. 24 Ma (95% HPD = 9.95–34.13 Ma), with subsequent rapid divergence of the haplotypes during the late Miocene  failed to identify population expansion. The mismatch distribution for cpDNA showed a multimodal distribution, consistent with a stable population size  provided a higher confidence estimate than did the DEC model (95% HPD = 45.25%), and its results indicated that the ancestral distribution area of the Q. franchetii complex was widespread in southwestern Yunnan and the Southern Himalayas . Then followed three vicariance events, which led to the divergences of the NPR lineage (Clade I) during the Oligocene (95% HPD = 99%) and the RR lineage (Clade II) during the late Oligocene episode (95% HPD = 98.97%), with an increase in the divergence of the HDM lineage (Clade III) and YGP lineage (Clade IV) during the early Middle Miocene . Annual Mean Temperature (bio1) was the greatest contributor , followed by Temperature Seasonality (bio4)  and Annual Precipitation (bio12)  in identifying the areas of occurrence for Q. franchetii complex populations. The maximum sensitivity plus specificity value 0.13 was used as the species absence/presence threshold. The current distribution of the Q. franchetii complex was similar to the predicted distribution, except for quite a few occurrence sites in southwestern and southeastern Yunnan and Southern Himalayas that were located in predicted unsuitable areas . In the future period, the higher the RCP value was, the more obvious of an expansion in the suitable area to the northwest was detected. The ratios of the present range to the future range under RCP2.6, RCP4.5, RCP6.0, and RCP8.5 were 1.07, 1.15, 1.04, and 1.18, respectively. Accordingly, the highly suitable habitat would be reduced by 7.2, 7.2, 16.5, and 40.1%, respectively . Ar was associated with both longitude and latitude (P < 0.05). Cluster A (CA) and GenPCoA1 were both associated with latitude (P < 0.05). However, He was not associated with NPre, NstabLGM, NLGM, latitude, nor longitude (P > 0.05)  .Q. franchetii complex , which is similar to the genetic diversity of other evergreen oaks in southwestern China, e.g., Q. schottkyana      , Quercusst Asia) , Q. mongn Japan) , Q. acutt China) . The dect China) . It is et China) . As a reQ. franchetii complex, with FST estimates for cpDNA and nSSRs of 0.87 and 0.369, respectively, which is very similar to that found in Q. delavayi, as the cpDNA of the both species show significant phylogeographic structure, IBD pattern, and high differentiation among the populations . Generally, the seed-mediated gene flow among populations is more restricted in the species with instant germination seeds comparing to those species that seed with a period of dormancy. Consequently, the typical recalcitrant seed species shows significant phylogeographic structure in cpDNA makers. Vice versa, as pollen of oaks can disperse long distance, population differentiation revealed by biparental markers was much lower than that in maternal makers  had similar genetic structures and distinct IBD pattern. The different genetic structures detected in the “young” and “old” species might reflect the outcomes of past geological events on the biota at the different epochs, as the ancient geological events may imprint a genetic structure pattern in an “old” and widespread lineages, but not in those of the “young” lineages. However, our hypothesis requires further investigation, as the molecular markers we used in previous phylogeographical studies on oaks of southwestern China are not all the same. Regardless, these markers are informative to reveal the spatial genetic structures of these oaks, but it contains potential bias when comparing across the species to reveal the underlying mechanisms that shaped the genetic structures of these oaks. Further studies using universal high-throughput markers to scan oak populations in southwestern China and incorporating seed functional traits analyses are necessary to reveal the underlying drivers lead to the contemporary genetic structure found in these oaks.Contemporary and historical factors shaped the genetic structure of organisms . Geologi 6.37 Ma , Q. kerrt 6–7 Ma , and Q. ca. 5 Ma  are lateCautleya, Roscoea (Badidae (Paini)  .cpDNA data showed that the Q. franchetii complex once had a wide distribution in southwestern China and the southern Himalaya regions during the early Oligocene, followed by three vicariance events. Among these events, the NPR (Clade I) was first diversified during the mid-Oligocene (ca. 30 Ma), and then, during the late Oligocene and the early Miocene, the RR lineage and HDM lineage were derived, respectively. Within the main lineages, the fast divergence of the cpDNA haplotypes occurred during the late Miocene  can be found below: MD and X-LJ conceived and designed the experiments and were responsible for field collections and specimen identification. S-SZ performed the experiments. S-SZ and X-LJ analyzed the data. MD, S-SZ, and X-LJ wrote and revised the manuscript. Q-JH gave much advice on the details of the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.