Aphis gossypii, one of the most important agricultural pests in the world, can cause serious economic losses in the main crop-producing areas. To clarify issues such as the genetic differentiation, genetic structure, and demographic history of A. gossypii populations, we used 10 nuclear microsatellite loci (SSR) and two mitochondrial gene sequences (COI and Cytb) to investigate genetic diversity and population structure of A. gossypii populations that were collected from 33 sampling sites in China from different climatic zones. SSR and mtDNA data suggested low to moderate levels of genetic diversity. A star-shaped network of mtDNA haplotypes indicated that the maternal ancestor of China cotton aphids likely originated in Xinjiang. The POPTREE, STRUCTURE and principal coordinate analysis (PCoA) revealed two genetic clusters: an eastern and a western region group. Isolation by distance (IBD) results showed a positive correlation between geographic distance and genetic distance in the vast eastern region but not in the western region. Neutrality testing and mismatch distribution analysis provided strong evidence for a recent rapid expansion in most populations. Genetic bottleneck was not detected in A. gossypii populations of China. The present work can help us to develop strategies for managing this pest. Aphis gossypii Glover (Hemiptera: Aphididae), is an important cosmopolitan pest that attacks numerous cultivated plants, such as Cucurbitaceae, Rutaceae and Malvaceae, causing serious economic losses in major crop areas around the world. Widely distributed in tropical, subtropical and temperate regions, it is the most common aphid and the primary target pest of cotton in the world1. Generally, adults and nymphs of this species feed on the underside of leaves or the growing tips of shoots, sucking juices from the plant, causing seedling death, leaf curl and withering, and serious yield loss. Aside from direct damage, this pest is an important vector of various viruses such as mosaic, crinkle, and rosette, and among others3. A. gossypii is a highly fecund, extremely polyphagous species, and host-adapted races have been identified6. In general, this species can be holocyclic with Hibiscus, Catalpa or Rhamnus trees as primary hosts in China, the United States and Japan. Its life cycle includes both parthenogenetic and sexually reproductive phases2. In spring, each egg gives rise to a wingless viviparous, parthenogenetic female, followed by several more parthenogenetic generations during the spring and summer7. At the end of autumn, parthenogenetic females give rise to a single sexual generation of males and females, and mated females lay fertilized eggs, which represent the overwintering stage8. To our knowledge, A. gossypii has developed resistance to all of the original major groups of synthetic insecticides in the last few decades and in virtually all cotton-growing areas worldwide12. Therefore, this pest has quickly become a major problem when chemical controls fail because of resistance. Thus, an effective control method is essential and urgent.The cotton aphid, 13. In recent years, many molecular markers have been used to infer the phylogeny and biogeography of species to understand their patterns of evolution14. Owing to their locus-specific codominant, high abundance and high rates of transferability across species, microsatellite markers have been widely used in population genetics of aphid species17. Of course, cytochrome oxidase subunit I (COI) and cytochrome b (Cytb) genes also have a clear evolutionary pattern and evolve at a relatively moderate rate, which makes it suitable for reconstructing species and population-level phylogenies19. Little information on the genetic variation and population genetic structure of A. gossypii has been available before the development of methods for identifying polymorphisms of microsatellites, which are now used for analyzing the population genetic structure of many organisms20. Previous studies have shown that genetic diversity of A. gossypii is structured by host plants, and its genetic diversity and host specialization are affected by natural selection pressure23. Distinct genetic differentiation was found in A. gossypii populations from its winter hosts and summer hosts25. In North China, this species has two host-specialized biotypes (cotton and cucumber)26. By contrast, many studies have documented that genetic differentiation between localities was not significant, whereas genetic differentiation was high between host plant families. Low genetic diversity and a lack of significant differentiation have also been found in France28. However, the studies had smaller sample sizes or a limited number of populations, which were insufficient for clearly assessing the genetic diversity and population structure of this species in different climatic zones28.A better understanding of population genetic structure could help to manage aphid populations by providing more reliable estimates of population dynamics and the risk of resistance genes arising. The population genetic structure of an organism is determined by various factors, such as geographical barriers, ecological difference, and historical processes, as well as the dispersal ability of speciesA. gossypii in China . We then discuss management strategies for this species based in the results, which also provide a theoretical framework for developing appropriate regional management strategies against this insect pest.Here, we examined the population genetics, including genetic variation, population structure and demographic history of 33 populations of ina Fig.  using tea = 0.05), and the studied loci were not linked with each other and were used in the subsequent analysis. The null allele frequency per locus was mostly less than 0.1  correction method was 0.224 and 0.213 using ENA per locus (Supplementary Table FST estimations did not differ significantly (P = 0.4813). Therefore, the presence of null alleles had little influence on the estimation of FST. The basic summary statistics of genetic variation among different geographic populations of A. gossypii in China based on the 10 microsatellite loci are presented in Supplementary Table Na) across microsatellite loci ranged from 3.900 in Tacheng (TAC) to 8.400 in Baicheng (BC) and Changchun (CC), and had a mean of 6.103. The effective number of alleles (Ne) values across microsatellite loci ranged from 2.013 in Heze (HZ) to 4.129 in Taian (TA) and had a mean of 3.223. The mean observed heterozygosity (Ho = 0.512) was similar to the mean expected heterozygosity (He = 0.574) in the total populations. The observed heterozygosity ranged from 0.291 in Liaoyang (LY) to 0.697 in Jinan (JN), whereas the expected heterozygosity ranged from 0.388 in HZ to 0.681 in TC. The unbiased expected heterozygosity (uHE) ranged from 0.395 in HZ to 0.698 in TC. Shannon’s information index (I) values across loci ranged from 0.748 in HZ to 1.537 in BC. Allelic richness (AR) across loci was 3.735, ranging from 2.455 (HZ) to 4.615 (BC).In the present study, a total of 782 individuals from 33 populations were genotyped using ten microsatellite loci. Fisher’s exact tests showed that 111 of the 330 locus–population combinations deviated significantly from Hardy–Weinberg equilibrium (HWE). Significant linkage disequilibrium was present in 50 of 1485 tests  and 629 bases of Cytb gene from 738 individuals (accession nos. KY069922–KY069936) were obtained. For subsequent analyses, we combined COI and Cytb and treated them as a single locus because of the lack of recombination in the mtDNA. Of the 1208 total characters, 1172 were conserved, and 36, that included 22 singleton polymorphic sites and 14 parsimonious informative sites, were variable. The distribution of haplotypes and genetic diversity among different populations of Aphis gossypii based on the combined COI and Cytb sequences was shown in Fig. A region of 579 bases in the mtDNA A. gossypii haplotypes of the combined COI and Cytb and to detect any groups that can be explained by their geographic distribution. ModelTest indicated that the best substitution model was HKY + G. High congruence was observed between the phylogenies derived from maximum parsimony (MP) and maximum likelihood (ML) analysis, and these analyses generated only one inclusive group, but simultaneously provided many unresolved branches  showed statistically significant genetic differentiation for 496 pairwise comparisons (P < 0.05)  grouped in a fourth cluster at K = 4  was greater than in the opposite direction (0.0016). Limited gene flow may be associated with moderate migration capability of the cotton aphid, habitat differences and lack of commercial trade between the east and the more isolated west, although potential dispersion routes (gene flow) from west to east are presented.The PCoA result Fig.  showed aA. gossypii are shown in Table P < 0.001) as inferred from above analyses. In contrast to SSR, mtDNA did not support the western–eastern pattern (−0.34%). Considering the limited variability (information) of the mtDNA here, which may due to the slower evolutionary rate of mtDNA and the especially rapid expansion of China cotton aphid populations (see the demographic results that follow), the AMOVA result from the SSR data showing moderate variability are more reasonable and can represent more recent events.Results of the AMOVA test on microsatellite and mtDNA markers in different populations and regional groups of A. gossypii. If the dispersal of A. gossypii is limited by distance, genetic and geographical distances should be positively correlated, producing a pattern of isolation by distance. Applying the Mantel test, significant positive relationships between genetic and geographic distances were found over the 33 populations of China cotton aphids based on both kinds of markers . Furthermore, the unimodal mismatch distribution of all populations and the eastern region group and western region group indicated a sudden demographic expansion  and classic “Star” shape of mtDNA haplotype network provide preliminary but strong evidence of expansion Fig. . In addiZ-tests of selection rejected the null hypothesis of strict-neutrality (dN = dS) in favour of purifying selection  rather than for positive selection .It is more likely that the mitochondrial loci reflect the patterns of resistance of other genes and are thus a by product of selection. When we scanned for signals of selection, the 31, presumably because of clonal selection. Permanent parthenogenetic clones have high heterozygosity as a consequence of accumulated mutations34. The FIS values of the 10 microsatellite loci evaluated in this study were a little less than zero, which indicated a deficit of heterozygotes (Supplementary Table He) was similiar to the observed (Ho). Note that a low Ho was obtained in some populations such as Liaoyang (Ho = 0.291), which suggested a high genetic identity within the population. These results could be attributed to the specificity of the geography and climate in Liaoning in northeastern China because aphids usually reproduce sexually at relatively high latitudes and low temperatures.Deviations from HWE were observed at multiple loci in multiple populations. Fisher’s exact tests showed that 111 of the 330 locus/population combinations deviated significantly from HWE. The specific reproductive mode of parthenogenesis might also result in deviation from HWE among the studied populations. In general, aphid population genetic studies have more often documented populations not in HWE because of heterozygote deficits A. gossypii through the use of molecular markers has shown that the clonal diversity is structured by its host plant35. In the present study, A. gossypii had low to moderate genetic diversity based on microsatellite (Supplementary Table He varied between 0.388 (in HZ) and 0.681 (in TC). Similar cases of moderate genetic diversity have been documented in this species; He ranged from 0.409 to 0.873 on the basis of eight microsatellite loci17. Within a glasshouse population of A. gossypii, clonal diversity declined significantly as the spring/summer season progressed22. Low levels of genetic diversity were also found previously for other aphid species such as Eriosoma lanigerum36 and Myzus persicae37, which resulted from the adaptation of the aphids to two heavy selection pressures, the distribution of host plants and the use of insecticides27. In general, a critical feature of aphid biology is their viviparous reproduction by cyclical parthenogenesis, which can result in numerous overlapping generations in a short period and an extremely rapid increase in population size, which could explain the low level of genetic variability detected in aphids as compared with other insects7. Additionally, aphids are also very sensitive to selection. A. gossypii is a particularly polyphagous species; it has been described on almost 300 host plants from various botanical families3. Because the eastern populations had more genetic diversity than in the western populations  in the western and eastern region might have prevented or slowed nuclear gene flow between the eastern and western populations, resulting in lineage differentiation. Furthermore, for the JY population in south China , there is one mtDNA haplotype (H29) which was more divergent from H10  and Northeast China  was not large though the two sites are separated by more than 3000 km , which was detected in all 33 sampled populations, is the strongest evidence of expansion. The large negative values of Tajima’s D and Fu’s FS statistic and the unimodal mismatch distribution are also evidence of population expansion in most populations of cotton aphids in China. SSR data indicated that these cotton aphids experienced a recent demographic expansion without a severe bottleneck in most regions in China. As noted already, the results will provide essential information for understanding possible local adaptation and dispersal patterns and for further clarifying the relationship between genetic variation and outbreaks of A. gossypii. In the future, we will focus on monitoring the population dynamics of this species and understanding its seasonal occurrence in the main crop-producing areas of China to provide more useful data for forecasting outbreaks and managing this species.Increasing our understanding of the population dynamics of A. gossypii. Unfortunately, A. gossypii has developed resistance to insecticides such as carbamate and organophosphates46, and the exponential growth associated with parthenogenesis in this aphid species favours rapid selection for insecticide resistance mechanisms as a consequence of intense insecticide use12. It is more likely that the mitochondrial loci reflect the patterns of resistance of other genes and are thus a byproduct of natural selection. However, we did not detect any signal for positive selection on COI or Cytb. Positive selection may act on detoxification enzymes and metabolic genes like P450 family. High gene flow among the cotton aphid populations may explain the rapid spread of insecticide-resistance genes, and many resistance characteristics also accumulate through long-distance migration. Therefore, to survey for populations of A. gossypii with insecticide resistance over large temporal and spatial scales and investigate the distribution of resistance genes relative to the genetic structure and level of gene flow among populations of A. gossypii in China, quantifying the level of resistance to insecticides will be important for devising application strategies for chemical pesticides. In addition to geography and climate, host plant specialization and insecticide treatment can also affect the distribution of genetic variation in aphid populations27.Over the past several decades, pest management programs, such as cultural controls, biological controls, and the use of insecticides have been used in China and have partially prevented the damage and spread of A. gossypii populations in the cotton-growing area in Xinjiang over several consecutive years would help to reveal the relationship between the low genetic diversity and the adaptation of the aphids to two heavy selection pressures, the distribution of host plants and the use of insecticides. Additionally, we also found evidence of differentiation between the Xiuyan population, which was collected from Rhamnus koraiensis, and other populations collected from cotton . Obviously, a broad-scale sampling strategy on different host plant families is needed. Therefore, future work should concentrate on the genetic differentiation between winter hosts and summer hosts through extensive sampling in China. Simultaneously, we will also focus on the relationship of the incidence of aphid populations on cotton, natural selection pressure , and genetic structure. Our genetic data suggest significant genetic differentiation among different strains of Wolbachia symbiosis, which is present between the eastern region and western region . Therefore, future experiments should shed light on the influence of the bacterium on the diversity and genetic structure of cotton aphid populations. Developing additional microsatellite markers and more extensive sampling, including samples at a finer distance scale, should improve the resolution of the understood population genetic structure and our understanding of intra- and inter-race diversity of this species.The study of the temporal dynamics of genotypic diversity of A. gossypii were collected from 12 provinces at 33 geographic locations in China, covering four climatic regions—mid-temperature zone (MTZ), warm temperate zone (WTZ), tropical zone (TZ), and subtropical zone (SZ)—from June 2012 to December 2015  according to the manufacturer’s protocols Aphis glycines (GenBank accession nos. JQ860254 and GU205350) and Aphis craccivora (GenBank accession nos. AB506714 and AM085376) were used as outgroup species in the phylogenetic analysis.All individuals of COI and Cytb. To amplify COI, we used published primers CIS (5′-ACCAGTTTTAGCAGGTGCTATTAC-3′) and CIA (5′-GTATATCGACGAGGTATACCATTT-3′)47 and for Cytb, CP1 (5′-GATGATGAAATTTTGGATC-3′) and CP2 (5′-CTAATGCAATAACTCCTCC-3′)48. Each PCR mixture contained 0.25 μL EasyTaq DNA polymerase (5 U/μL), 2.5 μL 10× Easy Taq buffer, 0.5 μL dNTP mixture (2.5 mM of each), 0.5 μL of each primer (10 pM), 1 μL of DNA template and 19.75 μL of distilled water, making a final volume of 25 μL. The reactions were performed in a GeneAmp PCR System 9700  under the following conditions: 94 °C for 5 min; 35 cycles at 94 °C for 1 min, 52 °C for COI or 48 °C for Cytb for 1 min, 72 °C for 1 min; and a final extension at 72 °C for 10 min. All specimens were sequenced on an Applied Biosystems ABI 3730 DNA sequencer (Applied Biosystems). Chromatograms, including sense and antisense, were edited and assembled using DNASTAR 5.0  to obtain a single consensus sequences.We sequenced two partial regions of the mitochondrial genes et al.49 for loci ago59 and ago89; Kim et al.50 for loci Agl2-6, AgI1-10, AgI1-11 and AgI1-2; and Gauffre & Coeur51 for loci AF-93, AF-45, AF-F and AF-beta, which were assigned unique fluorophores for fluorescent tagging of DNA in a PCR reaction. For these isolated microsatellite, each 20 mL reaction volume for the genotyping PCR contained 0.2 μL EasyTaq DNA polymerase (5 U/μL), 2.0 μL 10× Easy Taq buffer, 0.2 μL dNTP mixture (2.5 mM of each), 0.4 μL of reverse primers (10 pM), 0.4 μL of forward primer, which were labeled with fluorochromes (HEX or FAM) (10 pM), 0.5 μL of DNA template and 16.3 μL of distilled water. For the thermal profile, an initial denaturation step at 94 °C for 4 min was followed by 30 cycles of denaturing at 94 °C for 30 s, annealing at 58 °C for 30 s, and extension at 72 °C for 30 s; and a final 5 min extension at 72 °C. Following amplification, the products were visualized at Sangon Biotech Co., Ltd.  using an ABI 3730XL automated sequencer (Applied Biosystems). Microsatellite alleles were analyzed using GeneMapper 4.0 (Applied Biosystems).Ten microsatellite loci were used in this study using the methods of Michel 52 using the multiple alignment default parameters and corrected by hand. Nucleotide composition, conserved sites, variable sites, parsimony informative sites were analyzed with MEGA 653. Molecular diversity indices such as haplotype diversity (Hd) and nucleotide diversity (Pi) were estimated using DnaSP 4.054.The sequences were first aligned with CLUSTAL X 1.8155 using a heuristic search with 1000 random sequence repetitions and tree-bisection-reconnection (TBR) branch swapping. Consensus trees (50% majority rule) were obtained if more than one equally parsimonious tree was found. ModelTest 3.756 and the Akaike information criterion (AIC)57 were used to identify the appropriate nucleotide substitution models, and the selected models of sequence evolution were used for the ML phylogeny reconstruction. The ML analysis was performed in PAUP* 4.10b using a heuristic search strategy with 10 random additions of sequences and TBR branch swapping. Bootstrap analysis was performed under the same model with 100 pseudoreplicates, 10 random addition sequences per replicate and TBR branch swapping. Bayesian analysis was also carried out using the model selected by Modeltest 3.7. The analysis used a random starting tree and proceeded for one million Markov chain Monte Carlo generations, and trees were sampled every 100 generations. The first 2,500 generations  were later discarded as burn-in; 50% majority-rule consensus trees were generated from the remaining trees and posterior probabilities computed. Compared with conventional phylogenetic trees, haplotype networks can enhance the relationships among haplotypes and are preferable for intraspecific analyses. The median-joining network of the haplotypes of COI and Cytb were generated in Network 2.0 by a median-joining method58.Maximum parsimony (MP), maximum-likelihood (ML) and Bayesian inference (BI) phylogenetic analyses were used to identify major clades and to evaluate the relationships between the haplotypes separately. MP analyses were performed in PAUP* 4.10bFST) statistics between the populations were conducted in Arlequin 3.059. Mantel tests were performed to determine the significance between genetic and geographic distances.Analysis of molecular variance (AMOVA) and calculation of population genetic differentiation , positive (dN > dS) and purifying selection (dN < dS) using MEGA 653. Demographic history changes were analyzed for A. gossypii using two neutrality tests, Tajima’s D29 and Fu’s FS30, which were calculated from the total number of segregating sites and used to assess evidence for population expansion. Mismatch distributions were calculated between the observed and expected mismatch distributions used as a test statistic. According to coalescent theory, a population usually exhibits a unimodal mismatch distribution following a recent population demographic or range expansion60. To determine whether movement patterns were influenced by spatial scale, we conducted a spatial analysis of genetic structure by performing the isolation by distance (IBD) analysis for each site type using a Mantel test implemented in the web-based IBDWS 3.3261. An IBD analysis was used to determine the relationship between calculated pairwise FST and pairwise geographic distances (km) between collection sites.We performed codon-based 62 and exact probability tests. An exact test for HWE was conducted per locus and over all loci in each population using the same program. Corrections for multiple tests were performed by Bonferroni corrections. Null allele frequencies for each microsatellite locus were estimated following the expectation maximum algorithm of Dempster et al.63 using FreeNA41. The excluding null alleles (ENA) correction method was used to provide accurate estimation of FST in presence of null alleles. The population genetic diversity indices such as mean number of alleles (Na), effective number of alleles (Ne), Shannon’s information index (I); observed heterozygosity (Ho), expected heterozygosity (He), and unbiased expected heterozygosity (uHE) were assessed using GenAlEx 6.4164. Allelic richness (AR), fixation index (FST), inbreeding coefficient (FIS) among these regions were calculated in FSTAT 2.9.365. The levels of genetic differentiation between pairs of populations were also estimated in Arlequin 3.059.Genotypic linkage disequilibrium (LD) was tested among all pairs of loci across all populations using GenePop 3.464 and by using the Bayesian model-based clustering method implemented in STRUCTURE 2.2.366. We also used POPTREE 267 to construct a unrooted tree using the neighbour-joining method. PCoA was based on the covariance of the genetic distance matrix. STRUCTURE sorts individuals into K optimal region groups, according to their genetic similarity. The allele frequencies of different populations were assumed to be correlated due to historical migration and common ancestry68. In addition, the admixture model of individual ancestry was used to assign hybrid individuals into population clusters; no prior population information was used69. Ten independent runs for K values ranging from 1 to 10 were performed with a burn-in length of 30,000 followed by 1,000,000 Markov chain Monte Carlo replicates. To identify the optimal number of groups (K), measures of change in the likelihood function (ΔK) and FST (ΔFST) were calculated using the package CORRSIEVE671, and the plateau criterion was applied57. The software CLUMPP 1.172 was used for model averaging of individual ancestry coefficients across the 10 independent runs. Then clusters were visualized using DISTRUCT 1.173. The consistency of the clusters identified through the STRUCTURE approach was tested by a dissimilarity analysis, which was calculated from allelic data. The significance of the hierarchical partitioning of genetic structure among the geographic groups was examined using an analysis of molecular variance (AMOVA) in Arlequin 3.059. Three hierarchical levels were defined: (1) between regional groups (western and eastern region), (2) between populations within regional groups, (3) between individuals within populations. BayesAss 374 was used to estimate recent migration between regional groups. We ran the MCMC for 20,000,000 iterations, discarding the first 2,000,000 iterations and sampling per 200 iterations. We then adjusted the mixing parameters to ensure an acceptance rate ofabout 30% and carried out three independent runs with different random number seeds. Additionally, we conducted an IBD analysis between microsatellite and geographic distance data using a Mantel test implemented in IBDWS 3.2361.Population structure was estimated using principal coordinate analysis (PCoA) implemented in GenAlex 6.4175. A qualitative descriptor of the allele frequency distribution (mode-shift indicator), which discriminates between bottlenecked and stable populations was also used. Under the three models, the standardized differences test was removed from this study because this test is typically only used when at least 20 polymorphic loci are available.We tested the bottleneck effect at the population level to explore population demography using different models and testing methods implemented in Bottleneck 1.2.02, and the infinite allele model (IAM), two-phase model (TPM), and the strict stepwise mutation model (SMM) were applied with 10,000 replicationsRevised supplementary information files


Genome evolution is predicted to be rapid following the establishment of new (neo) sex chromosomes, but it is not known if neo-sex chromosome evolution plays an important role in speciation. Here we combine extensive crossing experiments with population and functional genomic data to examine neo-XY chromosome evolution and incipient speciation in the mountain pine beetle. We find a broad continuum of intrinsic incompatibilities in hybrid males that increase in strength with geographic distance between reproductively isolated populations. This striking progression of reproductive isolation is coupled with extensive gene specialization, natural selection, and elevated genetic differentiation on both sex chromosomes. Closely related populations isolated by hybrid male sterility also show fixation of alternative neo-Y haplotypes that differ in structure and male-specific gene content. Our results suggest that neo-sex chromosome evolution can drive rapid functional divergence between closely related populations irrespective of ecological drivers of divergence. Dendroctonus ponderosae.The evolution of new sex chromosomes potentially generates reproductive isolation. Here, Bracewell et al. combine crossing experiments with population and functional genomics to characterize neo-sex chromosome evolution and incipient speciation in the mountain pine beetle,  These patterns reflect the fundamental role that heteromorphic sex chromosomes play in the evolution of deleterious epistatic interactions underlying intrinsic reproductive isolation6, but the importance of these processes to the early stages of speciation has been debated7. For example, while sex-linked hybrid sterility generally evolves much more rapidly than inviability, both are thought to accumulate more slowly than other forms of ecological or behavioural reproductive isolation8. However, most speciation research has focused on animal systems with well-established and highly heteromorphic sex chromosomes9, resulting in a limited view of the contribution of sex chromosome evolution to the origin of species.Understanding how reproductive isolation evolves between populations is essential to understanding the origin of biodiversity. Nearly a century of research has established that hybrid incompatibilities usually manifest in the heterogametic  sex first  or through the fusion of an ancestral sex chromosome with an autosome . Given the surprisingly fluid nature of sex chromosome systems in some groups, there are at least three general stages of sex chromosome evolution—each with distinct evolutionary dynamics—that might contribute to the process of speciation 4 and various evolutionary dynamics  that can drive rapid sex-linked divergence6.It is now apparent that transitions to new (neo) sex chromosomes are common over evolutionary timescalesDendroctonus ponderosae) is the most important forest pest in western North America, and is the primary contributor to recent tree mortality on 6.6 million hectares in the western US, exceeding tree mortality caused by wildfire21. Previous studies have found that populations at the southern reaches of the beetle’s range  are the most genetically divergent and genetic variation follows an isolation-by-distance pattern around the Great Basin Desert22 . Despite very low levels of genetic differentiation between populations22, hybrid male sterility (HMS) has been detected between some mountain pine beetle populations consistent with cryptic speciation24. In addition, the mountain pine beetle shares a neo-XY chromosome system with its closest relative, the Jeffrey pine beetle (Dendroctonus jeffreyi), descending from a fusion between the ancestral X chromosome (anc-X) and the largest autosome in the common ancestor of these species25. There is no known karyotypic variation within either species25 and the degree of genetic, structural, or functional divergence between these nascent sex chromosomes has not been evaluated. However, several Dendroctonus species and many other beetles have distance-pairing heteromorphic sex chromosomes that do not synapse and are fully non-recombinant12. In these systems, the Y chromosome appears highly degenerate and is a small fraction of the size of the X. If distance-pairing and the associated loss of recombination carries over following an autosomal X chromosome fusion event, then neo-sex chromosomes may be immediately predisposed to the rapid evolution of heteromorphy in this system.The mountain pine beetle  and Southern California , as might be expected if hybrid incompatibilities are an indirect consequence of ecological divergence. For example, beetles from populations bordering the HMS boundary (OR and ID) were collected from the same host tree species (Pinus contorta), were morphologically indistinguishable, and do not differ in body size or development time27. Pheromone profiles were also found to be very similar between reproductively isolated OR and ID populations  that might contribute to asymmetric HMS in this system based on standard genetic and molecular diagnostics24. Thus, this distinct genetic architecture requires epistatic interactions involving genetic factors with uniparental inheritance3. In mountain pine beetles this equates to nuclear-cytoplasmic interactions involving mitochondrial DNA (mtDNA) and/or sex chromosome-linked interactions. To test this hypothesis and to evaluate the overall genomic context of these hybrid incompatibilities we first generated a range-wide population genomic data set by combining two published genomes29 (CAN) with 27 additional whole genomes from nine populations , but did not differentiate the autosomes from the neo-sex chromosomes29 which are thought to descend from an ancestral X-autosome fusion25 . These estimates of synonymous gene divergence are about two-fold higher than those found in D. miranda, an early neo-XY system  where signatures of specialization and degeneration are already apparent15. The age of the Dendroctonus neo-sex chromosomes are not clear, but the Jeffrey pine beetle and mountain pine beetle share the same neo-XY configuration. Assuming a simple mtDNA molecular clock30, the neo-sex chromosomes in the mountain pine beetle are at least 2–3 million years old (1 generation per year).These analyses revealed three important attributes of neo-sex chromosome evolution in this system. First, the sex chromosomes comprise ~22% of the assembled genome. This estimate is broadly consistent with karyotypic datame Table . Third, Fst = 0.01). Instead, genetic variation largely grouped populations based on geographic proximity  and low between-population genetic differentiation  and individual mtDNA genomes Fig. . On the ity Fig.  with lowIn contrast, we detected markedly increased genetic differentiation on the anc-X and neo-X and extreme differentiation of the neo-Y between reproductively isolated populations Fig. . On the 1, sterility evolves more rapidly than inviability2, and asymmetry precedes reciprocal isolation3. These evolutionary transitions have mostly been established through comparative analyses of crosses between different species pairs spanning a broad range of genetic divergences2. The existence of population-level variation in hybrid incompatibilities is now well established32; however, the striking progression in the strength and pattern of intrinsic reproductive isolation occurring between populations separated by such low levels of genomic divergence is highly unusual. Second, sex-linked incompatibilities are playing a central role in the evolution of reproductive isolation. Based on overall patterns of population genetic differentiation and HMS in reciprocal crosses, involvement of mtDNA can be unambiguously dismissed while the X and neo-Y chromosomes show moderate to complete differentiation between populations that are also isolated by hybrid male sterility . Alternatively, reproductive isolation may have evolved during an initial period of geographic isolation or allopatry followed by secondary contact. The low population differentiation and extensive network reticulation observed at autosomal loci Fig.  could re34, allowing us to differentiate these primary vs. secondary models of divergence-with-gene-flow35. We first calculated the f3 statistic34 for all possible three-way population comparisons using 1,269,066 high quality autosomal SNPs. This approach uses allele frequency correlations between populations to provide a formal test of admixture that is robust across a broad range of population histories34. We detected strongly negative skews in the f3 statistic in the centre of the range, indicative of secondary contact followed by extensive autosomal admixture between populations adjacent to the HMS boundary with signatures of admixture tailing off with geographic distance from the boundary .The population-frequency based 35, especially in systems where reproductively isolating phenotypes and their underlying genetic architectures are unknown. In mountain pine beetles, the distinct asymmetric architecture of HMS  and lower absolute sequence divergence (Dxy) between populations 37. Genetic drift is generally predicted to be stronger on the sex chromosomes due to differences in their effective population sizes. Assuming equal sex ratios, genetic diversity on the X and Y should be ~75 and ~25% of autosomal variation. However, we found that the anc-X, neo-X, and neo-Y all showed significantly reduced levels of genetic variation relative to these theoretical predictions  after accounting for variation in mutation rate  should be robust to the influence of selection within contemporary populations, but is sensitive to chromosomal differences in effective population sizes including the effects of selection within ancestral populations37. We observed lower absolute X and Y-linked divergence (Dxy) between reproductively isolated populations of beetles . Thus, patterns of diversity and divergence both indicate significant long-term reductions in the effective population sizes of the sex chromosomes in mountain pine beetles. Moreover, while more efficacious selection is common on sex chromosomes38, dramatic long-term reductions of neo-Y and neo-X diversity relative to the anc-X and the autosomes suggests that natural selection has been particularly intense40 during the ongoing evolution of the mountain pine beetle neo-sex chromosomes.Skews in mating sex ratios cannot account for reduced variation on both sex chromosomes, or for significant reductions in X-linked but not mtDNA diversity. Alternatively, recurrent positive and purifying natural selection can reduce chromosome-wide levels of genetic diversity, especially on non-recombinant Y chromosomeses Table  and thes42; therefore, we would only expect the persistence of strong X and/or Y differentiation over this fine geographic scale if HMS involves sex-linked interactions. Consistent with this, only the sex chromosomes clearly partitioned genetic variation between these neighbouring populations , elevated on the anc-X (0.15), and even higher for the neo-X (0.51)  of an additional 30 beetles from the neighbouring OR and ID populations , which showed very low autosomal differentiation and extensive admixture in our range-wide whole genome sequencing Fig. . Dispersons Fig. . Differe51) Fig. . The neo43 and our results indicate that fine-scale resolution of X-linked reproductive isolation in mountain pine beetles may be possible through expanded population sampling and a more resolved X-linked genetic map.Analyses of population structure in OR and ID revealed further important differences between the anc-X and neo-X in individual ancestry. While the neo-X showed a clear split between OR and ID populations with only two individuals showing low levels of mixed ancestry, several ID beetles showed substantial OR ancestry on the anc-X Fig. . This as44. For example, pollen and molecular data show that several Pinus host trees were isolated in distinct coastal and Rocky Mountain refugia during the Pleistocene and subsequently expanded northward as the climate warmed44.Collectively, these results indicate a period of allopatry leading to genetic differentiation and functional divergence between some populations followed by secondary contact and gene flow near the centre of the mountain pine beetle range. Consistent with this model, multiple host tree species show broadly concordant patterns of population isolation during Pleistocene range retractions and secondary contact within the same geographic region45. Asymmetric hybrid incompatibilities  chromosome49 and Y chromosomes are masculinized50, but the evolutionary tempo of this specialization remains unclear15. We characterized genome-wide expression patterns of multiple tissues  from beetles collected in Montana . The anc-X was enriched for ovarian expression  and depleted of testis-specific genes  in insects16. Dendroctonus and many other beetles appear to have fully non-recombinant sex chromosomes12, thus neo-sex chromosomes may be immediately predisposed to the rapid evolution of heteromorphy in this system and could account for the apparently rapid pace of structural degeneration.Given the overall pattern of neo-Y gene specialization Fig.  and dege29 within these deletions that were present in both East and Southeast beetles, five of which were highly expressed in testis or were male-specific in our expression analyses , which could further facilitate the persistence of reproductive barriers in the face of gene flow47. Here we have focused primarily on male fertility, but the observation of delayed development in some crosses raises the intriguing possibility that rapid neo-sex chromosome evolution impacts a broader range of developmental processes.Our results suggest that sex chromosome-autosome fusions can initiate rapid divergence between populations and that this divergence is likely to have fitness consequences in hybrids. Gene movement on and off of the sex chromosomes can play an important role in speciation54, and therefore could be a recurrent driver of population divergence and speciation.Although we observed the most striking patterns of population genetic and functional differentiation on the neo-sex chromosomes, our results do not rule out important contributions of the ancestral X chromosome to reproductive isolation in mountain pine beetles. Indeed, neo-sex chromosome systems derived from recent sex-autosomal fusions are particularly intriguing in that they are potentially subject to a broad range of processes  that could contribute to the evolution of hybrid male incompatibilities Fig. . Given t55 and beetles are by far the most diverse group of insects. Neo-sex chromosome transitions occur repeatedly across the beetle radiation12, providing ample opportunities for this non-ecological mode of speciation.That a rapid neo-sex chromosome speciation event appears to be unfolding in a phytophagous beetle irrespective of typical ecological drivers of divergence is all the more intriguing. Speciation in plant feeding insects is thought to be largely driven by host plant use24. Here we add new data from populations of an intermediate geographic distance  and the most genetically differentiated populations . For intermediate populations, we collected beetles from ponderosa pine (Pinus ponderosa) in the Lubrecht Experimental Forest, MT  and from a lodgepole pine (Pinus contorta) from Lassen NF, CA  in the same vicinity of the CA3 population used in Bracewell et al.24. For the most genetically differentiated populations, we collected beetles from the Pinaleño Mtns, AZ , from Southwestern white pine (Pinus strobiformis) and from near Big Bear Lake, CA , from singleleaf pinyon (Pinus monophylla).Previous crosses have established asymmetric HMS between Oregon (OR) males and Idaho (ID) females, while more geographically distant crosses showed symmetric HMS24, we conducted crosses between nCA and MT in 3 lodgepole pine bolts (tree sections) per cross  with 12–14 matings per bolt . The resulting inter- and intra-population offspring were collected and pooled by cross type. We tested the reproductive capacity of hybrids with reciprocal backcrosses to both parental populations . Mating pairs were replicated 18 times per combination and were randomly inserted in a 6.1-cm-wide strip of tree phloem that was caged to collect re-emerging parents. After 26 days, each strip was examined for eggs, the length of gallery (cm), and total number of eggs hatched.Following established husbandry protocols56. Experimental conditions were as above with a few minor modifications: all crosses were conducted in 4 (interpopulation) or 5 bolts (intrapopulation) per cross type with 7 matings per bolt. The sex and date of emergence was recorded for all offspring, which were then investigated for sterility by reciprocal backcrossing as above (14–35 replicates per each of 10 combinations).We investigated both fertility and developmental timing of hybrids in the AZ×sCA cross. Previous studies have explored development time of hybrids  and Bayesian GLMs in R (version 3.1.2). All post hoc pairwise comparisons were done with Tukey’s honestly significant difference (HSD) tests using the multcomp package. We excluded crosses with <10 cm of parent gallery, resulting in analysis of 129 nCA×MT crosses (90%) and 235 sCA×AZ crosses (79%). Total egg hatch was overdispersed count data with complete separation of some levels and was therefore modelled with a Bayesian GLM and quasipoisson error distributionTo assess hybrid male reproduction in more detail we first quantified sperm transfer to females. On their collection day, each female had her spermathecal pump and sac removed and transferred to a drop of distilled water on a microscope slide. Each structure was immediately scored under ×400 magnification on a scale of 0 (no sperm) to 4 . Differences in the sperm quantities were determined using Wilcoxon rank sum tests of all pairwise comparisons with a Bonferroni correction  and nCA males (n = 20) were dissected in 60 µl of phosphate buffered saline (PBS) and each testis, attached seminal vesicle, and accessory glands were isolated and transferred to 20 µl of PBS. Spermatozoa were released from the seminal vesicle into solution using fine dissection tools and 5 µl of homogenized solution was then transferred to a Makler Counting Chamber . Sperm lying ≥50% within the counting grid were counted, averaging counts across the two seminal vesicles for each male. Differences between hybrid males and intrapopulation males were determined using a Wilcoxon rank sum test  collected during the peak of emergence from rearing containers. Hybrid males  utilizing a covariance matrix. Compounds detected in no more than trace amounts in a sex were excluded from the PCA for that sex.Pheromone samples were obtained from adult beetles collected near OR and ID . Brood adults newly-emerged from logs cut from naturally-infested lodgepole pine were induced to mine into a freshly-cut lodgepole pine log in the laboratory. Solitary females  that had been mining <1 d and males that had been allowed to pair 1 d earlier in the entrance of a female from the same site were excised from the bark and held in chemical adsorbent-containing microvials for 1 d at 22 °C29  to identify putative X, Y and autosomal scaffolds using relative sequencing coverage between the sexes. We used SeqyClean version 1.8.10 to quality filter, adapter trim, and screen for common Acinetobacter contaminants . Filtered reads were then mapped to the reference male genome29 using BWA-MEM version 0.7.962 and PCR duplicates were removed using rmdup within samtools version 1.163.We first eliminated mountain pine beetle scaffolds that were short (<2 Kb including N’s) or were likely bacterial (identified using BLASTn against the NCBI nt database), resulting in 4,877 scaffolds  for further inquiry. We then took advantage of the large amount of raw sequencing data available from the male and female genome builds64 to test for significant per sex, per scaffold differences in coverage by performing exact tests with a false discovery rate of 5% for each of the 4,877 scaffolds. As a proof-of-principle, we verified that six previously identified ancestral X-linked scaffolds29 showed significant female-biased sequencing coverage  to the male genome build scaffolds. We then used edgeRDendroctonus jeffreyi) . DNA was extracted using either OMEGA E.Z.N.A Tissue DNA kits or Qiagen DNeasy kits. Sequencing libraries were prepared for fourteen samples from seven populations  at the University of Montana using 300 ng of genomic DNA and the NEXTflex DNA Sequencing Kit, and DNA Barcodes by Bioo Scientific . These libraries were sequenced at the Vincent J. Coates Genomics Sequencing Laboratory, Berkeley, CA. We sequenced an additional fifteen beetles including one male from each population above and two males and one female from two additional populations . These libraries were prepared by GENEWIZ and sequenced using 150 ng of genomic DNA, the New England Biolabs NEBNext Ultra DNA Library Prep Kit.We Illumina 100 bp paired-end (PE) re-sequenced whole genomes of two adult males and one female from each of nine populations of mountain pine beetle Fig.  and one 65 to call genotypes. All BAMs were processed using realignerTargetCreator to identify regions with potential insertion-deletion variation, locally realigned using indelrealigner, and genotyped using the GATK UnifiedGenotyper version 3.1–1. We then filtered SNPs with excess depth (>60×), eliminated all indels, sites flagged other than PASS, non-biallelic positions, and individual genotypes with a genotype quality score <30. The CAN male and female samples were previously sequenced to very high coverage29, resulting in universally high genotype qualities. To account for this high coverage bias, we only called CAN genotypes at sites that passed filters in all other individuals.All whole genome re-sequencing data was cleaned and mapped as above. We used the GATK Best Practices pipelinen = 19) and eliminated confidently called heterozygous positions. We further partitioned the X chromosome into ancestral-X (anc-X) based on previously reported synteny with the Tribolium castaneum X chromosome29 and treated any remaining scaffolds as putative neo-X. After these filters, there were 87,482 SNPs located on anc-X scaffolds and 75,450 SNPs on neo-X scaffolds. Retained SNPs had similar mapping qualities across partitions .Neo-XY systems present a number of genotyping challenges related to differences in sequence complexity and residual sequence similarity between homologous X and Y-specific regions. With these issues in mind, we conducted both site and interval-based analyses using a series of conservative filters to reliably identify SNP positions across all partitions. For site-based analyses, we excluded autosomal or unclassified positions where 14 (48%) or more individuals were missing confident genotype calls  resulting in 5,300,757 autosomal and 164,080 unclassified SNPs. To identify high quality neo-Y SNPs, we also filtered sites with confident genotypes called in any female, as well as sites called as heterozygote for any male. Both conditions should not occur on the neo-Y in males unless there are errors in the assembly and/or in read mapping. This rather conservative filtering resulted in us identifying 544 high quality SNPs  on the neo-Y distributed across 234 scaffolds . The neo-sex chromosomes were not repeat-masked in advance and a large portion of filtered SNPs occurred in repetitive regions or stretches of low sequence complexity based on variance in local sequencing coverage and lower mapping qualities. To identify X-linked SNPs, we restricted analyses to males . We tested K-values of 1–9 and ran 6 replicates per each K under an admixture model. We analysed 60,000 autosomal, 15,000 neo-X, and 25,000 anc-X SNPs with X-linked analyses using a haploid model (PLOIDY = 1). The best K for each chromosome category was determined using STRUCTURE HARVESTER69  and a Z-score value of 3 and above considered significant33. Phylogenetic NeighborNet trees were generated using SplitsTree471 . Relative node depth was calculated following Feder et al.39. To characterize the sex-linked genetic diversity relative to the autosomes, we first estimated divergence (Dxy) to the outgroup, D. jeffreyi. After accounting for mutational differences (diversity/divergence to outgroup) we calculated the sex chromosome/autosomal ratio38.Next we used interval-based analyses to evaluate diversity and divergence between West and East beetles across all genomic partitions. To accurately identify both variant and invariant positions, we re-analysed and re-filtered our whole genome data. For the anc-X, neo-X and autosomes, we first restricted analyses to high quality intervals ≥100 bp in length where all individuals had coverage ≥3x and ≤1 SD of their total estimated mean coverage. The purpose of these thresholds was to ensure that all individuals had adequate coverage to call a variant but not excessive coverage as would be expected if the interval was a collapsed repeat or originated from some other assembly error. We used the same absolute coverage thresholds (≥3×) for neo-Y intervals but also eliminated regions with female coverage. We then eliminated X and Y intervals that included sites where more than one male was confidently called a heterozygote (GQ ≥20) as these are likely repetitive regions or collapsed portions of the neo-X/neo-Y. This series of step-wise filters allowed us to find regions of the degenerate neo-Y where we felt confident that we were estimating our parameters over single copy regions that were free of assembly mistakes that plague mountain pine beetle neo-Y scaffolds. Mapping qualities were similar across filtered partitions . We genotyped either males or females using GATK’s UnifiedGenotyper, filtered as above, and estimated nucleotide diversity and DFst), PCA and phylogenetic trees to verify that qualitative patterns between partitions were consistent. Second, we repeated X-linked population genetic analyses on females  using autosomal filtering. All qualitative patterns were consistent across partitions under these conditions. For example, female estimates of diversity on the anc-X  and the neo-X  were similar to male-based estimates  and MITOS (http://mitos.bioinf.uni-leipzig.de/help.py) to annotate mtDNA genomes. We then genotyped all 31 individuals using the highest quality de novo mtDNA genome assembly as a reference. Using GATK UnifiedGenotyper, we called the most frequently encountered genotype at all sites to avoid errors originating from nuclear copies of mtDNA. We confidently assembled 14,783 bp of the mtDNA genome for each beetle, including 13 protein coding genes, 2 rRNA genes, and 19 tRNAs. MtDNA sequence divergence between the mountain pine beetle and the Jeffrey pine beetle was estimated to be 6.8% and we used the insect mtDNA molecular clock estimate from Papadopoulou et al.30.We used Velvet 1.2.1074 to identify ‘paralogous’ sequences within the draft mountain pine beetle genome. We restricted our set of genes to those where a single clear ‘paralog’ was present on a scaffold not identified as neo-Y and where at least 60% of the neo-Y gene could be aligned. Genes below this threshold were too fragmented to accurately align and were often only partial hits of dubious homology. We then downloaded coding domain sequences and aligned the putative neo-X and neo-Y gametologs using MAFFT75 and calculated Ks on 119 unambiguous neo-X/neo-Y homologs . We then used this list along with Ensembl and BioMartWe counted the number of reads mapping  to the 4,114 scaffolds. We then used edgeR to normalize and estimate RPKM (reads per kilobase of scaffold per million reads mapped) for each individual and to test for differences in per scaffold coverage between East, West, and Southeast beetles. We used the pheatmap package in R for clustering (hclust), restricting analysis to scaffolds with a minimum of two counts per million for at least eight individuals.77, with additional DNA cleaning steps and a slightly modified PE2 adapter (available upon request). DNA was cleaned, digested with PstI (New England Biolabs), barcoded adapters were ligated to the restriction cut sites, pooled across samples and sonicated using a Bioruptor . Sheared DNA was blunt-end repaired, a 5′ A was ligated to the fragment, and then a small Y-adapter was ligated to the A overhang. The indexing read barcode and Illumina specific adapter sequences were added to the fragment through PCR  and a final extension at 72 °C for 5 min). Libraries were pooled and 100 bp PE Illumina sequenced at the University of Utah Microarray and Genomic Analysis Core Facility, Salt Lake City, UT, or the Vincent J. Coates Genomics Sequencing Laboratory, Berkeley, CA.Approximately 400 ng total DNA from adult OR and ID beetles was used for the RADseq protocol. We developed a customized single restriction enzyme digest protocol similar to Etter and colleagues78 and default settings. The resulting paired end reads were then mapped to the male reference genome using the BWA aln and sampe pipeline version 0.7.5 and filtered for PCR duplicates as above. We compared coverage of RADtags positioned within 141 putatively neo-Y deleted regions  using samtools63 . We found Fst to be qualitatively similar although somewhat less pronounced . We further explored how removing all male-specific filters in males would influence these estimates and we found that again, the results were qualitatively similar, although less pronounced .Raw sequencing reads were processed and cleaned using the process_radtags program from Stacks version 1.18Adult beetles were collected from newly attacked lodgepole pine near Marias Pass, MT  in the fall of 2013. Tissues were dissected from live beetles in RNase-free PBS and RNA was extracted immediately using a Qiagen RNeasy kit. RNA integrity was verified using an Agilent 2100 Bioanalyzer (Agilent Technologies) and libraries were prepared using the Agilent SureSelect Strand-Specific RNA Library Kit . We generated 13 RNA-seq libraries, five of which consisted of pools of three individuals. These pools were of male heads, female heads (two replicates), ovaries and testes. Individual libraries were constructed from ovaries of four females and testes of four males. All libraries were initially Illumina sequenced in a truncated run (machine failure) of one lane of 100 bp PE and then re-sequenced with one additional lane at the Vincent J. Coates Genomics Sequencing Laboratory, Berkeley, CA. Analyses were performed on the combined data.29 using Tophat2 version 2.0.1079. We used default parameters, specified fr-firststrand, and used the mountain pine beetle annotation file  to assist in mapping. Because we had pooled data as well as individual data, we performed two separate analyses using Cufflinks version 2.2.180. To allow for gene discovery, we used Cuffmerge to merge published mountain pine beetle transcriptome data with the transcriptomes from our tissues80. For pooled samples, we only estimated relative gene expression levels. The individual data set had sufficient replication to allow tests for differentially expressed genes. In both analyses, gene expression values  were restricted to only those genes found in our 4,114 high-quality scaffolds. Next we used our pooled expression data to perform hypergeometric tests for tissue specific enrichment or depletion of counts of expressed genes across linkage groups. A gene was considered tissue specific if it was expressed in the focal tissue and not expressed in the three other tissues (FPKM = 0). These analyses were conducted with two different gene expression thresholds: focal tissue >1 FPKM and focal tissue >10 FPKM  to compare read counts of East+Southeast to West beetles over 781 annotated gene intervals on neo-Y linked scaffolds, 24 of which showed significant differences in coverage. We then identified a subset of 11 gene intervals that showed greater than 10-fold higher coverage in East+Southeast beetles with little or no coverage in West beetles. The remaining 13 significant gene intervals did not show complete loss of coverage in West beetles, and likely represent multicopy genes and/or genes with intronic regions harbouring regional indel variation.The M2-ACE-like gene stood out an interesting candidate for playing a role in hybrid male sterility. To determine if this gene might have a homolog on the neo-X, we used BLASTp to search the neo-Y protein sequence (accession ENN80010) against GENBANK and found the top two hits were to a protein on a different male mountain pine beetle scaffold, Seq_1102825, (accession ENN74047) and to a single protein in the female mountain pine beetle genome (accession ERL91207). The presence of only one other ACE-like gene in both the male and female genome builds provided some evidence that these versions might be on the neo-X. We then performed PCA of SNPs located on the putative male neo-X scaffold (Seq_1102825) and found that the first principal component split individuals by sex and explained 33.6% of the variation providing more evidence of X-linkage. Investigation of gene expression for the putative neo-X version of the gene suggested that in contrast to the neo-Y version Fig. , it is whttps://doi.org/10.6084/m9.figshare.5479594.v1). Any additional data that support the findings of this study are available from the corresponding authors upon request.Sequence and crossing data that support the findings of this study have been deposited at the NCBI Sequencing Read Archive, BioProject ID PRJNA306777 and at FigShare (Supplementary InformationPeer Review File


Allele frequency changes in candidate genes were used to detect loci under selection. Out of the 183 polymorphisms identified in the four candidate genes, twenty-five loci in the intron regions and four loci in coding regions were found to display a selection signature. All loci in the coding regions are synonymous substitutions. Selection in both directions were observed on polymorphisms that appeared to be under selection. Genetic diversity and linkage disequilibrium within the candidate genes were low. The recurrent divergent selection caused excessive moderate allele frequencies in the cycle 3 reduced lignin population as compared to the base population. This study provides valuable insight on genetic changes occurring in short-term selection in the polyploid populations, and discovered potential markers for breeding switchgrass with improved biomass quality.Switchgrass is undergoing development as a dedicated cellulosic bioenergy crop. Fermentation of lignocellulosic biomass to ethanol in a bioenergy system or to volatile fatty acids in a livestock production system is strongly and negatively influenced by lignification of cell walls. This study detects specific loci that exhibit selection signatures across switchgrass breeding populations that differ in  Over the last decade, biomass energy consumption has increased more than 60%, driven by biofuel production, mainly in the form of bioethanol . Switchgin vitro dry matter digestibility (IVDMD) in a livestock production system showed a strong genetic correlation between IVDMD and ethanol yield of r = 0.84 [Recent approaches to improving switchgrass biomass quality have focused on engineering genes involved in the lignin biosynthesis pathway. Switchgrass plants with down-regulated caffeic acid o-methyltransferase (COMT) evaluated in the field had biomass with 10 to 14% reduced lignin concentration, 34% greater sugar release and 28% higher ethanol yield compared to control plants . Despiter = 0.84 . This stForward genetic screening for causal alleles underlying the phenotypic variations in the natural populations can be carried out in light of high resolution of single nucleotide polymorphisms (SNPs) . Differebona fide CAD, is hypothesized to be correlated with the origin of lignin, based on evidence from phylogenetic distance of CAD genes and the lack of lignin found in the earliest plants without bona fide CAD genes [bona fide CAD [Numerous gene members in the monolignol biosynthesis pathway can affect lignin concentrations and forage digestibility in various species. COMT catalyzing methylation in the monolignol pathway , 21 is cAD genes . The CADfide CAD . Geneticfide CAD –27. Genefide CAD , CAD [28fide CAD , and 4-cfide CAD , each shThe switchgrass populations used in this study were generated by divergent breeding for decreased and increased in vitro dry matter digestibility (IVDMD) at the USDA-ARS grass breeding project at the University of Nebraska-Lincoln, Nebraska . The IVDTo identify polymorphisms responsible for the distinct differences of IVDMD, lignin concentration and ethanol yield in the breeding populations, candidate genes in the monolignol biosynthesis pathway were investigated in this study. We described the approach of detecting selection signatures in divergent selected switchgrass populations by AF changes. The identifications of polymorphisms under selection provided insight into the genetic basis of recurrent selection in switchgrass, and potential SNP markers to facilitate marker-assisted selection.A random sample of five generations of divergent selection for IVDMD (populations C-1 through C+3) was space-transplanted in the field in May 2006. The populations are described as NE Trailblazer C-1, NE Trailblazer C0, Trailblazer, NE Trailblazer C2, and NE Trailblazer C3 in the official release notification by USDA-ARS. For our purposes, Trailblazer is noted as C+1 and the other four populations are noted according to their cycle number from the original population . The sign refers to the direction of selection for IVDMD and the number refers to the number of selection cycles or recombination events. The breeding generation evaluation nursery was established in 2006 with a randomized complete block design in Lincoln, Nebraska . Within The dried leaf samples of five populations were pooled by population with 0.002g per individual. DNA extractions were made for each pool using the protocol described by . CandidaTo control the sequence quality, preliminary AF was calculated for each polymorphic site as the proportion of the minor polymorphism to the total reads across all five populations. The polymorphic sites with preliminary AFs lower than 0.05 were discarded. If a site has more than one minor allele and one of the preliminary AFs was greater than 0.05, the site cannot be discarded, but the haplotypes corresponding to the rare polymorphisms at this site were discarded. The AF per site per population were then calculated to be used in the statistical tests for selection signature.2 for both SNPs and InDels in R 3.2.0 [Nucleotide diversity and haplotype diversity were calculated using DNaSP , 34. Nuc R 3.2.0 . LD valuThe sequence data set were separated into the five divergent populations , and the AFs were calculated within each population. The allele frequencies in the C0 population are the initial frequencies. Only the loci common among all 5 populations were kept for the following statistic tests.The demographic scheme was simulated 10000 times with genetic drift only to build the null distributions for the statistic tests . For eacIn the AF change test, thresholds were decided using the null distribution unique to each initial AF. The p-values were calculated as the proportion of simulated allele AF excessing the observed AF in a total of 10,000 simulations. The polymorphisms significant for the AF change test were then processed through a second, independent, statistical test: linear regression of AF on the cycle numbers. The slopes of the linear regression for each locus were compared to slopes generated from the simulated distribution. The p-values of both tests were adjusted by the controlled false discovery rate . SelectiTwo family members of COMT (COMT1 and COMT2) and CAD2 and 4CL1 were sequenced using pooled genomic DNA from each population. The cDNA sequences of COMT2, CAD2, and 4CL1 in switchgrass were obtained from NCBI  , 30, 40.Gene structures of the resulted genomic sequences were inferred by comparing them to homologs from maize and sorghum . ThirteeThe number of SNPs and InDels among all five populations were summarized in Nucleotide diversity was estimated for each gene ranging from 0.0027 to 0.0060 . 4CL1 haThe phylogenetic tree of the haplotypes indicated that despite the number of haplotypes discovered, the haplotypes within each gene have no significant branching. The substituted amino acids were analyzed for the impact of substitution on protein function in SIFT . There wTo investigate the association of polymorphisms in the four genes with selection for IVDMD, allele frequencies and AF changes between the most extreme populations were analyzed. Minor allele frequencies of the SNPs/InDels were calculated within each population pool. The AF changes were calculated as the AF in C+3 population (high IVIDMD) minus the AF in C-1 population (low IVDMD).During short-term selections, two reasons could result in AF changes across the genome, genetic drift and selection, one causing random AF fluctuation, while the other producing directional frequency changes. The loci under constant selection would likely have bigger AF changes than the loci undergone only genetic drift, if the selection intensity is high or the trait under selection is highly inheritable. A demographic scheme was simulated  to reflevice versa. Even though CAD2 and COMT2 had medium levels of genetic diversity comparing, they have fewer loci with significant AF changes.In total, 36 SNPs and InDels were found significant for AF changes after adjusting p-values to control FDR . None ofThe SNPs/InDels significant in the AF change test were analyzed by regression of allele frequencies on selection cycles. Slopes of the linear regression in the observed data were compared with that calculated in the simulated data to determine p-values. Eighty percent of the significant polymorphisms from the AF change test were also significant in the regression test. As a result, 29 SNPs and InDels passed both tests as final significant polymorphisms associated with recurrent selection. All 7 polymorphisms that didn’t pass the regression coefficient test were from CAD2 gene. Significant loci were detected only in COMT1 and 4CL1.2) and the slopes (b) were plotted against their physical positions in each gene  and alfalfa (Medicago sativa) [Different monolignol genes in these divergent switchgrass populations showed low to medium nucleotide diversities. Nucleotide diversity of COMT genes in this study fell within the similar range as the estimations in maize ( sativa) , 62, 63. sativa) . Differe sativa) .The patterns of genetic variation in these candidate genes depicted the complexity of the octoploid switchgrass genomes. Majority of the significant loci have initial allele frequencies in the low range (<0.2) except for two loci in the COMT1 gene. This could be explained by that genetic diversity for low lignin and high IVDMD traits are not necessary for surviving in the wild habitat, sometimes might even be defective . Before The significant polymorphisms discovered in this study are potential candidates for QTL underlying biomass quality in switchgrass, and provided possible markers for marker-assisted selection , 70. DepS1 FigThe figure is adapted from data of Vogel and others  for illu(TIF)Click here for additional data file.S2 FigFrom the base population C0, one cycle of selection for low IVDMD and three cycles of selection for high IVDMD were conducted, resulting in four selected populations, C-1, C+1, C+2 and C+3. Population sizes are represented by n and the number of selected individuals by m for each group of selected individuals . The figure is adapted from data of Vogel and others  for illu(TIFF)Click here for additional data file.S1 TableSwitchgrass v3.1 genomic identifier were obtained from phytozome genome database by using our sequences as queries in BLAST.(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


Myodes glareolus) as a model system, we tested the hypothesis that approximately 50 generations of exposure to the Chernobyl environment has significantly altered genetic diversity of the mitochondrial genome. Using deep sequencing, we compared mitochondrial genomes from 131 individuals from reference sites with radioactive contamination comparable to that present in northern Ukraine before the 26 April 1986 meltdown, to populations where substantial fallout was deposited following the nuclear accident. Population genetic variables revealed significant differences among populations from contaminated and uncontaminated localities. Therefore, we rejected the null hypothesis of no significant genetic effect from 50 generations of exposure to the environment created by the Chernobyl meltdown. Samples from contaminated localities exhibited significantly higher numbers of haplotypes and polymorphic loci, elevated genetic diversity, and a significantly higher average number of substitutions per site across mitochondrial gene regions. Observed genetic variation was dominated by synonymous mutations, which may indicate a history of purify selection against nonsynonymous or insertion/deletion mutations. These significant differences were not attributable to sample size artifacts. The observed increase in mitochondrial genomic diversity in voles from radioactive sites is consistent with the possibility that chronic, continuous irradiation resulting from the Chernobyl disaster has produced an accelerated mutation rate in this species over the last 25 years. Our results, being the first to demonstrate this phenomenon in a wild mammalian species, are important for understanding genetic consequences of exposure to low‐dose radiation sources.Currently, the effects of chronic, continuous low dose environmental irradiation on the mitochondrial genome of resident small mammals are unknown. Using the bank vole ( The distribution of substitutions per site for each gene region across locality‐time points was assessed using F tests for equality of variances and Shapiro–Wilk's tests for normality . Genetic distances between time points within each locality were calculated, ranked and subsequently evaluated by power analyses.A series of population genetic statistics were calculated to compare levels of genetic diversity among locality‐time points. The statistics calculated were (i) the number of haplotypes serving as a basic measure of genome diversity; (ii) the number of polymorphic sites serving as a basic descriptor of nucleotide variability; (iii) both (i) and (ii) divided by locality‐time point sample sizes to control for sample size; (iv) gene diversity Nei, , as the n = 20) was randomly subsampled to the average locality‐time point sample size (n = 13) through 100 iterations, and number of polymorphic sites and gene diversity estimates were recalculated. Following Shapiro–Wilk's tests for normality, permuted subsampling distributions were compared to uncontaminated localities using Mann–Whitney U tests.To further identify any effects on locality‐specific statistics by sampling error the most common haplotype in the overall population (15.3% of the sample) was removed from the data set, and population genetic statistics mentioned above were recalculated for each locality‐time point. In addition, because sample size was the largest for Red Forest 1998, the total sample at this locality‐time point . Comparison of average substitutions per site estimated for contaminated and uncontaminated localities across the entire mitochondrial genome resulted in significantly greater substitutions per site at contaminated localities as compared to uncontaminated localities  = 2.64). Similar comparisons for each gene revealed apparently higher mean substitutions per site for contaminated localities for all gene regions except ND3 and Cytb. Subsequent statistical testing identified COI, COIII, ND1, ND4, ND5 and D‐Loop as having significantly greater substitutions per site at contaminated localities as compared to uncontaminated localities , polymorphic sites  and genetic diversity  at contaminated localities as compared to uncontaminated localities, while no statistically significant differences for any of these population genetic measures were found when years were compared (Table p = .05 and power (1−error probability) = 0.8.To further quantify differences in genetic variation across localities, a series of population genetic measures and statistical tests were evaluated Table . Consisted Table . Althougn = 20) to that of the average locality‐time point sample size (n = 13) resulted in a distribution of polymorphic sites with a mean and standard deviation of 176 and 18, respectively, whereas mean and standard deviation of uncontaminated sites were 104 and 25, respectively. These distributions were found to be significantly different from each other (Mann–Whitney U = 2, p < .01; Figure U = 7, p < .01; Figure To detect effects stemming from the spatial distribution of genetic variation on locality‐time point sampling error, the most common haplotype Figure  was remo4While evolutionary impacts have been suggested in other vertebrate and invertebrate species exposed to physical and chemical toxicants, the mitochondrial genome comparisons in this study are the first to detect a statistically significant difference in the genetic diversity of a native, resident mammalian species encountering multigenerational chronic exposure to radiation in any contaminated environment (Matson et al., In spite of the inferred genetic effect of chronic low‐dose radiation exposure, comparison of population sizes and health status (Baker & Chesser, The Dryad repository titled “Data from: Elevated mitochondrial genome variation after 50 generations of radiation exposure in a wild rodent” is now being processed by the curatorial team. The data package has been assigned a unique identifier, called a DOI. This DOI is provisional for now, but may be included in the article manuscript. It will be fully registered with the DOI system when your submission has been approved by Dryad curation staff.Data package title: Data from: Elevated mitochondrial genome variation after 50 generations of radiation exposure in a wild rodent.https://doi.org/10.5061/dryad.j11s7Provisional DOI: Data files: mtDNA gene alignments. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.


DNA sequences, nuclear DNA sequences, and microsatellite genotypes, we show that the benthic‐developing sea star, Parvulastra exigua, has lower levels of within‐ and among‐population genetic diversity, more inferred genetic clusters, and higher levels of hierarchical and pairwise population structure than Meridiastra calcar, a species with planktonic development. While both species have populations that have diverged since the middle of the second glacial period of the Pleistocene, most P. exigua populations have origins after the last glacial maxima (LGM), whereas most M. calcar populations diverged long before the LGM. Our results indicate that phylogenetic patterns of these two species are consistent with predicted dispersal abilities; the benthic‐developing P. exigua shows a pattern of extirpation during the LGM with subsequent recolonization, whereas the planktonic‐developing M. calcar shows a pattern of persistence and isolation during the LGM with subsequent post‐Pleistocene introgression.Life‐history traits, especially the mode and duration of larval development, are expected to strongly influence the population connectivity and phylogeography of marine species. Comparative analysis of sympatric, closely related species with differing life histories provides the opportunity to specifically investigate these mechanisms of evolution but have been equivocal in this regard. Here, we sample two sympatric sea stars across the same geographic range in temperate waters of Australia. Using a combination of mitochondrial  Multiple studies have shown that the mode of larval dispersal can predict relative levels of genetic and phylogeographic structure in species comparisons  larval stage and a short larval duration of 1–2 weeks  in southeastern Australia, whereas P. exigua unexpectedly did not  and 1,306 bp (P. exigua) respectively.Following the protocols in Keever et al. , we PCR‐a et al.  and was 2.3P. exigua at five microsatellite loci and samples of M. calcar at seven microsatellite loci, following the protocols in Keever, Sunday, Wood, Byrne, and Hart  loci as described in Puritz, Addison, and Toonen . Due to large amounts of INDEL variation and INDEL‐like errors of 454 sequencing, many alignments were realigned by eye. If heterozygous base pairs were found in the contig alignment, the alignment was then sorted by that base pair. The two most common haplotypes were then selected as the two allelic states for that locus.We sequenced approximately 20 individuals from 12 localities of d Toonen  and Purid Toonen . In shor2.5arlequin 3.5  selected the Tamura–Nei model as the best fit for genetic distance among mtDNA haplotypes. We then calculated all AMOVA and pairwise measures of population structure for mtDNA in arlequin 3.5 using this model, and 50,000 permutations for significance testing. We used genodive to calculate all AMOVA and pairwise measures of population structure for microsatellites using the standardized FST′ (AMOVA) and GST″ (pairwise) statistics with 10,000 permutations for significance testing  score and results displayed are from the single lowest DIC run from the K = 6 replicates, and other replicates were consistent with the presented results. For M. calcar, clusters were estimated with the BYM admixture model , with correlated allele frequencies, 100,000 burn‐in steps, 100,000 sampling steps from 1 to 7 groups, and three replicate runs per group. structure harvester 0.6.8  for coalescent analyses in IMa2p , within central NSW , within southern NSW , and within WA  were combined and randomly subsampled into representative populations. For M. calcar, only samples that had no missing data across the five nDNA loci and mtDNA locus were included; samples from within northern and central NSW , within southern NSW , within eastern TAS , within western TAS , and within WA  were combined and randomly subsampled down to 10 individuals with equal representation among localities.For each species, data sets were reduced to minimize the coalescent parameter space of six unlinked sequence loci . For P. exigua returned abnormally large estimates of the mutation rate scalar (~24) for the mtDNA locus, likely because of the large difference in the number of haplotypes for the nDNA loci compared to mtDNA locus. For the final run, a geometric mean of all mutation rate scalars was used instead. This scalar more closely matched the scalar estimated for M. calcar and produced results similar to using the mtDNA locus alone for estimates.20–30 preliminary runs for each species were conducted adjusting priors between runs to obtain the priors used in final analysis. MCMCMC analyses were run for burn‐in until flat trend lines appeared in all estimated parameters, and afterward 300,000 genealogies per locus were saved with 10 steps between genealogy saves. Joint peak locations and posterior probabilities were estimated with a single “L‐mode” run for each species. Initial runs of 2.10−6 mutations per gene per year for P. exigua and 1.026 × 10−5 per gene per year for M. calcar. These rates are based on the smallest average pairwise sequence divergence between geminate pairs of Neotropical sea urchin species (another echinoderm) separated by the Isthmus of Panama , assuming 3 million years of isolation, which gives a rate of 7.60 × 0−9 mutations per site per year. We use an average COI rate across the entire locus, although we recognize that the concatenated mtDNA sequences in our study include small portions of slower evolving tRNA sequences and large portions of the faster evolving control region of the mitochondrion .Across all sampled loci, s Figure ; Table 13.2The haplotype networks for both species revealed distinct regional partitioning of mtDNA genetic variation, but the details differ between the two.P. exigua, few haplotypes were shared between regions, and there was complete lineage sorting among SA, NSW, and WA. Alleles were shared between northern and central NSW as well as central NSW and southern NSW, but no haplotypes were present in all NSW populations  ; however, that ancestral lineage diverged from all other lineages nearly 20,000 ybp . The other four lineages all diverged approximately 8,000–15,000 ybp . The remaining four lineages showed a divergence pattern in contrast to geography  nonfeeding pelagic larval stage. In contrast, P. exigua has protandry‐like hermaphroditism and benthic larvae, presumably with more limited potential for dispersal. Thus, all else being equal, these differences in reproductive and developmental modes make clear predictions about the degree of gene flow and scale of population genetic structure expected in comparisons of these two species, but results of such comparisons have been somewhat mixed to date  when compared to M. calcar, a sympatric species with planktonic development. AMOVA and pairwise comparisons of all three marker types show consistently that the majority of genetic variation in P. exigua is partitioned among populations. This is not surprising, considering that populations of P. exigua can be strongly structured (FST ~ 0.6) on spatial scales of tide pools only meters apart  SA, based on phylogenetic networks and coalescence population size estimates, may be one of the older, larger, and more ancestral populations of M. calcar and this genetic cluster may include many shared ancestral polymorphisms; (2) the two dominant surface currents of southeastern Australia , when the ancestral lineage of northern and central NSW diverged from all other populations. The most probable estimate (MPE) corresponds with peaks in relative glacial periods , well after the end of the last glaciation and reestablishment of contemporary sea surface temperatures and sea levels around 11,000 ybp  between SA populations and populations from NSW and TAS. This timing falls between two of the greater glacial peaks during the Pleistocene , and yet have less than one effective migrant per generation between them. In contrast, northern/central NSW and southern NSW are not even inferred to be sister populations, diverging over 75,000 ybp, but have effective migrant numbers well over 35 effective migrants per generation in both directions. We hypothesize that SA populations of M. calcar were isolated from NSW and TAS during the majority of the Pleistocene and that populations in NSW and TAS were restricted to two ancestral refugia during the LGM; present day populations have ancestry from one of these two ancestral lineages, but are being homogenized by moderate levels of gene flow within NSW and TAS, with low gene flow between regions mediated by the contemporary sea surface currents.For M. calcar were significantly differentiated across the Strait, whereas genetic structure in P. exigua was entirely among populations, such that grouping populations into east and west of Bass Strait in a hierarchical AMOVA explained no additional molecular variance over populations alone. Our combined mtDNA, microsatellite, and nDNA results show that virtually all genetic variation is partitioned at the population, and not regional, level in P. exigua, clarifying the findings based on COI haplotypes alone. In addition, our coalescent analyses on the combined dataset help to explain previous findings because they show that populations east and west of Bass Strait all share a post‐Pleistocene origin. In that sense, the Tasmanian land bridge was not a barrier for P. exigua. This example highlights the value of multiple marker studies incorporating coalescent analyses to further our understanding of population connectivity in marine species. Our findings indicate that phylogeographic patterns in P. exigua and M. calcar are consistent with differences in their life histories, and this result is consistent with a previous meta‐analysis showing that the divergence between benthic and pelagic development was the primary correlate between life‐history differences and population structure .Raw 454 files, nDNA and microsatellite genotypes, mtDNA alignments, and IMa2 input and output files can be found on Figshare. Click here for additional data file.