Ganoderma boninense causes basal stem rot (BSR) and is responsible for substantial economic losses to Southeast Asia’s palm oil industry. Sarawak, a major producer in Malaysia, is also affected by this disease. Emergence of BSR in oil palm planted on peat throughout Sarawak is alarming as the soil type was previously regarded as non-conducive. Phylogenetic analysis indicated a single species, G. boninense as the cause of BSR in Sarawak. Information on evolutionary and demographic history for G. boninense in Sarawak inferred through informative genes is lacking. Hence, a haplotype study on single nucleotide polymorphisms in internal transcribed spacers (SNPs-ITS) of G. boninense was carried out. Sequence variations were analysed for population structure, phylogenetic and phylogeographic relationships. The internal transcribed spacers (ITS) region of 117 isolates from four populations in eight locations across Sarawak coastal areas revealed seven haplotypes. A major haplotype, designated GbHap1 (81.2%), was found throughout all sampling locations. Single nucleotide polymorphisms were observed mainly in the ITS1 region. The genetic structure was not detected, and genetic distance did not correlate with geographical distance. Haplotype network analysis suggested evidence of recent demographic expansion. Low genetic differences among populations also suggested that these isolates belong to a single G. boninense founder population adapting to oil palm as the host. Elaeis guineensis Jacq.) is a monoecious species of the Arecaceae family. Oil palm is the world’s leading source of vegetable oil and fat with palm oil and palm kernel oil accounting for approximately 81.85 million metric tons (40.2%) of the world’s vegetable oil supply in 2018 [Oil palm  and upper stem rot (USR) disease of oil palm [G. boninense infection at one metre from ground level and BSR as base level infection [Ganoderma species isolated from oil palm BSR as G. boninense. Subsequent studies reported that only three Ganoderma species, namely G. boninense, G. miniatocinctum and G. zonatum, associated with oil palm in Malaysia were determined to be pathogenic [Ganoderma applanatum, G. lucidum, G. pfeifferi, G. philippi and G. tornatum were proven to be non-pathogenic to oil palm through artificial infection studies [G. boninense species remains a major disease threat and limiting factor for oil palm productivity [The sustainability of oil palm is threatened by pests and diseases. Among major pathogens, oil palm ,4. It isoil palm . Upper soil palm ,7,8. Thenfection  identifithogenic . Ganoder studies . It is guctivity ,12,13.Ganoderma boninense is a weak competitor for available substrate and requires a large inoculum size to be able to infect a susceptible host [Reports on BSR disease in various soil types are alarming and geographical distribution mapping of disease incidence such as in Selangor, Malaysia did not show any relationship to different types of soil series . Althougble host . The modble host . Formatible host . Ganoderma species has been difficult as they are genetically heterogeneous with a wide range of genetic variations caused by distinct geographical origins and outcrossing over generations [Ganoderma basidiomata can also influence the descriptions for species identification [G. lucidum isolates [Ganoderma isolates can be resolved using molecular methods such as polymerase chain reaction (PCR) of internal transcribed spacers (ITS) region [G. boninense, G. zonatum and G. miniatocinctum [Identification of the erations ,20. Fielfication . Regionaisolates . Falsely) region . The mulocinctum ,25. HoweGanoderma isolates [Molecular approaches have been widely adapted in the field of biological science, including fungal identification in microbial ecology and plant pathology. Molecular methods were used to study and identify plant pathogens of many agricultural crops including oil palm . With adisolates .Ganoderma have arisen through nrDNA sequences analysis [Ganoderma could differ due to environmental factors so they are unreliable as the single criterion to distinguish diverse species. As such, the informative aspect of molecular characteristics should be investigated [Ganoderma isolates. It is essential to understand the current population structure of pathogenic Ganoderma spp. to predict the future population evolution and to resolve controversies regarding its taxonomic position. Likewise, alternative approaches to the taxonomic problems in analysis ,21. Morpstigated . As diseG. boninense ITS sequence data sets were obtained, representing four G. boninense populations in oil palm plantations. The combination of ITS1 and ITS4 primer amplified regions from the 3′ end of 18S rDNA to the 5′ end of the 28S rDNA included the ITS1, 5.8S rDNA and ITS2 regions. After sequence alignment and trimming, ITS1F primer read sequences were trimmed based on ITS1 primer reads. The final sequence length was 601 bp with six variable sites, five parsimoniously informative sites and 595 bp conserved sites were identified. The mean total nucleotide composition was 30% thymine (T), 23.7% cytosine (C), 22.1% adenine (A) and 24.1% guanine (G). Annotations for G. boninense were done based on information available in Bridge et al. [G. boninense sequences in GenBank® which consisted of a partial 18S small subunit gene and complete ITS1 region sequence revealed that our samples were missing 13 nucleotides from the 3′ terminus of the 18S small subunit gene. The length of our partial ITS1 region was 198 bp, the 5.8S was 155 bp and the complete ITS2 region was at 198 bp within the trimmed sequence. Sequences from ITS1F reads contained the complete ITS1-5.8S-ITS2 region. No sequencing errors or random substitutions were detected in the studied region. The haplotype search was carried out from 1st nucleotide position to 601. Seven haplotypes with one unique sequence and another six sequences shared among populations were distinguished based on single nucleotide polymorphisms of ITS region (SNPs-ITS)  . OverallNPs-ITS) . HoweverNinety-five isolates with GbHap1 corresponding to 81.2% of total individuals were found in all populations. Samples from Matu-Daro contained the highest number of individuals with GbHap1. Details on the haplotype distribution in each population are shown in Tomophagus colossus (KJ143923 and MG654429), which is closely related to Ganoderma genus in the Ganodermataceae family was included as an outgroup in the phylogenetic analysis [® that were used in phylogenetic analysis is shown in G. boninense is illustrated by the ML tree  and Japan (KJ143905 and KJ143906) within the G. boninense clade  to 0.002 (Miri). On the other hand, the haplotype diversity ranged from 0.343 (Matu-Daro) to 0.561 (Miri) (G. boninense and one outgroup (Tomophagus colossus) are showed in G. boninense, ranging from 0.2% to 0.7%. In contrast, higher genetic distance values (11.7–12.0%) were obtained with the presence of an outgroup. The pairwise FST values and results of the Chi-square tests for genetic differentiation among populations are displayed in ST values were obtained for Asajaya compared to all populations with p > 0.05. Negative FST values for Miri, Matu-Daro and Balingian with p > 0.05 indicated that these populations shared similar genetic material. High gene flow (migration index) with Nm > 4 suggested that these populations behave as a single panmictic unit. The AMOVA result revealed a variance of 100.254% within G. boninense populations whereas a negative percentage of variations (−0.254%) was presented among G. boninense populations  within the sampled populations.Each distinct haplotype was treated as an allele for population structure analyses. Nucleotide diversity among the seven 1 (Miri) . The nuculations . As no gGanoderma boninense population within Sarawak based on FST values, AMOVA, the Mantel test and spatial autocorrelation analysis of SNPs-ITS did not detect any genetic structure and no clear differences were found among populations within the sampling locations.The S values as negative with non-significant p-values  from Malaysia is identical to G. boninense (Accession No. MG200173). The record of G. zonatum ITS from Malaysia, however, was not publicly available for comparison with the generated G. boninense clade haplotypes. Phylogenetic analyses verified the inclusion of generated haplotype sequences within the G. boninense clade. Phylogenetic studies have also demonstrated that G. boninense and G. zonatum commonly formed a unique clade and are closely related to one another than other Ganoderma species [All samples collected in this study were identified as b et al.  identifiMalaysia ,12. Nucl species ,35. G. angustiporum, G. boninense, G. ryvardenii and G. zonatum , Cocos nucifera (coconut), Livistona chinensis  (var. boninensis and var. subglobosa) and Wodyetia bifurcate  [G. zonatum and the non-palm host records may have been inaccurately identified based on information from phylogenetic analyses. Ganoderma ryvardenii is a relatively new species compared to G. boninense and G. zonatum and has only been reported in oil palm plantation from Cameroon [Ganoderma angustisporum is a new species reported in 2018 by Xing et al. [G. boninense, G. zonatum and G. ryvardenii. It is plausible that the genus Ganoderma is relatively young and expanding its range to other hosts or climate zones [Ganoderma species divergence.Phylogenetic analysis revealed a strong support for clade containing  zonatum . However to palm . Our resil palm) . The firil palm) . Elliottil palm)  reportedCameroon . Ganoderg et al.  in Chinate zones  and manyGanoderma species and this can lead to species misidentification [Ganoderma spp. sequences in the NCBI GenBank® nucleotide database has revealed that 65% of these sequences were misidentified or incorrectly labelled [Ganoderma species such as G. boninense in the oil palm industry, accurate identification would influence disease management. Geographical regions and fungal-host relationships should also be considered in assessing monophyletic taxa in the Ganoderma genus [High morphological plasticity is common among fication ,23. A sulabelled . Hence, ma genus .Magnaporthe oryzae [Fusarium incarnatum [M. oryzae was indicative of genome plasticity for adaptation to different agro-climatic zones [F. incarnatum, however, were less informative and the genetic variation was better resolved through haplotype studies of translation elongation factor gene (tef1-α) sequences [G. applanatum var. gibbosum, G. fornicatum, G. japonicum, G. lucidum and G. neojaponicum [Application of ITS in haplotypes studies have been applied to fungal pathogen such as e oryzae  and Fusacarnatum . Variatiic zones . The DNAequences . Nuclearequences . Intra-saponicum ,46. SequG. boninense was further elucidated with analyses of SNPs-ITS  , high gene flows (Nm > 4) and genetic variations that were attributed to within the population (AMOVA). Current findings could indicate that these populations are from surviving individuals of G. boninense that were able to infect and adapt to oil palm. In this study, SNPs-ITS  and low SNPs-ITS . Furtherci (0–5) . The posG. zonatum [G. applanatum-australe [G. applanatum-australe complex between Southern and Northern Hemisphere samples [G. zonatum isolates throughout Florida, USA [G. boninense in this study did not detect the genetic structure as observed variation did not reveal any sub-population. This would suggest that only a single population of G. boninense can be found throughout the state of Sarawak. Population genetic structure analysis with ITS regions should, however, be interpreted with caution because ITS regions are multi-copy within the genome. Any variation which arises could potentially be credited to variation among repeats. Heterozygous sites within the ITS sequences could also potentially be chromosomal variation of paralogs and might not represent nucleotide variations from different chromosomes [Ganoderma genus and has been used to delineate isolates within a species into geographically distinct groups [G. boninense in Sarawak could be re-examined to determine precisely the inclusion of more polymorphic gene markers such as β-tubulin or tef1-α for inferring phylogeography and the population structure of fungi [Identical ITS haplotypes of  zonatum  and G. aaustrale  have bee samples  with a wida, USA  revealedomosomes . Howevert groups ,46. The of fungi .G. boninense as a calculation was made based on a single gene. Outcrossing is a common feature in basidiomycete sexual reproduction and the G. boninense gene flow inferred through microsatellites analysis which can extend to a regional level [G. boninense to evolve and establish itself. Oil palm monoculture expansion could influence indigenous G. boninense population size and accelerate natural selection for the oil palm specific pathogenic strain. In North America, G. zonatum were exposed to native and non-native palms cultivations. They were found to evolve accordingly through generations of outcrossing and increases in genetic diversity but they are maintained as a single population [G. zonatum species existence outside of the Americas continent to ascertain global demographic expansion. Founder populations of any fungi would possess low genetic diversity with traces of genetic drift, but the combination of sexual reproduction and an increase in host availability could likely increase pathogen evolutionary potential through recent population expansion, as reported for Alternaria brassicicola [G. boninense can also be found up to 700 km across regional geographical boundaries as reported in Merciere et al. [Although high gene flow (Nm > 4) was calculated between populations in Sarawak, caution should be practiced in interpretation as any indication of an outcrossing preference by al level . Fungi ial level . Hence, pulation . Howeversicicola . In addie et al.  which suG. boninense population in Sarawak was investigated using a neutrality test through Tajima’s D and Fu’s FS. Values were calculated as an individual population as well as a single population with both having negative values but were not statistically significant to conclude a demographic expansion  was not detected through their microsatellites analyses and molecular evidence was insufficient to establish the existence of any sub-population. As multiple individuals have the potential to cause further infection and propagate, population size would continue to expand through outcrossing and basidiospores spreading across long distances by nature or human activities [The demographic history of the xpansion . The neue et al.  through  Malacca . Specifitivities .G. boninense isolates are recent individuals adapted to oil palm as hosts with GbHap1 being the numeric dominant haplotype as the ancestral isolate. Nevertheless, the haplotype network  have been used within the Ganoderma genus, in elucidating phylogenetic analysis of closely related Ganoderma isolates to determine correct speciation and possible species complex formation [G. zonatum in Florida, USA, however, has only shown that rpb2 as the only gene region with significant differences while ITS region that was expected to contain higher genetic variability did not. Therefore, the multi-locus gene sequence for population genetic structure analysis may be relevant but not necessarily conclusive in describing the G. boninense population.In this study, the genetic structure based on a single-gene region was not found among the e et al. . This ise et al. . The ince et al. . Multiplormation ,41. ObseG. boninense basidiomata were collected from 107 BSR and 10 USR standing infected oil palm trees planted on peat in eight locations throughout Sarawak. These locations were then grouped together as local population representing their district or sub-district for analysis  as described by Ariffin and Idris [Ganoderma mycelia agar plugs were stored in sterile water as preservation by submersion in water would maintain G. boninense pathogenicity [nd Idris . Pure cugenicity .Cultures were grown for seven days in malt extract broth (MEB) before being harvested. The mycelia were snap-frozen in liquid nitrogen and ground to fine powder before DNA extraction . Total g® Flexi buffer, 2.4 μL of MgCl2 (25 mM), 2.4 μL of dNTPs (2.5 mM), 0.75 μL of each primer (20 µM), bovine serum albumin (BSA) (10 mgmL–1)  and 0.15 μL of GoTaq® DNA polymerase. The reaction mixture was then adjusted to a final volume of 30 μL with nuclease free water. The thermal cycling parameters set on the C1000 TouchTM Thermal Cycler  were as follows: Initial denaturation at 95 °C for 2 min; 30 cycles of denaturation at 95 °C for 60 s; annealing at 50 °C (ITS1-ITS4 pair) for 30 s or 55 °C (ITS1F-ITS4B pair) for 50 s; extension at 72 °C for 60 s; and a final extension at 72 °C for 5 min. The PCR products were subjected to electrophoresis on 2% (w/v) agarose gel, performed at 75 V for 30 min, stained with ethidium bromide and visualised under UV light through a Molecular Imager® Gel Doc™ XR+ Imaging System . The PCR products were purified using a QIAquick PCR Purification kit . A total of 107 samples were amplified with ITS1F-ITS4B and only 10 samples  were amplified using the ITS1–ITS4 primer pair. All purified PCR products were sent to First BASE Laboratories  for Sanger sequencing using the BigDye® Terminator v3.1 Cycle Sequencing Kit  with forward primer. Single directional sequencing was used as Sanger sequencing method is able to amplify the entire ITS1-5.8S-ITS2 region with ITS1F.The ITS region was amplified via PCR with the universal primer pair of ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) adapted from White et al.  as well Ganoderma boninense haplotypes were numbered in the order of their appearance in the analysis. Pairwise genetic distance between haplotypes was calculated in MEGA 7.0 with the Kimura 2-parameter evolution model (default setting) [Chromas 2.4.4 (Technelysium) software was used to view the sequences and chromatograms. Multiple alignments of the sequences were conducted using the ClustalX 2.0.10  and the setting) . G. boninense populations were examined by computing nucleotide diversity with Jukes–Cantor correction (PiJC) and the haplotype diversity indices (Hd) with DnaSP 6 [G. boninense populations was calculated via hierarchical analysis of molecular variance (AMOVA) implemented in Arlequin 3.5 [The levels of ITS variation within sampled  DnaSP 6 . The lev DnaSP 6 . Neutral DnaSP 6  and Fu’s DnaSP 6  were dev DnaSP 6 . Both tequin 3.5 . A Mantequin 3.5 .® corresponding to the accession number MK713555 to MK713561. Phylogenetic relationships were inferred by constructing a maximum likelihood (ML) tree using PhyML 3.1 [Sequences representing each haplotype were deposited in NCBI GenBankhyML 3.1  and BayehyML 3.1 , applyinhyML 3.1 . PhylogehyML 3.1 . AdditiohyML 3.1  and tcsBhyML 3.1 . AnnotathyML 3.1 .G. boninense isolates collected throughout oil palm plantations planted on peat located near coastal areas in Sarawak. Ganoderma boninense was identified as the causal agent regardless of BSR or USR origin. Analysis of variation in SNPs-ITS region showed a common major G. boninense haplotype distributed throughout Sarawak. Minority haplotypes were generated from this major haplotype based on haplotype network. Genetic structure was not detected among population with distribution of similar haplotype can be found up to the farthest distance of 504 km between sampling points. Haplotype diversity and the level of variations observed in the ITS region would suggest the possibility of only a single founder population of G. boninense existing in the collected samples throughout Sarawak. These findings, however, may only be related to the analysed gene of collected samples and should not be the representative patterns of the whole genome or taxa. Taking into consideration the disease cycle, subsequent resampling at the same or nearby location could also provide more information on population generation, population expansion and genetic differentiation of indigenous G. boninense isolates. Future application of comparing multiple gene regions such as rpb2 and tef1α could also be useful in resolving evolutionary relationships and population dynamics among phytopathogenic G. boninense or closely related species.This study shed light on the phylogenetic and phylogeographic relationships of


Cervus recovered many clades without whole genomic support.Previous investigations of phylogeny in C. unicolor, C. albirostris, C. nippon, C. elaphus and C. eldii) in the genus Cervus were analyzed using reduced-representation genome sequencing.In this study, the genetic diversity and phylogeny of 5 species , C. e. yarkandensis, C. c. canadensis and the other grouping the rest of C. canadensis from China. In the C. nippon clade, 2 well-distinct subclades corresponding to C. n. aplodontus and other C. nippon populations were separated. Phylogenetic reconstruction indicated that the first evolutionary event of the genus Cervus occurred approximately 7.4 millions of years ago. The split between C. elaphus and C. nippon could be estimated at around 3.6 millions of years ago. Phylogenetic ML tree of all samples based on individual SNP matrices, together with geographic distribution, have shown that there were 3 major subclades of C. elaphus and C. canadensis in China, namely C. e. yarkandensis (distributed in Tarim Basin), C. c. macneilli/C. c. kansuensis/C. c. alashanicus (distributed in middle west of China), and C. c. songaricus/C. c. sibiricus (distributed in northwest of China). Among them, C. e. yarkandensis was molecularly the most primitive subclade, with a differentiation dating back to 0.8–2.2 Myr ago. D statistical analysis showed that there was high probability of interspecific gene exchange between C. albirostris and C. eldii, C. albirostris and C. unicolor, C. nippon and C. unicolor, and there might be 2 migration events among 5 species in the genus Cervus.A total of 197,543 SNPs were identified with an average sequencing depth of 16 x. A total of 21 SNP matrices for each subspecies/population and 1 matrix for individual analysis were constructed, respectively. Nucleotide diversity and heterozygosity analysis showed that all 21 subspecies/populations had different degrees of genetic diversity. Cervus deer. In view of the current status of these populations, their conservation category will need to be reassessed.Our results provided new insight to the genetic diversity and phylogeny of The online version of this article (10.1186/s12864-019-5785-z) contains supplementary material, which is available to authorized users. Cervidae phylogeny has proven difficult to resolve, due either to rapid radiation in Pecorans (superordinate clade of Cervide) in the Mid-Eocene or ambiguity and conflict between different analyses [Cervus . The recent publication [Cervus: Elaphurus davidianus, Przewalskium albirostris, Rucervus eldii, and all species of Rusa, and sometimes Panolia eldii was wrongly included in the genus Rucervus, the genetic analysis demonstrated the significant evolutionary and systematic distance between Panolia eldii and Rucervus duvaucelii. They were approved by some important publications [Cervus, namely C. timorensis, C. unicolor, C. albirostris, C. nippon, C. elaphus, C. canadensis and C. eldii. Past taxonomic studies, based on morphology [The analyses –3, especlication , showed ications , 6. Whilications , 8. At prphology , karyotyrphology , 11, serrphology , mitochorphology –22 and/orphology , 24 haveC. unicolor was the most primitive species in the genus Cervus, followed by C. albirostris and C. nippon, and then C. elaphus, C. eldii has close relationship with C. unicolor [Cervus was found polyphyletic, C. eldii form sister group to the clade plus all other species of Cervus  [C. elaphus and C. canadensis were derived from a single common ancestor, while C. elaphus form a sister group to C. canadensis and C. nippon, C. canadensis share more nucleotide similarities with C. nippon [C. nippon is more primitive than C. elaphus and C. canadensis [C. elaphus probably originated from C. nippon in the middle Pleistocene [C. nippon is allied with C. elaphus in nuclear gene analysis [C. nippon, C. elaphus and C. canadensis were same in karyotype analysis [C. nippon, C. elaphus and C. canadensis were all spotted white, and there is no reproductive isolation between them. The above results imply that C. elaphus, C. canadensis and C. nippon could be very close-related species in the genus Cervus. Nonetheless, the classification proposed by all these studies are not in concordance with each other suggesting more work is needed to clarify these relationships.Karyotype analysis showed that unicolor . Based o nippon) . Some re. nippon , while onadensis , 16, 17,istocene , 25–27, analysis , the C-bCervus, (2) to test the genetic diversity in the Cervus populations, and (3) to investigate interspecific gene exchange between C. unicolor, C. albirostris, C. nippon, C. elaphus and C. eldii.The specific aims of our study were (1) to investigate the phylogeny of the genus Cervus, the divergence times, interspecific gene exchange and genetic diversity were estimated.To achieve these aims, SNP data from reduced-representation genome sequencing were used to provide phylogenetic relationships among 21 subspecies/populations derived from 5 species of genus All procedures concerning animals were organized to accord with the guidelines of care and use of experimental animals established by the Ministry of Agriculture of China, and all protocols were approved by the Institutional Animal Care and Use Committee of Institute of Special Animal and Plant Sciences, Chinese Academy of Agricultural Sciences, Changchun, China.Cervus , and 10 samples from Rangifer (R. tarandus) as out-group. The information about species distribution, classification, source, and number was collected , an anesthetic, was administered intramuscularly 1 ml per 100 kg body weight, peripheral vein blood of each sample were collected fresh and stored in − 20 °C until DNA extraction.We sequenced a total of 195 samples, including 185 samples from 21 subspecies/populations, representing the 5 described species of Genomic DNA extraction was carried out using whole blood genome DNA isolation kit . The degradation and contamination of the genomic DNA was monitored on 1% agarose gels, DNA purity was checked using the NanoPhotometer® spectrophotometer . DNA concentration was measured using Qubit® DNA Assay Kit in Qubit® 2.0 Flurometer .We carried out a pre-design experiment, the restriction sites of Mse I, Nla III and Hae III in the reference genome assembly (mhl.quiver-7-1) were predicted, the result showed that the three enzyme approach is more favorable than one enzyme or two enzyme method in library construction for the reduced-representation genome sequencing. The average enzyme capture rate was 97.0%  procedures in-house C scripts. QC standards as the following: (1) Removing reads with ≥10% unidentified nucleotides (N); (2) Removing reads with > 50% bases having phred quality < 5; (3) Removing reads with > 10 nt aligned to the adapter, allowing ≤10% mismatches. The assembly sika deer genome (mhl.quiver-7-1) was taken as reference genome (unpublished data). Comparison analysis of each sample was performed using BWA software (Parameter: mem -t 4 -k 32 -M -R).Population SNPs detection was performed with Stacks based on the clean data, the SNP datasets were filtered with the following criterion: maf = 0.01, mis = 0.7, dp = 2. For the population-based analysis, according to the clustering information (Table http://cmpg.unibe.ch/software/arlequin35/). Expected heterozygosity (He) and Observed heterozygosity (Ho) were calculated according to the formula provided by Nei [Nucleotide diversity (π) reflects population polymorphism, it was calculated using Arlequin software (d by Nei .6 steps were used in the Markov chain Monte Carlo (MCMC) iterations with sampling every 1000 iterations. Divergence times were estimated using MCMCTree 4.9e program (http://abacus.gene.ucl.ac.uk/software/paml.html), divergence time for R. tarandus and C. n. aplodontus 13.60–13.88 millions of years (http://www.timetree.org/home) were used as time correction point, the results were visualized using FigTree software.Phylogenetic analyses were conducted using maximum likelihood (ML) on inter-population SNP matrix and individual SNP matrix, respectively. All ML analyses were conducted using the on line version of RAxML program  with theC. eldii, C. unicolor, C. albirostris, C. elaphus and C. nippon were analyzed by D-statistics using AdmixTools software. D-statistics is a method included in AdmixTools to analyze the admixture of ancestral population. It infers the distances of genetic relationships between populations by determining gene exchange or gene infiltration between populations. This method usually takes four populations W, X, Y and Z as analysis objects, among them, Z is an exogenous group and Y is an ancestral group. If the analysis showed that there were gene flows between W and Y, X and Z, the BABA model is fitted, so the relationship between W and Y is closer; If there is gene flow between W and Z, X and Y, the ABBA model is fitted, so the relationship between X and Y is closer. In this study, for SNP locus i, the allele frequencies of different populations were w’, x’, y’, z’, respectively. Firstly, BABA was defined, which meant that the alleles of W and Y were the same, the alleles of X and Z were the same, and the alleles of W and X were different. Similarly, ABBA was defined, then gene exchange analysis was carried out accordingly. Population differentiation and mixing were estimated by TreeMix based on genome-wide allele frequency, 2–5 gradients were set respectively, combined with D statistical results, calculated the number of migration events among 5 species [Interspecific gene exchange of  species .The sequencing of 195 individuals resulted in a total of 84,231,376,992 bp raw de-multiplexed reads with an average 431,955,779 bp reads per individual. After strict filtration, high quality clean data were obtained. All the sequencing data have been statistical analyzed, including output, error rate, Q20, Q30, GC content  had the largest number of SNPs , C. eldii had the least number of SNPs  in Cervus, followed by C. n. aplodontus . In all populations, the number of SNPs in R. tarandus was the least . Individual analysis produced a matrix containing 197,543 SNPs.Under inter-population combined analysis, we generated a total of 21 SNP matrices for the phylogenetic analyses. The average number of SNPs among 21 subspecies/populations was 120,030  having the lowest (0.009) and C. c. alashanicus having the highest (0.022) π average . Subspecies/populations in C. elaphus  and C. c. xanthopygus showed relatively low average He  compared to other species of Cervus, and Ho was higher than other subspecies/populations in C. eldii and C. albirostris , while subspecies/populations in C. nippon (aodong) and C. nippon (xingkaihu) showed relatively low average Ho . Generally, all 21 subspecies/populations showed different degrees of genetic diversity. C. eldii, C. unicolor and C. albirostris showed relatively high He and Ho, while Ho in C. nippon was the lowest, indicating there was a certain degree of inbreeding rate in these subspecies/populations.Nucleotide diversity (π) ranged from 0.009 to 0.022 among 21 subspecies/populations, with Cervus clades based on the 21 SNP matrices showed that 5 clades that clearly separate C. eldii, C. unicolor, C. albirostris, C. elaphus and C. nippon from 21 subspecies/populations with the highest bootstrap support , C. e. yarkandensis, C. c. canadensis and the other grouping the rest of C. canadensis from China. In the C. nippon clade, 2 well-distinct subclades corresponding to C. n. aplodontus and other C. nippon populations were separated. Our phylogenetic reconstruction indicated that the first evolutionary event of the genus Cervus occurred approximately 7.4 millions of years ago. The divergence from a common ancestor of C. unicolor and C. eldii would have occurred at 2.7–6.1 millions of years (Myr) ago, so C. unicolor and C. eldii might be the most primitive Cervus deer. C. albirostris was the closest to them with divergence time of 3.3–5.8 Myr ago. The split between C. elaphus and C. nippon could be estimated at around 3.6 millions of years ago, the results were consist with previous report [C. elaphus, C. canadensis and C. nippon might have the same ancestor. It is surprising because C. elaphus  and C. e. yarkandensis were sisters to C. canadensis while usually C. canadensis turn out to be more closely related to C. nippon than to C. elaphus.Phylogenetic ML tree of all ort Fig. . Within C. elaphus clade, the divergence between C. elaphus  and the other would have occurred at 1.1–3.0 Myr ago, it was accordant with fossil record [C. e. yarkandensis and C. c. canadensis took place more recently, nearly 0.8–2.2 and 0.4–1.4 Myr ago. Whereas the bootstrap support of divergence was not high among other C. canadensis populations. In the C. nippon clade, there was a distinct divergence between C. n. aplodontus and other C. nippon populations, and the divergence time between them was 1.1–3.0 Myr. The divergence of other C. nippon populations was not obvious, so C. n. aplodontus might be the most primitive subspecies of C. nippon in our study.In the l record . The sepC. elaphus and C. canadensis were geographically widespread in history. At present, it was considered that there were 7 or 8 subspecies of C. elaphus and C. canadensis lived in mainland China, and they were very restricted in distribution. All the subspecies except C. c. wallichii were collected and analyzed in this study.The Cervus species based on the individual SNP matrices was basically the same as inter-population combined analysis, but some of the samples fell within their respective groups as anticipated , C. c. xanthopygus was split to 2 populations, one of which has close relationships with C. c. sibiricus and C. c. songaricus, and the other was grouped with C. c. alashanicus. Similarly, the C. nippon populations except C. n. aplodontus could not be well-separated. These results were probably caused by no geographical isolation between these local populations.Phylogenetic ML tree of all ted Fig. , such asC. elaphus and C. canadensis samples based on the individual SNP matrices, together with geographic distribution, have showed that, there were three major subclades in China, namely C. e. yarkandensis , C. c. macneilli/C. c. kansuensis/C. c. alashanicus , and C. c. songaricus/C. c. sibiricus , whereas there might be introgression in the current C. c. xanthopygus population , the higher the probability of interspecific genetic exchange events is. The result was showed in Table C. albirostris and C. eldii, C. albirostris and C. unicolor had more probability of interspecific genetic exchange; Group IV, V conformed to ABBA model, indicating C. unicolor and C. albirostris, C. unicolor and C. nippon had more probability of interspecific genetic exchange. Population differentiation and mixing could be explained by the estimation of migration events, which included both geographic migration and evolutionary process, and they might all be marked by the genetic evidences. In this study, migration events estimated by TreeMix was shown in Fig. Cervus: both migrations started from a common ancestor of C. eldii and C. unicolor, some of the ancestors migrated to C. albirostris, and another part migrated to the common ancestor of C. nippon and C. albirostris.Based on D statistical results, Cervus had close relationships, all the species could hybridize with each other. Little genetic markers could be used to disentangle phylogenetic relationships among the closely related species. A series of methods were used for these analysis, such as using more data to reconstruct phylogeny [In many studies, molecular markers were used to verify the accuracy of genetic resources –35, wherhylogeny , or devehylogeny , howeverhylogeny . Recentlhylogeny  was devehylogeny , restrichylogeny –44, genohylogeny  have beehylogeny . Among thylogeny . The GBShylogeny  and thenhylogeny . The twohylogeny –51. TherCervus. The sequencing depth is enough for SNP screening and analysis [C. nippon, C. elaphus and C. canadensis, the genetic varation was relatively low, this might be caused by the small population size and inbreeding effect. The result was accordant with other genetic diversity study of C. nippon and C. elaphus [Cervus to the sika deer genome resulted in a high mapping rate, providing a useful data set for the study of genetic diversity and phylogeny of Cervus deer. In comparison to previous studies, which used mitochondrial DNA [We report here the first genome-wide study on 21 subspecies/populations in the genus analysis . All the elaphus . More imrial DNA , 14, 54 rial DNA , 24, therial DNA .C. elaphus and C. canadensis are not conspecific [C. elaphus clade, the divergence between C. elaphus  and the other occurred firstly, form one group; then the separation of C. e. yarkandensis, form a second group; and C. c. canadensis and other C. canadensis populations form a third group.It is usually considered that specific , they cospecific , 57. OurC. e. yarkandensis and C. elaphus  were surprising in this study, because they were sisters to the C. canadensis while usually C. canadensis turn out to be more closely related to C. nippon than to C. elaphus [C. elaphus  in this study are in all likelihood descendants of European red deer, while the New Zealand sample might also be derived from C. c. canadensis, but in this study, C. elaphus  were not clustered with C. c. canadensis, so our New Zealand samples could be the descendants of C. elaphus. The estimated divergence of this artificial population from the indigenous Eurasian Cervus has a little value and does not show a real chronology of evolutionary processes. Apparently, the obtained time of divergence at Pliocene or Early Pleistocene time is an overestimation and may be related to the founder effect, artificial selection and other genetic phenomena usual in the case of an artificial introduced population. Since the present data are genomic and not just mtDNA or single nuclear loci, this is potentially very interesting as it would restore C. elaphus and C. canadensis monophyly to the exclusion of C. nippon. In the following studies, European red deer samples  would be included to see whether this pattern holds.Interestingly, the phylogenetic position of the  elaphus , 58. C. C. elaphus and C. canadensis was divided to western and eastern subclades, the western subclade comprised the C. e. yarkandensis and the European populations, the eastern subclade consisted of C. c. songaricus, C. c. sibiricus and populations from other Asian areas , and C. c. canadensis [C. e. yarkandensis was more closer to European red deer, meanwhile, C. c. canadensis were genetically closer to Asian populations in north China and Mongolia, supporting that C. elaphus might immigrated from northeastern Eurasia to North America through the glacier-induced land-bridge (Beringia) which had formed between the two continents after Late Pleistocene [C. e. yarkandensis might be a subspecies of European red deer, C. c. canadensis might originated from C. elaphus or C. e. yarkandensis, so they probably migrated from these regions. Other C. canadensis in China were clearly separated from C. e. yarkandensis, it might be interpreted by the independent evolution of C. e. yarkandensis in a relatively closed natural environment after they separated from other C. elaphus populations [Most of studies agreed with that the clade of nadensis , 18. It istocene , this waulations , 59.C. c. kansuensis and C. c. macneilli had the same genotypes [C. c. kansuensis, the populations in Qinghai should not be considered as a subspecies. Other studies based on mitochondrial DNA D-loop regions found that there were gene exchanges between C. c. sibiricus, C. c. songaricus and C. c. kansuensis, probably caused by hybridization between populations [C. c. songaricus and C. c. sibiricus, C. c. xanthopygus and other C. canadensis, however, difficult specimens in the genus may often be characterized as “hybrids” without adequate evidence of their hybrid status [The genetic analysis based on mitochondrial DNA cytb showed that, enotypes , 61, we ulations , in the d status .Cervus had wide range of distribution. Every evolutionary type, from primitive to modern, were preserved during the evolution. Among them, C. albirostris is unique to China, it has similar karyotype to C. unicolor and C. eldii [Cervus showed that C. albirostris had gene exchange with C. unicolor and C. eldii. It is consistent with previous research findings. C. elaphus and C. nippon are closely related, under natural conditions, they can hybridize with each other. But in this study, we didn’t find the evidence of gene exchange between the two species, we speculated that the similarity of gene frequencies between them is not necessarily due to the fusion effect of gene exchange, but to the same selection pressure. Gene exchange between species of the genus Cervus is a very interesting event, and it is still worth exploring in many different ways.The genus C. eldii , the chaCervus deer is increasing gradually. Many subspecies in the genus Cervus are extinct in the wild  [C. eldii, C. albirostris, C. unicolor), however, C. elaphus, C. canadensis and C. nippon were classified as “Least Concern” species, most of their subspecies are on the brink of extinction. As to C. canadensis in China, no distinction between C. c. songaricus and C. c. sibiricus, C. c. macneilli and C. c. kansuensis was evident from phylogenetic ML tree based on the individual SNP matrices. C. c. xanthopygus was split to 2 populations, one of which has close relationships with C. c. sibiricus and C. c. songaricus, and the other was grouped with C. c. alashanicus. Similarly, the C. nippon populations except C. n. aplodontus could not be well-separated. The conservation status of these populations will need to be reassessed, urgent protection measures were required to prevent introgression or extinction of these populations.Along with increasingly extensive and intensive human activities, the influence on darinus) . SeveralCervus, enabled us to undertake phylogenetic analyses to answer the initial questions: (1) C. unicolor and C. eldii might be the most primitive Cervus deer. C. albirostris was the most closest to them with divergence time of 3.3–5.8 Myr ago. The split between C. elaphus and C. nippon could be estimated at around 3.6 millions of years ago. C. elaphus, C. canadensis and C. nippon might have the same ancestor. There were three major C. elaphus and C. canadensis subclades in China, within them, C. e. yarkandensis was molecularly the most primitive subclade, with a differentiation dating back to 0.8–2.2 Myr ago. (2) All 21 subspecies/populations had different degrees of genetic diversity. C. eldii, C. unicolor and C. albirostris showed relatively high expected and observed heterozygosity, while observed heterozygosity in C. nippon was the lowest, indicating there was a certain degree of inbreeding rate in these subspecies/populations. (3) There was high probability of interspecific gene exchange between C. albirostris and C. eldii, C. albirostris and C. unicolor, C. nippon and C. unicolor, and there might be 2 migration events among 5 species in the genus Cervus.The high-performance and low-cost of reduced-representation genome sequencing provides numerous high quality SNPs in the genus Cervus deer. A revision of the current results based on comparison of phenotypic and molecular data is desirable for future research. In view of the current status of these populations, their conservation category will need to be reassessed.Our results provided new insight to the genetic diversity and phylogeny of Additional file 1:Table S1. Information on enzyme digestion results. (XLSX 21 kb)Additional file 2:Table S2. Statistics of output, error rate, Q20, Q30, GC content of all the sequencing data. (XLSX 24 kb)Additional file 3:Table S3. Summary of mapping rate and expected site coverage. (XLSX 29 kb)


Ustilago segetum (Pers.) Roussel tritici (UST) causes loose smut of wheat account for considerable grain yield losses globally. For effective management, knowledge of its genetic variability and population structure is a prerequisite. In this study, UST isolates sampled from four different wheat growing zones of India were analyzed using the second largest subunit of the RNA polymerase II (RPB2) and a set of sixteen neutral simple sequence repeats (SSRs) markers. Among the 112 UST isolates genotyped, 98 haplotypes were identified. All the isolates were categorized into two groups (K = 2), each consisting of isolates from different sampling sites, on the basis of unweighted paired-grouping method with arithmetic averages (UPGMA) and the Bayesian analysis of population structure. The positive and significant index of association (IA = 1.169) and standardized index of association (rBarD = 0.075) indicate population is of non-random mating type. Analysis of molecular variance showed that the highest variance component is among isolates (91%), with significantly low genetic differentiation variation among regions (8%) (Fst = 0.012). Recombination (Rm = 0) was not detected. The results showed that UST isolates have a clonal genetic structure with limited genetic differentiation and human arbitrated gene flow and mutations are the prime evolutionary processes determining its genetic structure. These findings will be helpful in devising management strategy especially for selection and breeding of resistant wheat cultivars. Ustilago segetum (Pers.) Roussel tritici Jensen (UST), is one of the most serious diseases on wheat (Triticum aestivum L.) globally. The disease is favored by moist and cool climate during anthesis , translation elongation factor 1-alpha (Tef1), beta-tubulin (Tub2) and actin (ACT) have been most frequently used for inferring phylogenetic relationships among fungi  analyze the genetic diversity of UST isolates of Indian origin based on geographic areas of collection by microsatellites and RPB2 gene sequence comparison (ii) investigate the possibility of random mating within their sampling sites using MULTILOCUS version 1.31  broth. The cultures were grown for 10 days in an orbital shaker (150 rpm) at 25 ± 1°C. The fungal mat harvested on sterile Whatman filter paper, frozen in liquid nitrogen, and ground to fine powder with pestle and mortar. Cetyl trimethyl-ammonium bromide (CTAB) method was used to extract genomic DNA as described by Kumar et al. . DNA wasRPB2) gene was amplified for all the 112 UST isolates using polymerase chain reaction (PCR) with primer RPB2F (5′- AACCACCGATTTGGAGCAGT-3′) and RPB2R (5′- ACTCATTAGATGGCGGGGAGA-3′). The primers were designed using the NCBI accession number DQ846896.1. PCR amplification was performed in Q cycler 96 . Each PCR reaction mixture (50 μl) consisting of 50 ng template DNA, 1.5 μM of each primer, 1.5 mM MgCl2, 0.2 mM of each deoxynucleotides, 1.5 unit of Taq DNA polymerase , and final volume of 50 μl was maintained by adding distilled water. The amplification was done as initial denaturing at 94°C for 4 min, followed by 35 cycles at 94°C for 1 min, annealing at 55°C for 1 min, extension at 72°C for 1 min and a final extension at 72°C for 7 min. The amplified PCR products were separated on 1% agarose gel, and the desired specific band was purified by Wizard SV Gel and PCR Clean-Up System  according to the manufacturer's instruction. DNA was sequenced commercially at the Eurofins Genomics India Pvt. Ltd. . A consensus sequence was obtained from the sequencing of both forward and reverse strands, and further data quality were checked using Chromas 2.32 (Technelysium Pty. Ltd.). BlastN search programme was used to compare the sequences available in National Center for Biotechnology Information (NCBI) databases. The sequences were aligned using MEGA 7 . PCR cocktail without template DNA was taken as control. PCR was programmed for initial denaturation at 94°C for 4 min; followed by 35 cycles of denaturation at 94°C for 60 s, annealing at 51, 52, 53, 54, and 55°C for 1 min, and extension at 72°C for 1 min; with a final extension step at 72°C for 7 min. PCR products were separated in 4% agarose gel stained with ethidium bromide along 100 bp ladder  to know the polymorphism. Test isolates were scored on the basis of amplification and non-amplification of SSR markers. The numbers of varoius alleles per locus, effective alleles per locus, private alleles and Shannon's Information Index were computed for each population using GenAlEx 6.5  in a total volume of 10 μl containing Promega™ PCR Master Mix, additional 0.5 mM MgCli is the frequency of the ith allele in the set.where k is the total number of alleles detected for a microsatellite; Pst) of the total populations and pairwise Fst among all pairs of populations were calculated to investigate population differentiation, and significance was tested based on 1,000 bootstraps. Gene flow among populations was calculated based on the number of migrants per generation (Nm) using the formula,The presence (1) and absence (0) of desired amplicom for each SSR marker in all the 112 isolates were treated as binary characters and was analyzed using the NTSYS-pc program V2.1 Rohlf, . All theK = 1 to K = 15 using five independent runs of 25,000 burn-in period length at fixed iterations of 100,000 with a model allowing for admixture and correlated allele frequencies. The optimum number of population was predicted using the simulation method of Evanno et al.  was also determined before sample clone-corrected. The value of π and Φw represents the average number of pairwise nucleotide differences and the total number of segregating sites in a set of DNA sequences, respectively. MULTILOCUS version 1.31 was used to measure linkage disequilibrium among SSR loci using the index of association (IA) and rBarD index .The Tajima's D value was estimated using the DnaSP program Tajima, . Number RPB2) gene sequences of all the 112 UST isolates were compared with those from known species of Ustilago available in the NCBI database  in UST populations of different wheat growing zones and provided evidence that the dominance of purifying selection and population expansion is operating in UST isolates . Similarpulation .To evaluate allelic diversity among UST isolates, 35 microsatellite markers were used. Twenty five SSR primer pairs produced clear single amplicons while rest did not amplify. Out of 25 primers, only 16  showed pa), effective alleles (Ne) and expected heterozygosity (He) averaged across all loci ranged from 1.824 to 2.0, 1.695 to 1.721, and 0.373 to 0.406, respectively for the four different populations . The NWPZ population had the highest Na values (2.000) while the CZ population had the lowest (1.765). Expected heterogyzosity (He) and effective Alleles (Ne) values were lowest in CZ population  and the highest in NWPZ population . Similarly, unbiased gene diversity (uHe) was the lowest in NHZ population (uHe = 0.381) and the highest in NWPZ population (uHe = 0.410), with an average of 0.396. The polymorphic loci (%) fell in the range of 88.24% (NHZ and NEPZ) to 100% (NWPZ), with an average of 88.35% (I = 0.589) population, whereas it was lowest in CZ (I = 0.531) population. Of all the analyzed populations, no private allele was found in entire population. The genetic diversity within populations (Hs) ranged from 0.0501 to 0.4943 with an average of 0.3367 (T = 0.3611). The proportion of the total genetic diversity attributable to the population differentiation (Gst) ranged from 0.0029 to 0.391 with an average of 0.0678 over all loci , isolates (Fis = 0.079) and within isolates (Fit = 0.090) was mentioned in st values of the genetic distance between different populations were low but significant  was distributed within UST isolates . Geneticm) was ranged from 0.00  to 29.131 (between CZ and NHZ). Pairwise estimates of gene flow (Nm) indicated that Nm value was more than 1.0 in most of the population pairs suggesting gene flow between populations, although with different magnitude except NWPZ and NHZ and NWPZ and CZ  followed by CZ and NEPZ pair . When population of NEPZ was compared with other populations for genetic distance (Fst) on the basis of SSR (0.009–0.037) and RPB2 gene loci (0.041–0.045) and gene flow (Nm = 4.934–14.989) indicating these populations are differentiated with low gene flow. Pair wise comparison of NHZ and NWPZ recorded the highest indices of Nei's unbiased genetic identity (0.9947), while CZ and NHZ showed lowest levels of genetic identity (0.9358) (The average gene flow among populations (NZ and CZ . The hig(0.9358) .K = 2 (ΔK = 113.96)   and K = clusters .A (0.991–2.034) and rbarD indices (0.066–0.134) in the association tests differed significantly from zero in all the UST populations , TPM (P = 0.00070) and SMM (P = 0.00132) model of evolution, indicating recent population expansion in CZ. Similarly, in NHZ and NWPZ population, significant H deficit in 1 of the 14 loci under IAM, TPM, and SMM model of evolution were observed (The results depicted in observed .P = 0.00002) was also observed in NEPZ population using IAM model. The SDT provided the T2 statistics equal to 4.024, 3.492, and 3.168 for IAM, TPM, and SMM, respectively in CZ population. The probability values were significant for IAM (P = 0.00003), SMM (p = 0.00024), and TPM (P = 0.00077). Thus, null hypothesis was accepted by all the models. The probability values with WT for one tail for H excess under three models IAM (p = 0.00006), TPM (p = 0.00009), and SMM (p = 0.00015) indicated acceptance of null hypothesis under all the models in CZ population. Thus, all the three tests  indicated the acceptance of mutation drift equilibrium (P > 0.05) in UST populations under all the mutation models for all CZ, NHZ, NEPZ, and NWPZ populations. Another powerful test of qualitative graphical method based on the allele frequency spectra detected a normal L-shaped curve, where the alleles with the lowest frequencies (0.03–0.3) were found to be most abundant in the entire wheat growing zones  in a recently bottlenecked population . In CZ png zones .Ustilago segetum tritici isolates were collected from four major wheat growing zones  of India. The genetic structure was analyzed by performing RPB2 gene sequence comparison and fingerprinting with newly developed SSR markers. The degree of nucleotide difference in the RPB2 region in UST populations is low. It may be due to action of concerted evolution leads to homogenizing effect. Furthermore, evidence of recombination (Rm = 0) in entire UST population was not detected.Loose smut is a monocyclic internally seed borne disease. The seed borne inocula leads to long distance rapid spread of the disease across the entire wheat growing zones of India. The st values (< 0.01 and < 0.05) and pair wise population differentiation among UST population from different zones indicate low genetic differentiation in the total populations (Fst = 0.012). The UST populations appear either of common origin or limited distribution, reproduces predominantly by asexual means, or experience substantial gene flow (from CZ to NHZ and NEPZ and later from NEPZ to NWPZ and NHZ) coupled with genetic drift. However in populations where mutation rates are high, Fst tends to fall back to zero (in case of CZ) as novel alleles are added to the population  showed that 91% of the total variation was due to differences among isolates within populations and the variation among populations reflected only 1% of the total variation. A low degree of differentiation among UST populations may be due to admixture among isolates from the different geographic regions. These results are also unswerving with the low Shannon's indices (0.531–0.589). The overall Shannon's index (I = 0.552) suggests that more than 50% of the genetic diversity explained by the differences between isolates. Therefore, all these results conclude that most of the genetic variation (91%) was distributed among isolates across the regions. The similar findings have been earlier reports in Ustilago maydis  values indicated low to moderate levels of diversity in different wheat growing zones. NWPZ revealed the maximum diversity based on the number of haplotypes . Few haplotypes were shared among different populations indicating role of asexual reproduction and long-distance dispersal. Contrary to this, sexual reproduction occurs at the site of infection on the spike, was apparently prevalent in Indian wheat growing areas. Besides this, two other reasons may explain the genetic variability among isolates, first there might be possibility of multiple founder populations that result in the admixture of populations. Secondly, population genetic expansion may took place due to the accumulation of different alleles in UST populations as evident from the total and shared mutations noticed in the presents study. All these are agreeable, as evidenced by the high levels of population admixture identified in Structure analysis. Mutation generates diverse regional populations of UST, creating a pool of mutants from which new, virulent isolates can emerge. Many haplotypes were shared among these populations supports multiple introductions of the same haplotype, which could be due to pronounced asexual reproductive phase, as the case with UST. Furthermore, the analysis revealed that all UST populations are admixed and contain haplotypes from multiple populations within a region or between the regions.Haplotype analysis performed in present study provides information on the number of haplotypes (h), their frequency and diversity, and genetic distances within and between * and F*, and Fu's Fs) were performed to examine the RPB2 sequence data for departure from neutrality. Significant and negative Tajima's D test statistics indicated that RPB2 locus is experiencing population bottlenecks, where the population is largely uniform and only a few sequences compose the new population. The biology of UST and its colonization could serve as a source of population bottlenecks. Similarly, significant and negative value of almost all D* and F* test statistics showed strong purifying selection. Overall, the RPB2 gene data displayed genetic divergence in the structure of the population among the four wheat growing zones analyzed and well-supported by the results from microsatellite loci. Moreover, 16 newly developed SSR markers used were polymorphic on all of the UST isolates. These results are comparable to earlier reports on SSR markers developed for other plant pathogens  is sufficient to maintain genetic similarity. The low levels of population differentiation were observed in corresponding high values of Nm. Nm is >1, reveals little differentiation among populations, and under such circumstances migration is more important than genetic drift  indicates that 21 isolates would need to be exchanged each generation among populations of different regions to achieve current degree of similarity. Highest gene flow was recorded between the CZ and NHZ (Nm = 29.131) populations. Regular gene flow and random mating among isolates from various populations could result in new pathotypes with improved pathological and biological fitness traits  indicated little genetic differentiation across UST populations. The corroborating results were also observed in American populations of P. nodorum   in north-central and northeastern North America  were observed. The genetic identities of the four populations evaluated in present study were close to 1. The moderate Gst (−0.0678) values indicated weak genetic differentiation and minimal geographic clustering among the populations from four different zones and yielded average Nm values 6.875 across all loci and populations, suggesting that the level of gene flow was approximately seven times greater than that needed to prevent populations from diverging by genetic drift. Moreover, absence of private alleles in all the four zone population may indicate that the observed migration levels reflect gene flow. The wind direction in India is generally from North West to North East during the wheat cropping season. This may cause migration and gene flow between populations lead to admixture among isolates from the diverse geographic origin as observed in present study. The pathogen is cosmopolitan in distribution, and telisopores are known to be disseminated over long distances by wind  indicated that the observed heterozygosity excess (He) found less than the expected excess heterozygosity (Hee) in all the four wheat growing zones. Thus, the lower magnitude of He with their respective Hee reflect absence of genetic bottleneck in UST populations. Further, the negative TD-value of the UST population indicates that the UST population is undergoing demographic expansion. Further support for this hypothesis is gained from lack of private alleles in UST populations collected from all wheat growing zones.Mutation is the main evolutionary mechanism that generates polymorphisms, and its implications to disease management are well-known (Jolley et al., o et al. , while sThe knowledge of population genetic structure of a pathogen provides information on its potential to overcome host genetic resistance (McDonald and Linde, The work was conceived and designed by PK and SK. The sampling survey was performed by PK, SK, PJ, and DS. Experiments were conducted by RT, PK, and RK. Data analysis was done by PJ and RT. The manuscript was drafted by PK and SK. The final editing and proofing of manuscript was done by DS and GS. RK and RT contributed equally. The manuscript was approved by all the authors.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.


Batrachochytrium dendrobatidis (Bd). Amphibian cutaneous microbiomes interact with Bd and can confer protective benefits to the host. The composition of the microbiome itself is influenced by many environment- and host-related factors. However, little is known about the interacting effects of host population structure, genetic variation and developmental stage on microbiome composition and Bd prevalence across multiple sites. Here we explore these questions in Amietia hymenopus, a disease-affected frog in southern Africa. We use microsatellite genotyping and 16S amplicon sequencing to show that the microbiome associated with tadpole mouthparts is structured spatially, and is influenced by host genotype and developmental stage. We observed strong genetic structure in host populations based on rivers and geographic distances, but this did not correspond to spatial patterns in microbiome composition. These results indicate that demographic and host genetic factors affect microbiome composition within sites, but different factors are responsible for host population structure and microbiome structure at the between-site level. Our results help to elucidate complex within- and among- population drivers of microbiome structure in amphibian populations. That there is a genetic basis to microbiome composition in amphibians could help to inform amphibian conservation efforts against infectious diseases.Amphibian populations worldwide are at risk of extinction from infectious diseases, including chytridiomycosis caused by the fungal pathogen  Multicellular organisms act as hosts to a diverse suite of bacterial communities, collectively referred to as the microbiome. Recent research across multiple taxa has highlighted that these bacterial communities perform beneficial functions for the host, including protection from infectious pathogens –3. DespiBatrachochytrium dendrobatidis; Bd) is causing mass mortalities and rapid population declines of amphibians around the world and is a major driver of the current amphibian extinction crisis [Bd, with particular bacterial genera conferring increased or decreased protection against Bd in experimental and field studies [Bd infection loads has been found across developmental stages of tadpoles [The amphibian skin microbiome represents a model system for understanding the ecological drivers of host microbiome structure and the role of symbiotic bacteria in protecting the host from pathogens. The lethal and globally distributed amphibian chytrid fungus  characterise the microbiome of A. hymenopus; (iii) examine the genetic population structure of A. hymenopus on the Drakensberg plateau; (iv) investigate predictors of microbiome structure among sites ; (v) investigate within-site predictors of microbiome structure ; and (vi) examine the roles of host microbiome and host genetics in influencing Bd infection loads.Here we use a disease-challenged high-altitude amphibian species in the Drakensberg Mountains of southern Africa to investigate within and among population predictors of microbiome structure that may have implications for disease susceptibility. Using microsatellite genotyping and 16S amplicon sequencing, we examine how population genetic structure, individual genotype, and developmental stage determine differences in microbiome structure among individuals. Specifically, we aim to: (i) quantify Amietia hymenopus) is the only amphibian found in most of the Mont-aux-Sources stream system, with the exception of the Kubedu river, where A. hymenopus is absent and A. umbraculata is present [A. hymenopus has a consistent history of Bd infection with associated mass mortalities [The Drakensberg Mountains of southern Africa are a vast mountain range reaching up to 3482 m above sea level (asl). Here, the Mont-aux-Sources plateau crosses north-east Lesotho and South Africa. On the South African side, four streams  flow 1–3 km across the plateau from multiple origins in the high peaks on the South Africa-Lesotho border, before dropping off a ~1000 m precipice into South Africa ; ‘Tugela’, ‘Tugela 2’ and ‘Tukelahed’ from the Tugela River (South Africa); ‘Khubnam’ from the Khubela River (Lesotho); and ‘Bilanjil’ and ‘Ribbon Falls’ from their respective rivers  and was used for all subsequent analyses.We collected 20–28 tadpoles per site (nine at Khubnam) along a ~150 m transect within an altitudinal range of 3010–3090 m asl to minimize variation in Bd infection intensity in triplicate using a 1:10 dilution of DNA with Bd standards ranging from 0.025 to 250 zoospores/µl. We calculated infection prevalence and intensity for each site.We constructed serial dilutions of DNA and conducted quantitative PCRs on a Qiagen Rotor Gene according to Boyle et al. . We quanAcinetobacter, Citrobacter, Enterobacter, Flavobacterium, Plantibacter, Pseudomonas and Serratia).We sequenced the microbial communities present on tadpole mouthparts using a modified version of the protocol in Kozich et al. and Bates et al. , 27. TraHalomonas and Pseudoalteromonas that are likely PCR mastermix contaminants. DADA2 identified 20 unique sequence variants in the sequenced mock community sample comprising 20 bacterial isolates. The final SV table, taxonomy table, and sample metadata were exported to phyloseq [We performed sequence processing in DADA2 v1.4.0  using the default pipeline (see Supplementary Information). Modal contig length was 253 bp once paired-end reads were merged. We filtered 145 sequence variants (SVs) with length >260 bp , and excluded 190 bimeras from the 22,010 input sequences. We assigned taxonomy using the SILVA v123 training set , 29. We phyloseq  in R v3.phyloseq .n = 19,395), yielding 2384 SVs. Results using all SVs are similar and not shown here. After filtering, mean library size across individuals was 20,160 reads . We rarefied all libraries to 13,156 reads per sample for alpha and beta diversity analyses. We calculated alpha diversity metrics (Shannon index) using the ‘estimate_richness’ command in phyloseq. We visualised differences in Bray-Curtis distance among samples using non-metric multi-dimensional scaling (NMDS) ordination of bacterial communities using the R package vegan [k to 3 to yield a stress value of <0.17. We also used the vegan function ‘vegdist’ to calculate Bray-Curtis distance between bacterial communities to compare to genetic distances among samples, and the ‘adonis’ function to compare bacterial community structure among populations using permutational MANOVA. We used the R package gplots [Following Longo and Zamudio , we exclge vegan , settinge gplots  to visuaA. hymenopus individual using Illumina 250 bp paired-end v2 chemistry. We characterised eleven novel tri- and tetra-nucleotide microsatellite markers according to Griffiths et al. [We sequenced DNA from a single s et al.   to study the effects of intraspecific genetic diversity at the population level on microbiome variation. We excluded Khubnam due to the low sample size (n = 9) compared with other sites (n = 20–28) to ensure sufficient power to detect differences among sites was retained. We created a pairwise matrix of Bruvo genetic distance [Details of quality control and summary statistics carried out can be found in  Supplementary Information. To assess levels of population differentiation, we calculated global d Estoup  with 100stoup [D  in Genodstoup [D  We used stoup [D  to calcudistance  between distance  in R to K) and assign individuals to them based on likelihoods. We inferred the most probable K using the Evanno method [We used Bayesian clustering program STRUCTURE v2.3.4 , 44 to ao method , 46, ando method  and Disto method  to proceD values and the standardised covariance method. We ran an analysis of molecular variance (AMOVA) in Genodive using the Infinite Allele Model. We tested for the presence of isolation by distance among all locations and within population clusters. We conducted a Mantel test in ade4 in R [FST values (FST/1–FST) and the logarithm of geographic distances. We used GENECLASS2 v2.0 [We conducted principal coordinates analysis (PCoA) in GenAlEx v6.502 , 50 usinde4 in R  in R witSS2 v2.0  to identSS2 v2.0 , 54.We used INEST v2.1  to identlme4 [n = 126 data points for which there were both microbial (Shannon) and host genetic (proportion of heterozygous loci) data). Gosner stage and proportion of heterozygous loci were z-transformed prior to model fitting. The most complex model contained a proportion of heterozygous loci × Gosner stage interaction. We assessed significance of terms using likelihood ratio tests between models estimated using maximum likelihood. To quantify differences in mean richness among populations, we extracted the posterior modes of the population random effects from a Bayesian version of the best-supported model, specified in the R package MCMCglmm [To examine the factors affecting Shannon diversity, we fitted a Gaussian Mixed effects model in the R package lme4  with Shalme4 ) as predMCMCglmm .microbiome package  to be used as a response in a Gaussian mixed effects model. The community structure model contained Gosner stage, proportion of heterozygous loci and their interaction as fixed effects, and population ID as a random effect. We calculated the r2 values of minimal models using the [MuMIn [A. hymenopus individuals  and Bray-Curtis dissimilarities among their microbial communities , while controlling for the effect of geographic distance. We used the ‘mantel.partial’ function in the vegan package, specifying the Pearson correlation statistic.To examine the factors predicting microbial community structure (beta diversity), we calculated the within-population ‘divergence’ metric of beta diversity using the sing the  method fe [MuMIn . We usedn = 6 sampling sites) to test the influence of allelic richness and Bd infection prevalence on alpha diversity. We did not fit both Bd prevalence and allelic richness in the same model to maximize residual degrees of freedom in the model. Significance of terms was assessed by likelihood ratio test.We fitted population-level regressions  zoospores, with an average prevalence of 10.91 (±3.92)% across populations. There was relatively low infection prevalence and intensity at all sites, with highest infection rates at Tugela 2 and a complete absence of infection at Ribbon Falls  were common to all sampling sites .We observed significant differences in Shannon diversity index among sampling sites Fig. . Tukelahχ2 = 2.61, p = 0.1), proportion of heterozygous loci , or their interaction .Shannon diversity of tadpole mouthparts was not affected by either Gosner stage of tadpoles . The best model explaining differences in beta diversity contained Gosner stage as the only fixed effect and had a marginal r2 of 4%.Gosner stage significantly influenced within-population divergence in beta diversity . There was no evidence that the Shannon diversity at a sampling site was affected by site-level allelic richness  or Bd infection prevalence .There was a positive correlation between host genetic distance calculated from microsatellites and microbial community distance when controlling for geographic distance  supported the presence of a Nampolice and Vemvane cluster. The PCoA and AMOVAs showed results consistent with the STRUCTURE analysis , and within the population cluster identified in STRUCTURE K = 2  . However, when tested within the Khubnam, Tugela, Tugela 2, Tukelahed and Bilanjil group identified in the PCoA and STRUCTRE K = 3, isolation by distance was not significant . Four putative first-generation migrants were found among the site: One in Nampolice originating from Vemvane (p = 0.009), one migrant each were exchanged between Tugela 2 and Bilanjil , and one in Tukelahed originating from Tugela 2 (p = 0.003).Isolation by distance was strong and significant when tested over all locations (p = 0.003), and deficiencies in M-ratios were found at Nampolice (p = 0.003), Vemvane (p < 0.001) and Ribbon Falls (p = 0.042).Bottlenecks were detected at three sites: Heterozygosity excess in respect to allelic richness was found at Nampolice . Althouyphemus; –66, inclyphemus; –69. Varismission , and theBd on post-metamorphic individuals. However, Bd primarily infects the mouthparts of larvae as keratin is localised to this area until the onset of metamorphosis when skin is remodelled to contain keratin. Although Bd infection intensities tend to increase as larvae develop due to increasing size [We detected a link between Gosner stage and mouthpart microbiome composition. Studies to date have predominantly used skin swabs to determine host microbiome of amphibians, as this is the region that becomes infected by ing size , we showing size , 72, whiing size . OntogenA. hymenopus has not arisen from contact between populations. A number of studies have now indicated that amphibians are able to select their microbiome based on functional traits [Despite these genotypic and ontogenetic influences on microbiome composition, populations shared a considerable core microbiome Fig. , which hl traits , 77. Thil traits . This mal traits , 79, 80.l traits . Individl traits  found baBd infection prevalence in the Mont-aux-Sources region, ranging from ~0 to 100% [Bd infection and microbiome diversity, although this has been demonstrated in other studies , 84Bd inA. hymenopus were seen at Ribbon Falls and Vemvane in September 2006 during routine monitoring (every 2–4 months) of Vemvane, Tugela, Bilanjil and Ribbon Falls ([Bd infections in 100% of the dead metamorphic A. hymenopus (n = 10) collected at each site. Further dead post-metamorphic individuals (n = 1–12) were encountered on most of the subsequent surveys, all infected with Bd [Bd infection prevalence at Ribbon Falls is generally low compared to other rivers in the region  with higher heterozygosity had higher Bd infection intensity, which they attribute to increased dispersal of heterozygous individuals leading to increased spread of Bd. Therefore, it is possible that the lack of immigrants to Ribbon Falls is also limiting the spread and maintenance of Bd at this site.Ribbon Falls, Nampolice and Vemvane showed evidence of recent population bottlenecks, and these sites also have some of the lowest allelic richness. Considerable mortalities of n Falls , and Ris et al.  showed mBd prevalence was low in the year we sampled, but we found lower genetic diversity and signals of genetic bottlenecks in sites where mass mortalities have been recorded in previous years, demonstrating the vulnerability to and persisting effects of disease in amphibian populations. Together these results demonstrate the key role of host genotype on microbial colonisation, which may be important in pathogen susceptibility in the amphibian-chytridiomycosis system. Identifying individuals based on this characteristic may aid amphibian conservation efforts against this lethal pathogen.Here we have shown that the microbiome composition of amphibians is determined by genetic and ontogenetic variation, as well as geographic site, indicating within and among population predictors of microbiome diversity. Supplementary material


Alternanthera philoxeroides recently expanded its range northwards in China. It is unknown if the range expansion has a genetic and/or epigenetic basis, or merely an environmental basis due to a warming climate. To test these possibilities, we used an RNAseq approach with a common greenhouse design to examine gene expression in individuals from the northern edge and central portion of alligator weed range from China to determine if there were differences in their responses to cold temperatures. We hypothesized that if the recent range expansion was primarily environmental, we would observe few differences or only differences unrelated to low-temperature adaptations. We assembled over 75,000 genes of which over 65,000 had long open reading frames with similarity to sequences from arabidopsis. Differences in expression between northern and southern populations that were both exposed to low temperatures showed similar expression among genes in the C-REPEAT/DRE BINDING FACTOR (CBF) regulon. However, gene set and sub-network enrichment analysis indicated differences in the response of photosynthetic processes and oxidative stress responses were different between the two populations and we relate these differences to cold adaptation. The transcriptome differences in response to cold between the individuals from the two populations is consistent with adaptations potentiating or resulting from selection after expansion into colder environments and may indicate that genetic changes have accompanied the recent northward expansion of A. philoxeroides in China. However, we cannot rule out the possibility of epigenetic changes may have a role in this expansion. Biological invasions are becoming a major global environmental and economic problem , and intAlternanthera philoxeroides , has recently expanded it range northward by nearly 5° of latitude beyond what was its predicted range in China  belongs to family Amaranthaceae  , 500 km north of the Shanghai in northern China (A. philoxeroides in China ( E121.5) , and cul E121.5) , causing E121.5) . This we E121.5) . A recenrn China , 2012. Tin China . It is uCBF2)  production through reduced photosynthesis from unadapted thylakoid membranes, or induction of free radical scavenging enzymes such as APX1, SOD1, NPX1, etc. . Many coCBF2) . ZAT12 iand CRY2 . ZAT12 hand CRY2 .Camellia sinensis)  , and Chrkingense  highlighkingense . AdditioA. philoxeroides between three individuals each from two populations, one from the northern edge of its range in Jinan, North China and the other from the central portion of its range in Shanghai, South China. If the recent range expansion was primarily environmental, we would expect few differences in gene expression between cold-treated plants from the northern and central populations, or only differences unrelated to low-temperature adaptations. We test this hypothesis by looking for differences in transcriptome responses indicative with a response to cold using a common garden experimental design. Thus, any such differences, if observed, would be attributed to genetic and/or epigenetic differences between the individuals from the two populations. Additionally, we expected to develop a database of expressed sequences tags (ESTs) for A. philoxeroides and eventually identify polymorphisms that have potential as genetic markers for more cold-adapted populations.Here we examine the differences in cold-responsive gene expression in A. philoxeroides plants were collected in November 2013 from local rivers of Jinan and Shanghai. Jinan, the provincial capital of Shandong, is located in North China in the lower reaches of the Yellow River, the second longest river in China. The average annual temperature in Jinan is 14.7°C with an average of -0.4°C in January and 27.5°C in July. The average annual rainfall is 672.7 mm. Its location is N36.6, E117.1. Shanghai is located in South China in the lower reaches of the Yangtze River, the longest river in China. The average annual temperature in Shanghai is 16.6°C with an average of 4.7°C in January and 28.0°C in July. The average annual rainfall is 1184.4 mm. Its location is N31.4, E121.5. The mentioned meteorological data from China Meteorological Data Sharing Service System (cdc. cma.gov. cn), and also see our paper  following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase RNaseH-. Second strand cDNA synthesis was subsequently performed using DNA polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3’ ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150∼200 bp in length, the library fragments were purified with AMPure XP system . Then 3 μl USER Enzyme  was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95°C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on an Agilent Bioanalyzer 2100 system.The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq SR Cluster Kit v3-cBot-HS (Illumina) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina Hiseq 2000/2500 platform and 100 bp paired-end reads were generated.E-value < 10-5.Primer sequences were trimmed from raw sequence reads and the resulting files were trimmed for high quality reads using the program Sickle-quality-based-trimming  in the iThe program Bowtie  was usedA. philoxeroides. Over 212 million one hundred base, paired end RNA sequencing reads were obtained from 3 cold-treated individuals from both central and northern populations. Sequences ranging from 29 and 48 million fragments were obtained after trimming the raw reads based on sequence quality scores with a minimum read length of 70 bases. Fewer than 8% of the fragments were removed by the quality trimming  to the arabidopsis TAIR10 database. These blast hits were used for the primary functional annotation of the transcripts  associated with biochemical pathways from the AraCyc database when the entire gene set was subjected to GSEA  was used to identify over-represented terms from the AraCyc  and GO  ontologi to GSEA . All but to GSEA . An anal to GSEA . Again,  to GSEA .p < 0.01, we identified 12, 6, and 5 terms when all of the genes were analyzed or when the genes were limited to those preferentially expressed in the central and northern populations, respectively.The pathway studio program maintains a database of transcription factor targets, interacting proteins, and proteins/chemicals regulating cellular processes . The resA. philoxeroides populations. One population from Jinan in northern China and appear to have gained the ability to flourish in a more northern climates previously uninhabitable by this weed. The other individuals were from a central population near Shanghai in the center of its established range. The goals of these experiments were to develop a sequence database of genes from A. philoxeroides, and to determine if these two populations differ in their transcriptomic response to cold temperature treatments. The cold treatments did not appear to result in obvious physical differences between the two populations, but transcriptome differences were detected between the tested individuals from the two populations. Further studies are needed to determine if either population is better able to survive and reproduce in the more northerly latitudes. However, differences in gene expression were observed between the individuals from these two populations and the changes in gene expression are consistent with cold resistance mechanisms. These observations are most consistent with the hypothesis that the northward expansion of A. philoxeroides into Northern China is not simply the result of global warming, but is likely due to evolution of adaptations to colder environments.We performed an RNAseq analysis following a cold treatment on three individuals of two Heveabrasiliensis) indicated that 17% of the assembled transcripts were from non-plant sources  indicated that ∼25% might result from contamination of the samples from other organisms (mostly arthropods). A search of the literature suggests that most RNAseq assemblies contain about 1–3% non-plant sequences, although one study in rubber tree  between the central and northern populations in response to the cold treatment. The most notable differences were involved in photosynthesis, which appear repressed in the tested individuals from northern populations. This observation is supported from both an examination of the most differentially expressed genes and from the gene set and sub-network enrichment analyses. One of the primary causes of damage due to chilling stress is the production of oxidative radicals produced when the electron transport chain is disrupted by altered membrane fluidity that prevent proper association of the proteins involved in the electron transport in the chloroplast stroma  among the contigs and genes that were assembled. However, we observed up-regulation of other cold-responsive regulons in the northern population. Genes such as a COR27-like and RAV1-like that in arabidopsis are modulated through circadian clock and ABA signaling (In our study, very little evidence points to alterations in the CBF regulon between the northern and central populations. Neither “neighbors of CBF” or “response to cold” were indicated as over-represented ontologies by SNEA or GSEA. Likewise, no known targets of CBF were indicated as significantly differentially expressed – despite the presence of putative sequences of targets (such as a ignaling  were fouIn addition to non-CBF regulated cold inducible circadian-responsive genes, GSEA identified numerous genes in biotic defense responses, such as salicylic acid and flavonoid biosynthesis, as up-regulated in the northern population. These processes have been associated with growth inhibition in Arabidopsis and other plants . LikewisA. philoxeroides in the central portion of its range in South China and individuals from a population in North China which has recently expanded its range northward. These results are most consistent with the development and/or selection of genetic and/or epigenetic differences that enhance the cold-tolerance of the northern populations. Further work using a population genetics approach will be needed to determine if the differences observed in these individuals is representative of the whole population, and to identify loci associated with these differences. However, the differences we observed in gene expression point towards alteration in the cold-responsive regulon controlling circadian/ABA signaling rather than alterations in the CBF regulon. Importantly, our results also provide the first large-scale database resource of A. philoxeroides gene and transcript sequences. Such sequences should serve as a rich source for markers needed to examine the population genetics of a species that has proven invasive on at least three continents.We have demonstrated there are differences in gene expression associated with response to cold treatments between tested individuals from populations of LD, LP, and LW performed the experiments and collected samples. DH performed primary data analysis and carried out bioinformatics analysis. LD and DH designed the experiments and wrote the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.