To fully understand cell type identity and function in the nervous system there is a need to understand neuronal gene expression at the level of isoform diversity. Here we applied Next Generation Sequencing of the transcriptome (RNA-Seq) to purified sensory neurons and cerebellar granular neurons (CGNs) grown on an axonal growth permissive substrate. The goal of the analysis was to uncover neuronal type specific isoforms as a prelude to understanding patterns of gene expression underlying their intrinsic growth abilities. Global gene expression patterns were comparable to those found for other cell types, in that a vast majority of genes were expressed at low abundance. Nearly 18% of gene loci produced more than one transcript. More than 8000 isoforms were differentially expressed, either to different degrees in different neuronal types or uniquely expressed in one or the other. Sensory neurons expressed a larger number of genes and gene isoforms than did CGNs. To begin to understand the mechanisms responsible for the differential gene/isoform expression we identified transcription factor binding sites present specifically in the upstream genomic sequences of differentially expressed isoforms, and analyzed the 3′ untranslated regions (3′ UTRs) for microRNA (miRNA) target sites. Our analysis defines isoform diversity for two neuronal types with diverse axon growth capabilities and begins to elucidate the complex transcriptional landscape in two neuronal populations. Next Generation sequencing applied to the transcriptome (RNA-Seq) is a transformational technology that uncovers vast amounts of novel information about transcript expression and identity. With its advent the rich complexity of the mammalian transcriptome is beginning to be fully appreciated A detailed knowledge of the expression of cell specific isoforms is crucial to understanding cellular diversity. This is especially obvious in the nervous system, with its enormous variety of cell types with distinct functions and characteristics. Previous RNA-Seq studies of nervous system tissues have involved mixed populations of neurons with other cell types We found enormous diversity of isoform expression between DRG neurons and CGNs, with over 8,000 differentially expressed isoforms. We scanned the promoters and 3′ UTRs of differentially expressed isoforms for cis-elements involved in transcription regulation and identified transcription factors (TFs) and miRNAs potentially involved in the control of isoform specific expression. Known interactions between predicted TFs and miRNAs were used to generate regulatory networks that may be driving isoform specific expression differences. Thus we have identified new intracellular targets that may affect neuronal type specific transcript expression.Fragments (aka: reads) Per Kilobase of exon per Million mapped reads (FPKM). There were over 50,000 transcripts mapping to annotated areas of the genome and over 135,000 transcripts mapping to genomic regions lacking annotation. For the purpose of this report we focused only on transcription events at the level of known, active loci . DRG neurons were used because of their robust axonal growth in culture, and because they represent a well-established model for understanding axonal regeneration The vast majority of transcripts mapping to an annotated locus (95%) had an FPKM that was lower than 1% of the FPKM of the highest expressed isoform  indicating that the majority of transcripts fall into a relatively low expression fraction. This observation is consistent with previously described global gene expression patterns . To inteBecause DRG neurons and CNS neurons such as CGNs are highly distinct in their developmental origins, integration into circuits, and axonal growth properties DRG neurons not only expressed higher levels of transcripts but also expressed approximately 25% more unique isoforms . The 5328 cell type specific genes in DRG neurons produced 8483 isoforms, while CGNs had 4358 cell type specific genes that produced 6778 isoforms. Interestingly, the isoforms produced by DRG neurons were much more diverse, as a group, than those produced by CGNs. For example, isoforms expressed by DRG neurons utilized 1999 different transcription start sites (TSSs) compared to 710 used by CGN exclusive genes. Similarly, DRG neurons not only use a greater number of coding sequences  but also a greater number of 3′ UTRs (1828 vs. 676), compared to CGNs. Overall, DRG neurons exhibited a nearly 3-fold increase in the number of TSS, CDS, and alternative 3′UTRs compared to transcripts found in CGNs. Thus DRG neurons not only express more isoforms, but each isoform differs, on average, in more positions than in CGNs.To begin to understand the nature of the gene expression differences between these two neuronal types, we chose 7 gene classes relevant to neuronal functions such as cell adhesion, vesicle transport and neurotransmitter expression . CGNs expressed a greater number of neurotransmitter receptor genes. In all other categories DRG neurons expressed the largest number of isoforms ; Table 2Aplp1) and ATPase, Na+/K+ transporting, alpha 3 polypeptide (Atp1a3) because the expression of these isoforms was estimated to be similar between the cell types; activating transcription factor 3 (Atf3) and the phosphatase and tensin homologue (Pten) were chosen because they gave rise to a host of unique isoforms; tissue inhibitor of metalloproteinase 1 (Timp1), member RAS oncogene family (Rab5b), gamma synuclein (Sncg), and solute carrier organic anion transporter family, member 3a1 (Slco3a1) had high expression in DRG neurons compared to CGNs; and CAS1 domain containing 1 (Casd1) had high expression in CGNs compared to DRG neurons  and , occurs when FPKM 1- FPKM 2 and qPCR 1- qPCR 2 have the same sign, where the subscript indexes the observation. Under the null, the probability of concordance is 0.50. All of this data supports the idea that RNA-Seq faithfully represents transcript expression To evaluate the reproducibility of our results, we used qPCR to assess gene expression from 9 genes and compared these levels to those estimated by RNA-Seq  and 4. W neurons . To char neurons ; Table 2Atf3, Pten, and matrilin2 (Matn2) are regeneration-related genes Three of these genes, Atf3 is involved in peripheral nerve regeneration Atf3isoforms were identified in our analysis . We validated the existence of Pten J2 through both PCR amplification and sequencing  and serine/threonine protein kinase Akt, key regulators of neurite outgrowth 3 to such a degree that Akt mediated neurite outgrowth is affected.Another gene of interest in neuronal regeneration is quencing . Pten J2analysis . The truanalysis . Availabanalysis –C. We hyRNA-Seq data can be used to identify precise exon locations that in turn allow the determination of TSSs for each expressed isoform. Scanning individual isoform promoters for TF binding sites allows prediction of TFs regulating specific isoforms RNA-Seq identifies not only TSSs, but also the full 5′ and 3′ untranslated regions (UTRs) of transcripts. The 3′UTR is thought to be the main target region for miRNAs, which bind to mRNAs and mediate their degradation or inhibit their translation Groups of 3′UTRs were submitted to TargetScan, which allowed us to scan for known and registered MREs SRF;Srf a prime candidate for activation because it has the potential to regulate numerous genes simply by its ability to impact the expression of multiple miRNAs in a cell type (DRG neurons) exhibiting robust neurite growth both in vitro and in vivo after an axonal injury. Indeed while there is no change in the expression of serum response factor (Srf), we find that a novel isoform of serum response factor binding protein (Srfbp1) is significantly overexpressed (up to 10-fold higher) in DRG neurons  and dorsal root ganglion (DRG) neurons. While a few recent studies have utilized RNA-Seq on nervous tissue Atf3 and Pten produced multiple isoforms  typically is reactive to mitogenic stimuli Atf3 isoform with the highest FPKM in DRG neurons was J2 and its promoter is completely novel , but it is also possible that PTEN J2 exists solely as a regulatory noncoding RNA (ncRNA), which could potentially function as a miRNA sponge to participate in the fine regulation of the main transcript Another point of isoform diversity occurs with alterations in the CDS and 3′UTRs. Changes in CDS may lead to the most obvious alterations in function as whole protein domains with specific functions could be present or absent. We examined numerous other transcripts associated with neuronal regeneration and found that many harbored changes that would affect the CDS . One sucal cells . The preal cells . We wereCepba, Irf1, Myc, Pax4, Rel, and Tead). Some differences may be attributed to the TFBS matrices used. Smith et al. used TRANSFAC to examine specific binding sites, while in this study we employed the JASPAR database, which examines TFBS matrices. In addition, different cutoffs for differential gene expression between the two studies likely account for some of the differences observed. It will be interesting to test whether the 6 TFs common to both analyses may be network hubs driving DRG neuron specific gene expression.Why analyze differentially expressed isoforms for specific TFBSs and for miRNA response elements? If one hopes to truly understand the regulation driving isoform diversity, it is necessary to identify the transcription factors and miRNAs that direct their expression. The idea that this approach identifies novel regulatory networks is supported by a comparison between the present study and a previous study performed in our lab. In that study, TFBS analysis was performed on differentially enriched genes between DRG neurons and CGNs after subtractive hybridization and microarray analysis In addition, network analysis could identify novel and functionally relevant pathways. In fact, we found that numerous interactions first identified in muscle tissue All procedures using animals were approved by the University of Miami Animal Care and Use Committee.5 cells were grown for 16hrs on tissue culture dishes coated with 100ug/mL poly-d-lysine (Sigma-Aldrich) and 5ug/mL laminin . Two biological replicates for each CGN substrate were prepared for RNA-Seq. For qPCR validation, three additional biological replicates were prepared exactly as for the RNA-Seq experiment.DRGs and CGNs were cultured from P8 C57bl/6j mice. CGNs were isolated as previously described by our laboratory For DRG isolation, each ganglion was trimmed of its axons, and then incubated in a solution of dispase , trypsin , and collagenase . Fetal bovine serum was used to quench the trypsin. Cells were centrifuged at 80G and then resuspended in L-15 media (Invitrogen) and DNase  followed by trituration. Ganglia from two mice were combined for each biological replicate. Three biological replicates were prepared in total. The DRG media was prepared as previously described RNA was extracted using Trizol Reagent (Invitrogen) following standard methodology. The RNA Integrity Number (RIN) was greater than 9.5 for each biological replicate. Next Generation Sequencing was performed at the Hussman Institute for Human Genomics Sequencing Core Facility . RNA was prepared for Next Generation Sequencing following the Illumina mRNA Sample Preparation Guidelines . Each sample was run over two lanes and subjected to 52 sequencing cycles on the Illumina Genome Analyzer II (Illumina). Image analysis and base calling were performed using Genome Analyzer II Pipeline v1.5 (Illumina). Read alignment using the Efficient Large-Scale Alignment of Nucleotide Databases (ELAND) algorithm is part of the Illumina pipeline and standard service at the sequencing core. 80% or more of the reads aligned to the mouse reference genome (mm9) which is similar to what was found in our analysis using Bowtie http://www6.miami.edu/miami-magazine/featurestory2.html).All of the bioinformatic analyses were run on the “Pegasus”, a Linux based supercomputer with 5000 central processing units . For this purpose we used the Tophat software, version 1.0.13 Fragments Per Kilobase of exon per Million fragments mapped) together with confidence intervals. A different part of the software, named Cuffcompare (version 0.8.3) classified the reconstructed RNA-species as novel or known according to how they map back to the provided reference annotation Aligned reads were assembled into the different RNA-species by the Cufflinks software (version 0.8.3). At first Cufflinks uses the aligned reads in the dataset to describe a set of transcripts starting from the reads that span splice-junctions. We ran this step using a non-annotated reference genome because without an annotation the software will assemble novel transcripts and isoforms. After transcript assembly, normalized expression levels are estimated and reported as FPKM  relating the probability that an isoform was reliable to log FPKM.TSSs were defined as the beginning of the first exon. A change in CDS was defined as any change that occurred from exon 2 to the second to last exon when compared to the conventional isoform. 3′ UTRs were defined as the last exon. The number of differentially expressed isoforms associated with a CDS in the reference annotation was greater than 93% indicating that the vast majority of transcripts analyzed in this dataset are in fact messenger RNAs and not noncoding RNAs.t method Three additional biological replicates were created for both DRGs and CGNs. RNA from these replicates was used as input (450ng) for a reverse transcription reaction using oligo d(T) primers following the manufacturer's recommendations . In all cases primers designed for qPCR spanned exon-exon boundaries. Real time PCR was performed using 2X SYBR Green master mix (Applied BioSystems) on the Gene Rotor System . Relative expression was calculated using the delta delta CAtf3 and Pten isoforms. Primers for validation can be found in Sequences for each set of isoforms were retrieved from the UCSC Genome Browser after uploading a user supplied GTF with the coordinate locations of each exon in the Pten and Pten J2 CDS's were cloned into a modified pAAV-MCS plasmid  containing the 2A peptide  were overexpressed from the pAAV-2A plasmids. Transfected cortical neurons were grown for 3 days, fixed and immunostained with Hoechst dye to mark nuclei and for neuronal specific tubulin . Neurons were grown at densities such that processes could be accurately imaged and traced without substantial overlap between cells. Neuronal imaging and tracing was performed using the Cellomics ArrayScan VTI automated microscope (Thermo Fisher). Neurite length was determined using the Neuronal Profiling Algorithm v3.5. Data analysis was performed in Spotfire Decision Site Software . Transfected neurons were determined by calculating a background fluorescent intensity in neurons that did not receive plasmid and then a threshold was set, above which a neuron was deemed transfected  peptide . The 2A Differentially expressed transcripts were grouped according to their expression profiles. Promoter regions -1000 to + 300 bp from the TSS were isolated for each single transcript. We took advantage of individual start sites as identified by sequencing to define RNA-species specific promoters.Matrix models for Vertebrate Transcription Factors Binding Sites (TFBS) from the JASPAR database were then used to scan the promoter regions http://www.targetscan.org/mmu_50/; The same groups of transcripts used for the TFBS analysis were tested for abundance of target sites for specific miRNAs. MiRNAs are thought to target primarily 3′UTR's of messenger RNAs although recent reports have demonstrated that functional target sites can be found even in the coding sequence as well as in the 5′UTR of the transcripts To test for significance of over or underrepresentation of target sites in the different groups the approach described for the TFBS analysis was used. Briefly, frequencies of target sites per base pair were calculated for each group as well as for a background of 5000 randomly chosen 3′UTRs. Frequency in each group was compared to the frequency in the background to calculate the expected frequency. Observed and expected frequencies were then used to calculate a Z-score representing over or underrepresentation of target sites in each individual group. This approach was previously described by Sui and colleagues Figure S1Atf3 transcripts in the UCSC Genome Browser. Labels are directly above each track display. Thick black bars are exons and the thin line indicates introns. Arrows indicate direction of transcription (Atf3 is on the minus strand). Atf3, Atf3 J1, and Atf3 J2 have all been reported before. Atf3 J2 is a novel isoform. In total three promoters are active: P1, P2 and a novel promoter, P3.(TIF)Click here for additional data file.Figure S2Pten isoform analysis.A, B, pAAV-2A-Pten or -Pten J2 over expression plasmids. C, The 2A peptide bridge sequence which mediates a ribosomal pause resulting in two independent proteins . D, Pten and Pten J2 amino acid sequences were put into protein BLAST. Pten J2 predicted protein lacks the phosphatase domain. E, Total neurite length is not changed compared to control (Oxr1). Doublecortin (DCX) is a positive control as it is known to increase neurite length. ***P<0.005.(TIF)Click here for additional data file.Table S1Isoforms tracking file. The nearest reference ID column refers to associated transcript ID given by UCSC Known Genes, RefSeq, or Ensemble database. A _j indicates a novel isoform. FPKM estimation for each individual sample with confidence intervals is shown. The biological replicates FPKM information was generated in the Cuffdiff run and used to differential expression analysis.(XLSX)Click here for additional data file.Table S2Transcription factor names and Z-scores that went into creating the  heatmap. The transcription factors are from the JASPAR database (release 10/2009). The Z-score for each TFBS in the promoters of DRG or CGN enriched isoforms is shown.(XLSX)Click here for additional data file.Table S3MiRNA names and Z-scores that went into creating the  heatmap. The Z-score for each TFBS in the 3′ UTRs of DRG or CGN enriched isoforms is shown.(XLSX)Click here for additional data file.


Differential expression of maternally and paternally inherited alleles of a gene is referred to as gene imprinting, a form of epigenetic gene regulation common to flowering plants and mammals. In plants, imprinting primarily occurs in the endosperm, a seed tissue that supports the embryo during its growth and development. Previously, we demonstrated that widespread DNA demethylation at remnants of transposable elements accompanies endosperm development and that a subset of these methylation changes are associated with gene imprinting. Here we assay imprinted gene expression genome-wide by performing high-throughput sequencing of RNA derived from seeds of reciprocal intraspecific crosses. We identify more than 200 loci that exhibit parent-of-origin effects on gene expression in the endosperm, including a large number of transcription factors, hormone biosynthesis and response genes, and genes that encode regulators of epigenetic information, such as methylcytosine binding proteins, histone methyltransferases, and chromatin remodelers. The majority of these genes are partially, rather than completely, imprinted, suggesting that gene dosage regulation is an important aspect of imprinted gene expression. The correct expression of imprinted genes, in which maternally and paternally inherited alleles are differentially expressed, is required for successful reproduction in both plants and animals Arabidopsis thaliana endosperm is hypomethylated not just at imprinted genes but at thousands of sites throughout the genome when compared to the embryo and to vegetative tissues de novo methylation during endosperm development, and/or increased methylation in the embryo Differential DNA methylation is an important aspect of the control of imprinted gene expression. For several imprinted genes the maternal allele is less methylated than the paternal allele in the endosperm MEA and FIS2 are maternally expressed imprinted Polycomb group genes that restrict endosperm cell division. However, since the identity, functions, and expression patterns of many imprinted genes are likely still unknown it is presently unclear how many of the imprinted genes will reasonably fit under the umbrella of the kinship theory. Other theories suggests that in species where the mother provisions or cares for the offspring, expression of maternal alleles is favored due to an increase in the adaptive integration of maternal and offspring genomes  Apart from the mechanistic basis of imprinted gene expression, parental conflict between maternally and paternally inherited genomes of offspring over maternal resource allocation is a popular explanation for why imprinted gene expression is evolutionarily advantageous  Arabidopsis thaliana embryo and endosperm, as well as information on endosperm and developmental expression patterns We previously used knowledge of differences in methylation between Arabidopsis thaliana accessions, Ler and Col-0. This strategy allowed us to distinguish transcripts derived from the maternally inherited or paternally inherited allele for a portion of expressed genes with Ler/Col-0 single nucleotide polymorphisms (SNPs). We identified >200 genes with parentally biased expression patterns. Our experimental strategy is particularly robust for identifying paternally expressed imprinted genes, as transcripts derived from the paternal genome must come from one of the fertilization products. Over 40 genes are predominantly paternally expressed, including a large number of transcription factors and chromatin related proteins. Most of the imprinted genes we identify exhibit parentally biased expression rather than complete monoallelic expression, suggesting that dosage regulation is an important factor in gene imprinting.Here we assay imprinted gene expression by performing high throughput sequencing on poly-A selected RNA (RNA-seq) from embryo and endosperm derived from reciprocal crosses between two er plants were grown in 16 hour days at 21° C. Stage 12c flowers er×Col, RNA was pooled from dissected seeds from 21 siliques (∼800–1000 seeds). For Col×Ler, RNA was pooled from seeds dissected from 14 siliques (∼560–700 seeds).Wild type Col-0 and Ler embryo and Col×Ler endosperm and two lanes each of Ler×Col embryo and Ler×Col endosperm at 50 bp, and one additional lane of each library at 36 bp. Sequencing reads are deposited as fastq files in GEO record GSE30511 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE30511).RNA-seq libraries were created by using the Illumina mRNA-seq kit and following the sample preparation guide protocol (Illumina). Briefly, 1 µg of total RNA was poly-A selected twice, fragmented, and converted into double-stranded cDNA. The cDNA was end repaired, adenylated, and adapters ligated. Adapter-ligated DNA approximately 200 bp in length was gel purified and amplified using Illumina PCR primers PE1.0 and PE2.0 with 15 amplification cycles. The library preparation method does not retain strand-specific information. Libraries were sequenced on two separate runs of Illumina GAII machines, including one lane each of Col×LSequencing reads were aligned to the TAIR 9.0 version of the Arabidopsis reference genome (Col-0) using TopHat er resequencing data downloaded from TAIR  p1 and p2 using the Storer-Kim method www.r-project.org) 1p is defined as the Col portion for a given locus in Col×Ler crosses, and 2p is the portion of Col in Ler×Col crosses. For the embryo we tested if 1p = 2p = 0.5 and for the endosperm if 1p = 22p = 0.67, because the ratio of maternal to paternal genomes in the endosperm is 2∶1. As read coverage increases smaller and smaller degrees of imprinting can be detected. We therefore computed an imprinting factor to determine the magnitude of imprinting. For each locus we determined the 95% confidence interval around the Col/Ler read ratio from each sample. The imprinting factor is the low value of the high confidence interval divided by the high value of the low confidence interval for the reciprocal crosses. In essence, the imprinting factor is a variable where we have high confidence that the ratio of Col to Ler reads is at least that many times greater in one of the reciprocal hybrids than in the other reciprocal hybrid.For each locus in the embryo or endosperm we determined a p-value for the null hypothesis of no imprinting and corrected for multiple testing using the Benjamini-Hochberg method. Specifically, we followed the method of Wang er allele, independent of the direction of the cross. We computed the p-value for the observed cis-effect given the null hypothesis of no Col vs. Ler effect and corrected for multiple testing using the Benjamini-Hochberg method. The cis effect factor is computed in a similar manner to the imprinting factor.In addition to imprinting effects, biased expression patterns can result from strain-specific effects on gene expression. For example, a Col allele might be more highly expressed than a Let al (2008) et al (2010) th percentile read count across the two experiments, where loci with zero read counts in both experiments were excluded when computing the 75th percentile. This method has been shown to outperform total read count normalization cis effect factors.RPKM (reads per kb per million mapped reads) values were determined using the method of Mortazavi er embryo and endosperm samples was reverse transcribed with oligo dT using the Retroscript Kit (Ambion). qPCR was performed using a StepOne Plus qPCR machine and Fast SYBR Green reagent (Applied Biosystems). qPCR primers were designed using QuantPrime Quantitative PCR (qPCR) was performed on the embryo and endosperm cDNA samples and the Illumina libraries to confirm enrichment for genes preferentially expressed in the embryo and endosperm and to determine if the sequencing libraries faithfully represented the original cDNA population. For qPCR, DNase I (Invitrogen) treated RNA from Col×LSDG20 imprinting in embryos, oligo dT primed cDNA was created from the embryo RNA samples used to create the Illumina sequencing libraries and a set of independently isolated reciprocal F1 embryo RNA. SDG20 was amplified with primers MG581 (5′-GCTGACCAGCTTATCAAGCAAGG-3′) and MG586 (5′-CCTTCTCCAAATCAGTAGAGCCGCTA-3′) for 40 PCR cycles. RT-PCR products were gel purified, cloned into the pCR 2.1 TOPO vector (Invitrogen), and individual clones subjected to dideoxy sequencing. Col/Ler SNPs at TAIR9 chromosome 3 positions 941,455 (A to C) and 941,585 (G to C) indicated whether clones were derived from Col or Ler alleles.To assay AT3G03750/http://david.abcc.ncifcrf.gov/) er SNP and at least 15 informative reads. Categories with EASE scores (a modified Fisher's exact test used by DAVID) less than 0.01 are presented along with p-values obtained after correction for multiple testing by the Benjamini-Hochberg method.Functional annotation of the 165 maternally expressed imprinted genes and 43 paternally expressed imprinted genes was performed separately to test for enrichments in GO_FAT terms, KEGG pathways, or INTERPRO domains using DAVID Bioinformatics Resources version 6.7  or endosperm (AT4G21680 and AT1G49770/RGE1/ZOU) er vs. Ler×Col) had only minor effects on gene expression, with high correlations between embryo samples and between endosperm samples . Embryo and endosperm expression profiles were clearly distinct .We performed a genomic analysis of imprinted gene expression in er SNP. Data from reciprocal crosses also allows parent-of-origin effects to be distinguished from strain specific biases in gene expression. We used information on previously identified SNPs  r = 0.81 for embryo and r = 0.77 for endosperm). To determine a reasonable cutoff for removing genes likely to be affected by maternal seed coat contamination, we examined the difference between LCMD seed coat and endosperm expression for the 82 potential endosperm PEGs with a p-value less than 0.01 and Affmetryix expression data. Maternal seed coat contamination cannot result in false positive PEGs, only false negatives. The average difference in seed coat and endosperm expression was −1.1 for potential endosperm PEGs and 1.2 for potential MEGs . After these final filtering steps we identified 18 genes with biased expression in the embryo  and 208 genes in the endosperm  . Increasing sequencing depth could reduce false negatives.As read coverage increases it is possible to detect smaller and smaller degrees of imprinting with statistical significance. Genes with a large number of informative reads can have very low p-values, even if the parental bias is intuitively not very strong. For example, locus AT2G05990 has 15,636 informative reads, 37% of which are paternally derived . This gene is identified as paternally biased with a p-value of 3.65×10ocus see  and furt is 1.25 . We also43 PEGs)  and S7. d MYB3R2  [6]. TheAlthough all genes in our list pass the same statistical criteria, we consider the identification of PEGs more robust than the identification of MEGs. The MEG list is more likely to contain false positives than the PEG list because any contamination from maternally derived tissues will make genes appear more maternally expressed than expected. The only source of the paternal genome is from the products of fertilization, embryo and endosperm, and thus contaminating RNA from maternally derived tissues cannot create false positives, only false negatives.in situ analysis will be required to conclusively determine if these genes truly exhibit parent-of-origin specific expression in the embryo. One embryo gene, SDG20, was identified as a PEG, which could not be due to maternal contamination. We attempted to independently validate paternally biased expression of this gene by performing RT-PCR on the original RNA samples used to create the embryo libraries and on a set of independently isolated F1 RNA. After sequencing cloned RT-PCR products, we identified 15 Col clones and 11 Ler clones from the original Col×Ler F1 embryo sample and 17 Col clones and 7 Ler clones from the original Ler×Col F1 embryo sample. Independently isolated samples had 18 Col clones and 11 Ler clones in a Col×Ler cross, and the reciprocal indicated 14 Col clones and 14 Ler clones. Combining the data, in each F1 genotype 60% of the reads are derived from the Col allele, indicating a bias towards the Col allele independent of the parent of origin. The bias was also observed in dCAPS analysis of RT-PCR products (data not shown). Therefore, SDG20 is not imprinted in the embryo. We consider it likely that all of the 18 embryo genes are false positives.The data indicate that imprinting is primarily endosperm-specific at this stage of seed development. One embryo-imprinted locus has been identified in maize Most endosperm-imprinted genes (140 of 208) are more highly expressed in the endosperm than in the embryo , which iHDG9, MYB115, MYB77, MYB3R2, NF-YC12, and several zinc finger and leucine zipper genes. Additionally, MEGs were strongly enriched for genes involved in cell wall modification, particularly pectinesterases  biosynthesis, a zinc finger transcriptional repressor, STZ/ZAT10, that regulates JA biosynthesis genes, BR6OX, whose product generates the active form of brassinosteriods, and two genes involved in ethylene biosynthesis and response, EIN2 and MKK9. In addition, two auxin biosynthesis genes, YUC10 and TAA1, are paternally expressed imprinted genes  (previously identified regions of endosperm hypomethylation in the top 0.5% of differences) 5′ or 3′ . This is5′ or 3′ . The ass5′ or 3′  are asso5′ or 3′ . Overall5′ or 3′ .PHE1 and MEAet al.HDG3, HDG9, and SUVH7  are important for maintaining imprinted gene expression. These two mechanisms can act independently or in concert at a locus. The PcG group complex consisting of FIE/FIS2/MEA is required to maintain imprinted gene expression at several loci, including nd SUVH7 . We alsomea/fis1 , the PEGer/Col SNP and sufficient read coverage. The average distance between imprinted genes was not significantly different from the average distance between randomly selected informative genes. However, we identified 10 regions in the genome where two or three imprinted genes were within 10 kb of one another, including 6 instances in which adjacent genes were imprinted  mprinted . These met al.er reciprocal crosses. Using different statistical analyses and criteria, they identified 116 MEGs and 10 PEGs in the endosperm. Fourteen of the MEGs are in common with the 165 MEGs we identified, as are 6 PEGs , although almost all of these reads are paternal. We also analyzed their Col/Ler read counts for each gene using our statistical pipeline. The highest overlap between our set of imprinted genes was with their LCMD endosperm dataset. Applying our pipeline to their read counts, we find 137 imprinted genes in their dataset , more than half of which (n = 74) are in common with our set of 208 imprinted genes  (Hsieh e 6 PEGs . While t18 PEGs)  and S11.Arabidopsis thaliana endosperm. Our analysis identified dozens of new imprinted genes involved in transcriptional regulation, epigenetic processes, hormone biosynthesis and reception, and cell wall function. The function of most of these genes during seed development is unknown and the data represent a rich source for further understanding endosperm development, the mechanisms of gene imprinting, and the selection pressures driving its evolution.We have used high-throughput mRNA sequencing to identify genes imprinted in MEA, FWA, and PHERES1, did not arise in our analysis because of lack of SNPs or low expression. Furthermore, our list is specific to a particular stage of seed development, and we expect that different sets of imprinted genes are active at earlier stages. Moreover, it should be noted that our assay necessarily only reports on steady-state transcript levels, which could be impacted by a number of processes in addition to transcription itself, including maternal deposition of RNAs , transport of RNAs from other tissues, and transcript degradation. The most stringent test of imprinting is to show that transcription itself is differential between alleles While we have performed a genomic analysis of gene imprinting, it is important to emphasize that our list of imprinted genes is not comprehensive. Some known imprinted genes, like Our initial analysis of gene imprinting based on DNA methylation profiling discovered a subset of imprinted genes associated with TE-derived differentially methylated regions Arabidopsis thaliana genes exhibit parent-of-origin specific biased expression patterns? One possibility is that partial imprinting is an evolutionary echo of complete imprinting that existed at these genes when A. thaliana possessed a different mating system. A. thaliana is primarily self-fertilizing, with low but variable rates of outcrossing observed in the wild A. thaliana seeds . However, A. thaliana is estimated to have been self-fertilizing for a short amount of evolutionary time – perhaps only 400,000 years Striking features of our data are that most genes exhibit partial imprinting rather than strict monoallelic expression and that MEGs are more numerous than PEGs. The kinship theory of imprinting predicts that monoallelic expression  is the evolutionary stable strategy for genes in which the maternally and paternally derived alleles favor different optimal levels of expression A. thaliana will help test these ideas.An alternative, non mutually exclusive, possibility is that the partially imprinted genes do not reflect a record of past conflict but are instead imprinted as a form of gene dosage regulation. Many of the imprinted genes encode transcriptional regulators and chromatin modifiers – proteins that function in macromolecular complexes that can be dosage sensitive. But why would dosage regulation be subject to parent-of-origin effects? It may be that these genes are taking advantage of existing molecular differences already tied to one parent – namely demethylation of the maternal genome before fertilization. Because the presence or absence of DNA methylation can influence gene expression levels, demethylation provides a built-in mechanism of dosage regulation that is specific to the parent-of-origin. We expect that the parent-of-origin specific effects on gene expression are due to some combination of parental conflict, maternal-offspring coadaptation, and dosage regulation, with different evolutionary pressures possibly acting at different loci. Genomic analysis of imprinting in outcrossing relatives of Table S1Col/Ler SNPs used in this study.(TXT)Click here for additional data file.Table S2Gene expression in the endosperm.(XLS)Click here for additional data file.Table S3Gene expression in the embryo.(XLS)Click here for additional data file.Table S4Expression of transposable elements in endosperm.(XLS)Click here for additional data file.Table S5Expression of transposable elements in embryo.(XLS)Click here for additional data file.Table S6Endosperm imprinted genes.(XLS)Click here for additional data file.Table S7Embryo imprinted genes.(XLS)Click here for additional data file.Table S8Validation of candidate imprinted genes from Gehring et al. 2009.(XLS)Click here for additional data file.Table S9Subset of endosperm imprinted genes also identified as imprinted in Hsieh et al. 2011.(XLS)Click here for additional data file.Table S10Analysis of Hsieh et al. read count data for LCMD pure endosperm using the statistical pipeline described in this study.(XLS)Click here for additional data file.Table S11Genes identified as imprinted when Hsieh et al. LCMD pure endosperm read count data is analyzed using the statistical criteria of this study.(XLS)Click here for additional data file.Table S12qPCR primers used in this study.(XLS)Click here for additional data file.


By mass spectrometry analysis of mouse Cystic Fibrosis Transmembrane-conductance Regulator (mCFTR) expressed in yeast we have detected 21 phosphopeptides accounting for 22 potential phospho-residues, 12 of which could be unambiguously assigned. Most are conserved in human CFTR (hCFTR) and the majority cluster in the Regulatory Domain, lying within consensus sequences for PKA, as identified in previous mammalian studies. This validates our yeast expression model. A number of phospho-residues were novel and human conserved, notably mouse Ser670, Ser723, Ser737, and Thr1467, that all lie in acidic sequences, compatible with their phosphorylation by protein kinase CK2. Thr1467 is localized in the C-terminal tail, embedded in a functionally important and very acidic sequence (EETEEE) which displays an optimal consensus for protein kinase CK2. Herein, we show that Thr1467, homologous to human Thr1471 is readily phosphorylated by CK2. Indeed a 42 amino acid peptide encompassing the C-terminal segment of human CFTR is readily phosphorylated at Thr1471 with favorable kinetics (Km 1.7 µM) by CK2 holoenzyme, but neither by its isolated catalytic subunit nor by other acidophilic Ser/Thr kinases . Our finding that by treating CFTR expressing BHK cells with the very specific CK2 inhibitor CX4945, newly synthesized wild type CFTR (and even more its Phe508del mutant) accumulates more abundantly than in the absence of CK2 inhibitor, supports the conclusion that phosphorylation of CFTR by CK2 correlates with decreased stability of the protein. Cystic fibrosis (CF) is caused by mutations affecting a polytopic integral membrane protein, termed Cystic Fibrosis Transmembrane-conductance Regulator (CFTR) functioning as a phosphorylation stimulated, ATP-dependent anion channel. Pathogenic mutations of CFTR either impair its anion channel functions or make the protein unstable determining its premature degradation. This latter event is promoted by the commonest mutation of CFTR, a deletion of the amino acid residue phenylalanine 508 (Phe508delCFTR), which is found in 70–90% of CF patients. Phe508delCFTR is only partially functional when it is induced to traffic to the cell membrane In an attempt to disclose molecular events that may underlie deregulation of CFTR functionality and/or stability, attention has been focused on post-translational events, with special reference to phosphorylation. These studies clearly demonstrated that multiple phosphorylation of the regulatory domain of CFTR by PKA and probably also by other basophilic protein kinases such as PKC is essential to confer the full channel functionality to CFTR. The same residues phosphorylated in vitro by PKA Intriguingly however CFTR also includes several potential phospho-acceptor sites that display the consensus for the most pleiotropic acidophilic protein kinase, CK2  is abundantly expressed and where a methodology for the expression of CFTR by a protocol that overcomes proteolysis problems has been recently set up By MS analysis of CFTR expressed in yeast we have now been able to show that besides all the “canonical” PKA sites concentrated in the R domain (whose detection demonstrates the reliability of the “yeast” approach) we additionally observe that some novel phospho-residues previously never found in CFTR from animal cells, are present in mouse CFTR expressed in yeast. One of these is the best predicted site for CK2, Thr1467 that is conserved in human CFTR (Thr1471): this residue is also readily phosphorylated in vitro with favourable kinetics by CK2 holoenzyme once included in a recombinant fragment of human CFTR encompassing its C-terminal segment. This outcome is described in the present report together with pharmacological evidence supporting the view that CFTR phosphorylation by CK2 may correlate with accelerated degradation of the protein.33P]ATP (3000 Ci/mmol) as well as HRP (horseradish peroxidase)-conjugated antibodies were purchased from PerkinElmer. Mouse monoclonal anti-CFTR antibody (10B6.2) that recognizes a specific epitope on NBD1 domain was from CFTR folding consortium (www. cftrfolding.org). Complete protease inhibitor cocktail tablets were from Roche Diagnostic. Okadaic acid was purchased from Alexis Biochemicals. Methotrexate was from Santa Cruz Biotechnology. CX4945 was from SYN|thesis med chem.All chemicals, cell culture media, anti-β tubulin antibody, and phosphatase inhibitor cocktails 2 and 3 were from Sigma-Aldrich. ATP . The reaction was started by the addition of the indicated protein kinases. The reaction mixtures were incubated for 10 minutes at 37°C and stopped by the addition of 2× concentrated Tricine sample buffer  glycerol, 4% (w/v) SDS, 0.0025% (w/v) Coomassie Blue G, 0.0025% (w/v) Phenol Red and 105 mM DTT). Samples were separated on Tricine-SDS-PAGE following the Schägger’s protocol 33P incorporation was evaluated by gels autoradiography on PerkinElmer’s Cyclone Plus Storage Phosphor System. Initial rate data were analyzed with the program Prism .Peptides derived from human CFTR C-terminal region were phosphorylated by incubation in a 20-µl volume containing 50 mM Tris/HCl, pH 7.5, 10 mM MgClStructural model of the last 42 aminoacids of CFTR was built using ab initio Rosetta tool Protein-protein docking analysis was performed using two FFT-based docking software PIPER Molecular dynamics (MD) simulations of the final complex (parameterized with AMBER99) were performed with NAMD 2.8 Saccharomyces cerevisiae was subjected to tryptic digestion followed by TiO2 phosphopeptide enrichment prior to MS analysis as detailed in the Experimental section. From the MS spectra (Xenopus oocytes Full length mouse CFTR (mCFTR) expressed in  spectra  and S2 i spectra  leading  spectra . In a feSeveral phospho-residues identified by our analysis however were not previously reported to be phosphorylated in vivo: one or more are located in the N-terminal segment (Ser45 and/or Ser50) and at the end of the R domain (Ser808 and Ser811), most of which are conserved in hCFTR. Also conserved is Ser670 that lies at the end of the R domain, whose in vitro phosphorylation by both PKA Phospho-Thr1467, close to the C-terminus and sitting in a region which is 100% conserved in hCFTR (Thr1471), attracted our attention for three reasons: i) it conforms to the optimal sequence recognized by CK2, ii) it is adjacent to the PDZ interacting motif which is essential for interaction with AMP-activated protein kinase To corroborate the implication of CK2 as a phosphorylating agent of CFTR Thr1471 a 42 amino acid-long recombinant fragment encompassing the C-terminal segment of hCFTR was generated and tested for its phosphorylation by CK2. As shown in We also determined whether other acidophilic Ser/Thr protein kinases, besides CK2, are able to phosphorylate Thr1471. While protein kinases CK1 and the genuine Golgi apparatus casein kinase (GCK), recently identified with FAM20C protein Given the location of Thr1471 close to the PDZ-interacting motif that favors retention of CFTR within the cell by interacting with Golgi associated CAL  To provide evidence about the implication of CK2 in processes affecting the stability of CFTR and/or of its Phe508del mutant which is the commonest cause of Cystic Fibrosis, advantage has been taken of the most selective cell permeable CK2 inhibitor available, CX4945. In order to evaluate the efficacy of CX4945 on the level of de novo synthesized CFTR, protein synthesis was inhibited by two hours cell treatment with cycloheximide (CHX). Blockade was then removed by washing off CHX and newly synthesized CFTR was quantified after 2, 4, and 8 hrs incubation either in the absence or presence of CX4945. As shown in It can be concluded from the data of Expression of CFTR in yeast was primarily aimed at producing sufficient amount of the protein for structural studies Based on this supposition we wanted to see if CFTR expressed in a foreign environment like yeast, where CK2 (as well as PKA and many other important Ser/Thr protein kinases) are abundant, but where the homeostatic mechanism leading to CFTR degradation is lacking and proteolysis has been prevented by a number of devices, CFTR is phosphorylated, and at which residues, by CK2.Xenopus oocytes The study presented here led to the identification of 22 potential phospho-residues (12 of which were unambiguously assigned) in mouse CFTR expressed in yeast, all of which, with just a few exceptions, are conserved in human CFTR. Seven of these coincide with phospho-residues previously identified in human CFTR expressed in mammalian cells and/or To the best of our knowledge the other phospho-residues detected by us in CFTR were not reported before: one however (Ser670) was previously reported to undergo in vitro phosphorylation by both PKA At variance with the previously identified CFTR phospho-sites, which are almost exclusively located in the R domain and display the PKA consensus, several of the newly identified ones described here are located close to acidic residues which may suggest their targeting by CK2 and some are located outside the R domain: in particular one or two are located in the N-terminal segment, but could not be assigned with good confidence to individual positions see , though Our attention was mainly focused on phospho-Thr1471 (human numbering), since besides being highly conserved in human CFTR and displaying an optimal sequence for CK2 phosphorylation, it is adjacent to the PDZ interacting motif which is essential for interactions with the scaffold protein NHERF-1, anchoring CFTR to the cytoskeleton To get clues about the possible consequences of CFTR phosphorylation by CK2, advantage has been taken of the most selective CK2 inhibitor available to date, CX-4945 By treating CFTR expressing BHK cells with cycloheximide, in order to block protein synthesis and then restoring protein synthesis by washing away the inhibitor we observed that the re-appearance of CFTR reached higher levels if cells were incubated with the CK2 inhibitor CX-4945, as compared to control. This indicates that inhibition of CK2 results in larger accumulation of newly synthesized CFTR, consistent with diminished degradation of the protein. Interestingly such an effect is definitely more pronounced in the case of Phe508delCFTR with a two to three-fold increment of band B upon treatment with the CK2 inhibitor see , suggestAlthough additional work is needed in order to understand the significance of CFTR phosphorylation by CK2, with special reference to the enigmatic role of Thr1471, our data disclose the importance that a number of previously undetected phosphoacceptor sites may have in determining the stability and the functionality of CFTR. Our experiments also highlight the role of the master kinase CK2 in these processes, providing, from an empirical stand point, a new strategy to increase the yield of functional CFTR in CF cells, possibly by combining CK2 manipulation with usage of correctors and/or potentiators.Figure S1The sequence of mouse CFTR with all phosphopeptides identified is reported together with peptide identification confidence and phosphorylation site probability that are indicated with a color code. Table at the end of the file summarizes the data, by listing all identified phosphopeptides with their corresponding phosphorylation sites.(TIF)Click here for additional data file.Figure S2Annotated fragmentation spectra of the phosphopeptides identified in this study.(PDF)Click here for additional data file.Figure S3MS/MS spectrum of phosphopeptide EETEEEVQDTR from Human CFTR.(TIF)Click here for additional data file.Figure S4BHK cells expressing WT, WT/T1471A or Phe508delCFTR were exposed to either cycloheximide  or Brefeldin A . CFTR synthesis was blocked by two hours treatment with CHX as it is shown by the attenuation/disappearance of the ER-resident band B of CFTR. On the contrary the fully mature band C is not affected by CHX treatment due to its slower turnover. Conversely, when the escape from ER is prevented by using Brefeldin A, band B increases [a]. [a] Glozman R, Okiyoneda T, Mulvihill CM, Rini JM, Barriere H, Lukacs GL (2009) N-glycans are direct determinants of CFTR folding and stability in secretory and endocytic membrane traffic. J. Cell Biol. 184, 847–862.(TIF)Click here for additional data file.Table S1Phosphopeptides identified from mouse CFTR. For each peptide the following details are reported: aminoacid sequence, modifications, phosphorylation site/s, pRS score, pRS probability and pRS site probability, q-value, PEP score and number of missed-cleavages.(DOC)Click here for additional data file.Material S1(DOC)Click here for additional data file.


Atelomastix bamfordi, found on five of these Banded Iron Formations at two mitochondrial loci and 11 microsatellite loci. Strong phylogeographic structuring indicated the five populations became isolated during the Pleistocene, a period of intensifying aridity in this landscape, when it appears populations have been restricted to pockets of moist habitat provided by the formations. The pattern of reciprocal monophyly identified within the mtDNA and strong differentiation within the nuclear microsatellite data highlight the evolutionary significance of these divergent populations and we suggest the degree of differentiation warrants designation of each as a conservation unit.The Yilgarn Banded Iron Formations of Western Australia are topographical features that behave as terrestrial islands within the otherwise flat, semi-arid landscape. The formations are characterised by a high number of endemic species, some of which are distributed across multiple formations without inhabiting the intervening landscape. These species provide an ideal context for phylogeographic analysis, to investigate patterns of genetic variation at both spatial and temporal scales. We examined genetic variation in the spirostreptid millipede,  Islands provide a unique context for studying the evolutionary processes that drive species distributions. They are natural laboratories, allowing examination of processes of speciation, and patterns of colonisation, persistence, dispersal and extinction Inferring the evolutionary history of terrestrial island endemics can be difficult given that isolation is not always absolute, either spatially and temporally. Dispersal events can occur across valleys and lowlands for mountain-top endemics or over mountains for valley floor endemics, depending on the vagility of the organism and their environmental requirements Eucalyptus caesia, endemic to granite outcrop ‘islands’ of Western Australia. This species has high levels of genetic divergence with little geographic pattern to the distribution of chloroplast haplotypes, reflecting long-term isolation and persistence without connectivity and emphasising the role of genetic drift in this landscape Restricted gene flow between island populations over long periods of time, will result in substantial genetic divergence due to genetic drift and/or, local adaptation The Yilgarn Banded Iron Formations (BIFs) of south-western Australia are ancient, island-like rock outcrops that provide an ideal system in which to investigate island population dynamics. They border the transitional rainfall zone, a biogeographic region separating the high rainfall forest region from the arid interior and are characterised by high species endemism and richness Historically, the altitudinal relief of BIFs and their complex structure, including deep valleys and shaded gullies as well as exposed outcrops and talus slopes Atelomastix bamfordi, known to occur on five BIFs on the Yilgarn Plateau in Western Australia. Millipedes lack the waxy cuticle that protects other arthropods from desiccation and are reliant on moist environments for survival Atelomastix from the family Iulomorphidae, is a predominantly south-western Australian genus with many species distributed across the wetter parts of the southern and south-western coastline and mesic inland Ranges, including the Stirling and Porongorup Ranges 2) Atelomastix bamfordi is the most easterly occurring member of this genus. It inhabits moist habitats provided by BIFs, such as the base of rocky slopes, under large rocks and under dense leaf litter. The life cycle is probably annual with adult millipedes dying prior to, or during the harsh summer months (pers. comm. M Harvey). The species has been reported as abundant on the large Koolyanobbing Range We investigated phylogeographic pattern in the spirostreptid millipede A. bamfordi may have repeatedly come into contact through population expansion associated with historical climate change in this landscape. If this were the case, we would expect periods of gene exchange between neighbouring BIF populations to result in a pattern of isolation-by-distance, where proximate populations are more genetically related, or, if gene exchange has been extensive, to genetic homogeneity of populations Although unconnected today, BIF populations of A. bamfordi to investigate evolutionary relationships and patterns of genetic diversity and structure across BIF populations. Our goals were:We evaluated the genetic variation present in To determine patterns of genetic differentiation among the five known BIF populations and to use those data to infer the impact of historical processes on the distribution of millipedes across the landscape.To assess genetic diversity and structure to determine how limited dispersal capabilities affects structure within BIF millipede populations.To use patterns of genetic differentiation, both within and among BIFs to inform conservation and management of this species.A. bamfordi had been recorded from four of the Yilgarn BIFs: Koolyanobbing (KOO), Windarling (WIN), Mt Jackson (MTJ) and the Die Hardy Range (DH)  A. bamfordi . We also searched any water-gaining or densely vegetated sites between BIFs and although these recovered other millipedes from the genus Antichiropus, A. bamfordi was not found. Thus, to the best of our knowledge, A. bamfordi populations are restricted to BIF habitat and are not presently connected by populations in intervening habitat. The field study did not involve any endangered or protected species and none of the field sites from which we sampled (i.e. excluding KOO and WIN) required access permission, with the exception of Mt Jackson  and Mt Manning ). A license to take fauna for scientific purposes was obtained prior to sampling from DPaW. GPS coordinates of the sampling locations are provided in In total, 77 individuals from five BIFs were sequenced at two mitochondrial regions  and genotyped at 11 nuclear microsatellite loci. DNA was extracted from the legs or from the crushed heads of juvenile specimens stored in 100% ethanol using a Qiagen DNeasy Kit, or a salt-based DNA extraction protocol 5′TAAACTTCAGGGTGACCAAAAAATCA3′) 5′CGCCTGTTTTTCAAAAACAT3′) and 16Sbr (5′CCGGTTTGAACTCAGATCATGT3′) 2, 200 μM dNTP's, 0.4 μM of each forward and reverse primer, 0.25 μg of Bovine Serum Albumin (Fisher Biotech), 0.3 U of Platinum Taq DNA Polymerase (Invitrogen) and approximately 20 ng/μl DNA. The thermal cycler profile for both CO1 and 16 S involved an initial denaturation at 94°C for 3 min, followed by 35 cycles of 94°C for 30 sec, 46°C for 30 sec and 72°C for 30 sec followed by a final extension at 72°C for 2 min. PCR products were purified using an UltraClean PCR Cleanup kit  and the cleaned product was quantified using a Nanodrop (Nanodrop Technologies). Sequencing reactions (1/8) were carried out in 10 μl volumes containing 1 μl of Big Dye Terminator, 1.5 μl of 5× sequencing buffer, 1 μl of 3.2 pmol primer and approximately 40 ng of purified PCR product. Thermal cycling conditions for sequencing reactions involved an initial denaturation at 96°C for 2 min, followed by 25 cycles of 96°C for 10 sec, 46°C for 5 sec and 60°C for 4 min. Sequenced products were cleaned prior to electrophoresis using a standard ethanol precipitation method (Applied Biosystems). Products were sequenced on an Applied Biosystems 3730 capillary sequencer (Murdoch University).We sequenced two partial regions of the mitochondria, the Cytochrome Oxidase Subunit 1 (CO1) and the large Ribosomal Subunit 16 S. For amplification of CO1 we used the Folmer primers LCO1490 (5′GGTCAACAAATCATAAAGATATTGG3′) and HCO2198 , and nucleotide divergence (Jukes and Cantor) between populations were calculated in DnaSP v. 5.10 To test for isolation by distance within BIF populations using the combined sequence data, we conducted mantel tests in R using the Vegan package A. bamfordi. This was confirmed with all populations showing significant deviation from Hardy-Weinberg equilibrium, with the exception of Windarling, where individuals had been sampled in close proximity to one another. Rarefied allelic richness provided a better estimator of genetic diversity, given the sampling, and was calculated in HP-Rare v 1.0 Genotyping of the 11 microsatellite markers was carried out according to Nistelberger et al. C) To further reconstruct the phylogenetic relationships of populations using microsatellite data we generated Cavalli-Sforza chord distances . The variation was composed of 54 transitions, eight transversions and two multistate substitutions. The 16 S region yielded 24 haplotypes from the 77 individuals sequenced and included 21 transitions, one transversion and one single base pair indel. Combining the two regions resulted in a total, aligned sequence length of 1114 bp and 48 haplotypes. The most prevalent haplotype (Hap 47 WIN) had a frequency of 23%, followed by Hap 35 (MTJ) and Hap 39 (HA) with frequencies of 8%, and Hap 33 (MTJ) and Hap 40 (HA) with 6%. The remaining haplotypes were only found in one or two individuals. Measures of genetic diversity were consistently lowest in the Windarling population and highest in Koolyanobbing. Haplotype diversity ranged from 0.295 (WIN) to 0.986 (KOO) with an average of 0.794. Nucleotide diversity ranged from 0.06% (WIN) to 8.5% (KOO) with an average of 4%. No populations departed from the neutral model of evolution except for Windarling (D = −1.775) . MeasureThere was a large and highly significant degree of genetic structuring of the mitochondrial genetic diversity, with most variation (AMOVA) occurring among BIF populations (71.56%) rather than within (28.4%). The greatest genetic divergence occurred between the Mt Jackson and Helena Aurora populations (approx. 40 km apart) and the least between the two most spatially isolated populations, Koolyanobbing and Die Hardy (approx. 100 km apart) , Fig. 1.Microsatellite genetic diversity was low, both within and amongst BIF populations. Rarefied allelic richness ranged from 1.89 (HA) to 3.28 (KOO) with an average of 2.47. There was a highly significant degree of structuring of the microsatellite diversity with the majority of variation (68.11%) maintained within populations, compared to (31.89%) among populations.A. bamfordi, with minimal sharing of CO1 and 16 S haplotypes amongst individuals within BIF populations and no sharing of haplotypes between populations. Bayesian analysis identified five genetic clades in which populations were reciprocally monophyletic, with the exception of Koolyanobbing and Helena Aurora for which some samples were paraphyletic  or gene flow (migration). The three models with the highest AIC support all indicated there was no historical migration between the two populations .Mantel tests of pairwise sequence differences against geographic distance indicated significant isolation by distance within the Koolyanobbing (r = 0.740 p = 0.0001), Mt Jackson (r = 0.225 p = 0.0375) and Helena Aurora (r = 0.399 p = 0.0025) populations.Bayesian cluster analysis identified four genetic clusters in the microsatellite data. The first corresponded to the Die Hardy and Mt Jackson populations, and the remaining clusters to each of the three other populations  . FurtherA. bamfordi has revealed strong genetic differentiation between BIF populations, with mtDNA genetic lineages corresponding to each of the five BIF populations except for six individuals from Koolyanobbing that were paraphyletic with the Helena Aurora Range due to incomplete lineage sorting. Microsatellite data were broadly consistent with this result, identifying four genetic clusters, the first of which was further broken down into individual populations.Phylogeographic analysis of A. bamfordi, these studies emphasise the role of genetic drift in driving population differentiation. For example, mitochondrial and nuclear sequence data revealed extraordinarily strong phylogeographic structure in populations of trapdoor spiders (Moggridgea tingle) in south-western Australia, often situated just kilometres apart. Molecular dating suggested these populations became isolated in the mesic valleys and uplands of the Porongorup and Stirling Ranges following the onset of aridity in the late Miocene A. bamfordi, reciprocally monophyletic lineages across three geographic locations highlighted the reproductive isolation of M. tingle populations but the pattern emphasized a more ancient division, with monophyly observed in both mtDNA and the more slowly evolving nuclear genome Geocrinia lutea and G. rosea, also described patterns of extremely low dispersal and long-term isolation between habitat ‘islands’, with divergence attributed to isolation via range changes resulting from climate change rather than geographical barriers Habronattus pugillis occupies woodland habitat at altitudes above 1400 m but is absent from the lowlands. Using mtDNA, Masta A. bamfordi, smaller populations of H. pugillis formed monophyletic clades whereas larger Ranges showed evidence of paraphyly. Like A. bamfordi, this was ascribed to incomplete lineage sorting rather than resulting from migration The lack of correlation between genetic divergence and geographic distance, with the two most spatially isolated populations showing the least genetic divergence, suggests there has been little, to no connectivity of BIF populations following their separation. This result was emphasized in the Bayesian analysis which showed high support for genetic clades but poor support for the relationships between clades . It is aA. bamfordi clades at 0.7 to 1.1 million years ago during the Pleistocene. The timing coincides with known increases in aridity in the transitional rainfall zone (TRZ) due to climatic fluctuations associated with Milankovitch cycles during this period Molecular dating with the arthropod clock rate has placed the separation of The process of genetic drift acting independently on each BIF population is reflected in different patterns of genetic divergence and diversity seen in the mtDNA data. The smaller populations, Mt Jackson and Windarling, and the larger population Helena Aurora, which had less suitable millipede habitat, showed lower genetic diversity and higher levels of genetic divergence than the larger populations (KOO and DH). These three BIFs have probably harboured historically small effective population sizes, owing to the limited availability of moist sites, increasing their susceptibility to forces of genetic drift driving divergence Rapidly evolving microsatellite markers can provide a more contemporary view of BIF population dynamics. The information we gained from these markers was limited by a lack of Hardy-Weinberg equilibrium within our BIF populations yet we still identified patterns in genetic diversity that appear to support the impact of genetic drift on populations: lower genetic diversity in BIF's likely to have had small effective population sizes . Signals of isolation by distance (IBD) were seen in some of the BIF populations , but these were weak and likely to be heavily influenced by limited sample sizes. The small pairwise distances between samples at Windarling explains the lack of IBD seen in this BIF, but we were surprised to see evidence of IBD in the Helena Aurora and Mt Jackson individuals. Here, we expected limited gene flow to result in a pattern of ‘islands within islands’, where significant isolation has resulted in genetically divergent populations within BIFs that do not correlate with geographic distance. An increased number of samples from more locations would be required to determine whether IBD is really occurring at these spatial scales.The evolutionary significance of geographically isolated genetic lineages has long been recognised and conservation biologists have attempted to formalise this by delineating conservation units within a species such as Evolutionary Significant Units (ESUs) and Management Units (MUs) st century indicate increasing temperature, decreasing rainfall and a greater incidence of fire Although it is difficult to predict future climate change at such fine scales, broad-scale models for this region over the 21This is the first phylogeographic study conducted on a species inhabiting multiple Banded Iron Formations in this landscape and provides evidence that the disjunct populations have experienced long-term isolation following their separation during the Pleistocene. The divergence of populations during this period coincides with increases in aridity and emphasizes the role of climatic instability along with topographic features in driving differentiation and ultimately speciation in this landscape. We consider the strong phylogeographic structure and evidence of restricted gene flow between BIF populations warrant the designation of all five populations as distinct conservation units.Table S1Specimen information, including spatial coordinates from UTM zone 50 J and haplotype number based on combined CO1 and 16 S mtDNA data. Samples from Windarling and Kooylanobbing (bold) are from specimens lodged at the Western Australian Museum, registration numbers shown (WAM_T).(DOCX)Click here for additional data file.Table S2The three, best-fit models determined in IMa2 for tests of migration versus isolation between the paraphyletic populations Koolyanobbing and Helena Aurora. Results are based on mtDNA variation.(DOCX)Click here for additional data file.


There is no proven medical approach to attenuating expansion and rupture of abdominal aortic aneurysms (AAAs). One approach that is currently being investigated is the use of doxycycline. Despite being primarily used as an antimicrobial drug, doxycycline has been proposed to function in reducing AAA expansion. Doxycycline is effective in reducing the formation in the most commonly used mouse models of AAAs when administered prior to the initiation of the disease. The purpose of the current study was to determine the effects of doxycycline on established AAAs when it was administered at a dose that produces therapeutic serum concentrations.ex vivo measurements of maximal width of suprarenal aortas and AAA volumes. Also, doxycycline did not reduce AAA rupture. Medial and adventitial remodeling was not overtly changed by doxycycline as determined by immunostaining and histological staining.LDL receptor −/− male mice fed a saturated-fat supplemented diet were infused with AngII  via mini-osmotic pumps for 28 days. Upon verification of AAA formation by noninvasive high frequency ultrasonography, mice were stratified based on aortic lumen diameters, and continuously infused with AngII while also administered either vehicle or doxycycline (100 mg/kg/day) in drinking water for 56 days. Administration of doxycycline led to serum drug concentrations of 2.3±0.6 µg/ml. Doxycycline administration had no effect on serum cholesterol concentrations and systolic blood pressures. Doxycycline administration did not prevent progressive aortic dilation as determined by temporal measurements of lumen dimensions using high frequency ultrasound. This lack of effect on AAA regression and progression was confirmed at the termination of the study by Doxycycline administration did not influence AngII-induced AAA progression and aortic rupture when administered to mice with established AAAs. Abdominal aortic aneurysms (AAAs) represent a progressive disease state with a life-threatening but unpredictable risk for rupture MMPs are a family of zinc-dependent endopeptidases that are expressed in many cell types. MMPs have been linked to the development of AAAs due to their ability to degrade many extracellular matrix proteins, including elastin and collagen. This associative link of MMPs to AAAs has been enhanced by the detection of many different MMPs in human and experimental aneurysmal tissues, including MMP-1, -2, -3, -7, -8, -9, -12, -13, and MT1-MMP All three of the commonly used mouse AAA models . Mice were housed under barrier conditions and fed normal rodent laboratory diet and water ad libitum. One week prior to mini-osmotic pump implantation, all mice were fed a diet containing milk fat (21% wt/wt) and cholesterol .Mini-osmotic pumps  were implanted subcutaneously to deliver AngII  as described previously Lumen diameters of suprarenal aortas were measured using a Vevo 660 ultrasound imaging system in a real-time pattern as described previously Systolic blood pressures were measured on conscious mice using noninvasive tail-cuff systems  as described previously Serum cholesterol concentrations were determined using an enzymatic assay kit  as described previously During termination, aortas were excised after pressure perfusion at 100 mmHg with 10% neutrally buffered formalin and injected with 3% (wt/vol) agarose to maintain patency. AAAs were quantified by measuring ex vivo maximal diameter of suprarenal aortas using Image-Pro Plus software  Abdominal aortas containing AAAs were serially cross-sectioned (10 µm thick/section) from the proximal to the distal as described previously t test for normally and equally distributed data and Mann-Whitney Rank Sum analysis for data having failed either normality or equal variance test. Weekly body weight, systolic blood pressure, and aortic diameters measured at selected time points with ultrasound were analyzed using two way repeated measures ANOVA. Aortic rupture rate during prolonged AngII infusion (Days 28–84) was compared between the two groups (Vehicle versus Doxycycline) using LogRank survival analysis. A P<0.05 was considered to be significant.Data are presented as means ± standard error of means (SEM). SigmaPlot version 12  was used for statistical analyses. Two-group comparisons were performed using Student's All mouse studies were performed with approval of the University of Kentucky Institutional Animal Care and Use Committee (IACUC protocol number: 2006-0009).Forty-one male LDL receptor −/− mice were infused with AngII  for 28 days before these mice were administered either the vehicle or doxycycline. During the 28-day infusion with AngII, 9 mice (22%) died of aortic rupture. AAA formation was confirmed in 25 of the remaining 32 mice (78%) by ultrasound at day 24 of AngII infusion. Subsequently, mice with established AAAs (N = 25) were stratified to receive vehicle (N = 11) or doxycycline (N = 14) 28 days after AngII infusion. Doxycycline given in drinking water was well tolerated as determined by daily observation and body weight measurements on a weekly base. Oral administration of doxycycline at a dose of 100 mg/kg/day led to serum drug concentrations of 2.3±0.6 µg/ml as measured using reverse-phase high performance liquid chromatography . In miceProtracted AngII infusion led to progressive luminal expansion of suprarenal aortas as monitored by ultrasonography , which wPathologies of AngII-induced AAAs in advanced stages are highly heterogeneous, exhibiting complex features and differing markedly along the length of a single aneurysm Doxycycline suppresses formation of experimental AAAs as demonstrated in both rat and mouse models While doxycycline is a widely used antibiotic, it is also a well-recognized broad-spectrum inhibitor of MMPs. It has been reported that doxycycline reduces experimental AAAs via inhibiting MMP activation AAA diameter is the most commonly used parameter to monitor the progression of AAAs and is also used to represent the risk for aortic rupture in patients Although many studies have demonstrated that some drugs and genetic deletions ameliorate the initiation of AAAs The effects of doxycycline on AAAs have previously been reported in humans. A small clinical trial involving 32 patients received either placebo or doxycycline (150 mg daily) for 3 months and AAA diameter was followed for 18 months In conclusion, although doxycycline prevents the initiation of AngII-induced AAAs, it does not retard or obviate the progression of AAAs, or prevent rupture once AAAs have established. While MMPs may play a divergent role in the initiative and progressive stages of AAAs, further studies are necessary to define the molecular mechanisms that are responsible for the progression of AAAs before effective therapeutic strategies may be explored for established AAAs.


Linaria with special emphasis on the Mediterranean sect. Supinae (44 species). We revealed extensive highly supported incongruence among two nuclear  and two plastid regions . Coalescent simulations, a hybrid detection test and species tree inference in *BEAST revealed that incomplete lineage sorting and hybridization may both be responsible for the incongruent pattern observed. Additionally, we present a multilabelled *BEAST species tree as an alternative approach that allows the possibility of observing multiple placements in the species tree for the same taxa. That permitted the incorporation of processes such as hybridization within the tree while not violating the assumptions of the *BEAST model. This methodology is presented as a functional tool to disclose the evolutionary history of species complexes that have experienced both hybridization and incomplete lineage sorting. The drastic climatic events that have occurred in the Mediterranean since the late Miocene, including the Quaternary-type climatic oscillations, may have made both processes highly recurrent in the Mediterranean flora.We examined the phylogenetic history of  Besides this, species complexes that underwent rapid speciation also represent a major challenge for molecular systematics. In those groups species relationships could be obscured by the ancestral polymorphisms retained through speciation events as a consequence of incomplete lineage sorting Both polyploid and homoploid hybrid speciation might represent a large fraction of the source of plant biodiversity on Earth Linaria Mill.) constitute the largest genus within the snapdragon lineage (tribe Antirrhineae). Linaria comprises c.150 species that are widely distributed in the Palearctic region, but the genus is most diverse in the Mediterranean basin. The origin of the genus has been placed in the Miocene Linaria has been suggested based on nrDNA (ITS) sequences of eight species representing all sections Linaria have been proposed Linaria, Speciosae, Diffusae, Supinae, Pelisserianae, Versicolores and Macrocentrum) Supinae (Benth.) Wetts. (hereafter Supinae) is a clear example of the systematic complexity within Linaria because of the disagreement in taxonomic treatments (Supinae comprises 44 diploid (2n = 12) Supinae species are distributed in the temperate regions of Europe, northern Africa and western Asia (circum-Mediterranean distribution), with the highest diversity found in the Iberian Peninsula (40 species) Toadflaxes , Supinae species (13 successful crosses of 20 assayed), followed by clearly lower values in inter-sectional crosses (four successful crosses of 14) Supinae species is then suggested. Despite this, external barriers such as allopatry do exist at the present time within Supinae as few species have overlapping distributions. However, such geographical barriers may have not existed during glaciations.In Linaria may affect phylogenetic reconstruction in this genus. Nonetheless, incomplete lineage sorting cannot be discarded as a cause of phylogenetic incongruence. Both processes can be difficult to distinguish, but may also occur simultaneously UAGrpl32-trnL and trnS-trnG) regions are herein sequenced for Linaria, with special emphasis in Supinae species. Our aims are: (i) to test for the presence of reticulation signals by simulations under the coalescent model using the method of Maureira-Butler et al.Supinae by exploring the presence of incomplete lineage sorting and/or reticulation events that may have occurred during the course of the evolution of this plant group.The high chance for hybridization in UAGrpl32-trnL, trnS-trnG) Linaria species plus one individual of Antirrhinum and one individual of Chaenorhinum. In particular, we used one species of sect. Macrocentrum , three species of sect. Versicolores , five species of sect. Linaria , four species of sect. Speciosae , seven species of sect. Diffusae  and 24 of the 44 species of section SupinaeLinaria verticillata group” and the “Linaria alpina group” L. almijarensis Campo & Amo Individuals were collected in the field and dried in silica gel or obtained from herbaria  . Total gL. bubanii) ITS haplotypes were not inferred as sister (or very closely related) sequences in the gene trees. As the phase posterior probability for this individual was low (0.41), we empirically confirmed the L. bubanii ITS haplotypes by sequencing the PCR product using allele-specific primers as described in Scheen et al.More than one allele was found in both AGT1 and ITS in Sanger sequenced PCR amplicons. To decipher these, we first estimated the gametic phases of the sequences using Arlequin 3.5.1.2 Recombination was tested within ITS and AGT1 datasets using RDP 3.44 The haplotype sequences obtained from the three datasets  were analyzed by Bayesian Inference in MrBayes 3.1.2 Antirrhinum and Linaria (13.33–27.32 Ma) from a previous estimate obtained in a relaxed molecular-clock analysis of tribe Antirrhineae . This analysis was in turn calibrated with five Lamiales fossils and a divergence time between Oleaceae and Antirrhineae modeled as a normal distribution with mean = 74 Ma and Std = 2.5 Ma, on the basis of a relaxed molecular clock analysis of angiosperms Linaria clade to estimate the dates of the internal nodes with a penalized likelihood procedure implemented in r8s 1.71 k) was done using increments of k of 0.1, from k = −3 to 3, repeated for two trees from the stable posterior distribution of each gene; the smoothing values of both trees were very similar so we used the value with lower χ2 error. After cross-validation we set the smoothing parameter to 1.5 for ITS, 3.2 for AGT1 and 0 for cpDNA and rate smoothed 20 trees drawn from the posterior distribution after burn-in to obtain the chronograms that were used in the coalescent simulations.For time calibration, we used the divergence time between et al. We used simulations under the coalescent model following Maureira-Butler e) used in the coalescent simulations were derived from cpDNA haplotypes and obtained via θw = 2µe,N with theta (θw) and mutation rate per generation (µ) taken from data of three Linaria species with contrasting range sizes  , L. elegans (endemic to northern Iberia) and L. simplex (distributed across the Mediterranean basin). The effect of Ne estimates in the coalescent simulations was explored by repeating the set of simulations using the three Ne values separately (see Effective population size estimates (Nting Ne) : L. glace values used (see e. The nine replications (three datasets x three Ne) alleviated the non-reproducible effect of taxon exclusion due to the stochastic nature of simulations. The last step was to average the nine independent estimates obtained for each analyzed taxon.The detection of potential hybrids was addressed by examining the effect of taxon deletion on the observed and base line distributions. Theoretically, the potential hybrids detected by the test were the set of individuals that, after exclusion, retrieved overlapping observed distributions (pairwise tree-to-tree distances within their 95% HPD) and base line distributions , thus the null hypothesis of incomplete lineage sorting alone was no longer rejected. Here, this approach was difficult to apply as the results were very dependent on the Nused see . We idenSupinae. This was done to assess whether the incongruence  was exclusively explained by hybridization (as putative hybrids were excluded) and inference limitations, or whether additional processes generated real gene tree differences (in this case incomplete lineage sorting). In order to calculate support for the monophyly of Supinae we used two approaches: (i) the Shimodaira and Hasegawa 1H vs. H0) −2 to −6 as positive evidence against 1 in favor of HH0; 2xlnBF(1H vs. H0) −6 to −10 as strong evidence against 1 in favor of HH0; and 2xlnBF(1H vs. H0) <−10 as very strong evidence against 1 in favor of HH0We used AGT1 and cpDNA datasets (one haplotype per sequence) with hybrids excluded to test support for the monophyly of UAGrpl32-trnL and trnS-trnG) sequences. We used Sutton’s species delimitation L. almijarensis Campo & Amo Linaria and Antirrhinum was constrained to 20 Ma ±4 as a normal distribution, following date estimates obtained for the tribe Antirrhineae . A Birth-Death process −4-5×10−2 and 1×10−4-1×10−2 substitutions per site per Ma (s/s/Ma) for the nuclear loci and the plastid locus respectively. These rate constraints include previous estimates for herbaceous plant ITS rates (1.7–8.3×10−3 s/s/Ma) −3 s/s/Ma) Gossypium genes, 3.5–7.3×10−3 s/s/Ma, −3 s/s/Ma, After excluding potential hybrids (to not violate the species tree model assumptions), we used the allelic data  and (ii) highly differentiated alleles have not been obtained in the Arlequin analyses (excluding L. bubanii), being all sister or closely-related in allelic-gene trees, we then considered that the two ITS haplotypes detected by Arlequin were good representatives of the existing ITS alleles per sample.The Arlequin analysis gave us the two most probable haplotypes from the unphased genotypes of AGT1 and ITS sequences. For AGT1, we obtained haplotypes of 50 individuals with posterior probabilities (PP) above 0.95 and haplotypes of four individuals with PP below 0.95. For ITS, we obtained haplotypes of 34 individuals with PP above 0.95 and haplotypes of 20 individuals with PP below 0.95. The AGT1 phased data retrieved for the four individuals with low PP were empirically confirmed by amplicon cloning, recovering exactly the same allelic data that Arlequin inferred. As ITS is a multi-copy locus marker, there would be more than two copies for each unphased ITS genotype. This may have affected the haplotype detection, thus giving low support for the ITS haplotypes obtained. But (i) as one haplotype with low probability and differential position in the ITS allele-tree has been confirmed empirically (−2) but when contrasting the UPGMA trees of the recombinant and non-recombinant regions it showed almost the same topology with both potential parents separated in the tree from the potential recombinants. Additionally, evidence for recombination was not considered convincing if it only was detected by a single method as done in Poke et al.Recombination could not be detected in ITS by any of the five methods used. AGT1 showed one recombination event affecting several sequences that was detected by SiScan . In ITS, relationships within Supinae were not clearly related to morphological features  Ne estimates , the pairwise distances of gene trees lay inside the base line distribution of ITS and AGT1 genes , only one individual needed to be removed in order to retrieve overlapping baseline and observed distributions (not shown), and therefore only one potential hybrid could be considered robustly detected. In contrast, when using simulations with the smallest Ne values , even after removing all the individuals with incongruent positions, we still had non-overlapping distributions (not shown), and consequently all species with incongruent positions (17 spp.) were identified as potential hybrids. Therefore, our Ne estimates showed all possible scenarios: (i) gene tree incongruence is explained by incomplete lineage sorting alone (L. simplex Ne), (ii) gene tree incongruence is explained by both incomplete lineage sorting and hybridization (L. elegans Ne) and (iii) gene tree incongruence is explained by hybridization alone . These results clearly illustrated the high dependence on Ne estimates in order to obtain the exact number of individuals of hybrid origin. We assumed that a reliable number of potential hybrids lay between the two extreme values obtained in (ii) and (iii).When using simulations obtained with medium NThe effect of the deletion of each incongruent individual on both the observed and base line distributions is shown in Supinae for these genes was statistically rejected. The BF test  sect. Versicolores (1 PP), (ii) four species of sect. Diffusae (1 PP), (iii) a group formed by: three species of sect. Diffusae, four species of sect. Speciosae and five species of sect. Linaria (0.9 PP); and (iv) all sect. Supinae species (1 PP). Therefore sect. Supinae was retrieved as a monophyletic group with high support and was divided in three clades: one clade was represented by three annual species  with small corollas (2.5–9 mm) and a thick-wide seed wing . A second clade was represented by five annual or perennial species  with medium-sized corollas (6–18 mm) and a thick-wide seed wing or narrow wing  . The third clade contained eight perennial species  with large corollas (16–31 mm) and a membranous-wide seed wing    retrieveSup) see .L. bubanii position in the ITS tree and *BEAST species tree); from the AGT1 dataset we detected deep coalescence at medium depth branches  and at deeper branches ; in cpDNA we also detected deep coalescence at the deepest branches .The * BEAST species tree detected that incomplete lineage sorting has affected all gene trees analyzed. In the ITS dataset we detected deep coalescence at medium depth branches (see Supinae was placed in the late Pliocene-early Pleistocene (0.87–3.28 Ma), the TMRCA of ssArv was located in the middle-late Pleistocene (Ionian-Tarantian) (0.08–0.72 Ma), the TMRCA of ssSax in the early-middle Pleistocene  (0.39–2.08 Ma) and the TMRCA of ssSup in the early-middle Pleistocene  (0.31–1.58 Ma) (see The time to the most recent common ancestor (TMRCA) of  Ma) see .ssSup) and a clade with moderate support  within Supinae. Out of ten reticulation events that have been presumed to occur, one was produced within the ssSup clade, six within the ssSax+ssArv clade and three between these two clades. One of the six potential hybridization events within ssSax+ssArv clade is reflected in L. tursica, a species with morphological traits typical from both ssSax and ssArv clades , the variation generated by incomplete lineage sorting alone could explain the totality of incongruence observed between genes . Hence, the results of Maureira-Butler’s test suggested that incongruence among genes was difficult to explain by incomplete lineage sorting alone, indicating that hybridization may also account for the gene tree inconsistency. However, the exact number and identity of individuals that may have hybrid histories is not clearly established here, because of the sensitivity of the test to Ne estimation. We consider, instead, that the test has provided a probable set of individuals that may adversely affect the *BEAST analysis and that a cautious approach  is preferred here, rather than risking a spurious species tree inference.Here, we performed simulations under coalescence following the method of Maureira-Butler ee genes , whereasen genes . We consThe hybrid detection test  and the Linaria *BEAST species tree retrieved three well supported clades that agreed with previous classifications  four species of Sect. Diffusae and (iii) Sect. Supinae. It also retrieved a group that was incongruent with previous taxonomic treatments. This latter group contained three species of Sect. Diffusae, four species of Sect. Speciosae and five species of Sect. Linaria. In this analysis Supinae was monophyletic, as found in the ITS phylogeny. Furthermore, Supinae was divided into three morphologically-based subclades consistent with life-form, corolla size and seed wing shape (Supinae (ssSup), subsect. Arvenses (ssArv) and subsect. Saxatile (ssSax). These results are strikingly consistent with some earlier hypotheses, despite the incongruence observed among gene trees. ssSup contained eight species that were grouped together in several previous morphological classifications, ssArv contained three species that were also previously grouped in a taxonomical entity, whereas ssSax contained five species that were historically placed in several distinct taxonomic groups   as an independent section and divided Supinae in three subsections according to life form and seed wing shape: (i) subsect. Supinae (ssSup): perennial plants with membranous seed wings, (ii) subsect. Amethystea: annual plants with thick seed wings and (iii) subsect. Saxatile: annual or perennial plants with somewhat thin wings.The ications : (i) Secng shape , as founSupinae. This is supported by the fact that the species with very low investment in flower structures  are all self-compatible, whereas species with a high investment in flower formation  are all self-incompatible, mainly pollinated by large bees and with low pollinator diversity . Geography appears to have played a role in structuring the diversity within Supinae as the diversity of ssSax is located in the northern part of the Iberian Peninsula (three out of the five species are northern Iberian endemics), whereas the diversity of ssSup is located in southern Iberia (five out of eight species are southern Iberian endemics). The timing of divergence of the three subclades  excluding L. chalepensis (2n = 24)). Despite speciation via homoploid hybridization has been historically hard to detect  Supinae.We found that historical hybridization has been likely during the course of  sorting . Simple ssSup lineage, three between two distant parental lineages (ssSax+ssArv and ssSup) and six within the ssSax+ssArv lineage. Crosses between the two distant parental lineages retrieved in the analysis (ssSax+ssArv and ssSup) were also supported by morphology, given that those three taxa  presented morphological key traits from both clades . Taking into account the effect of low phylogenetic resolution that obscured the detection of ages in parental lineages (thus considering the maximum age of hybridization at deeper nodes), the present results lead us to affirm that all potential hybridization events detected but one may have occurred during the Pleistocene climatic oscillations. During the Quaternary, hybrid zones were established in contact zones  of interglacial northward colonization routes from the temperate regions of Europe Linaria and many other southern European plant groups  suggested that ancestral populations may have not been extremely large (see Linaria has diversified since the late Miocene-early Pliocene (3.57–12.14 Ma)  Linaria.Several studies have claimed incomplete lineage sorting as a major cause of gene tree incongruence and non-monophyly in Mediterranean plants . FailureThe basis underlying phylogenetic incongruence may vary depending on the plant group under study, but the flora of the Mediterranean is formed, in part, by many genera that similarly display numerous species generated in short periods of time that also may have suffered secondary contacts in short term cycles (20.000–100.000 yr.). In these groups incomplete lineage sorting and hybridization appear to be the rule rather than the exception.Figure S1Total evidence analysis. The 50% majority-rule consensus tree obtained in the Bayesian analysis of the concatenated ITS, AGT1 and cpDNA datasets. Numbers above branches are Bayesian posterior probabilities. Colors represent the systematic nomenclature for Supinae as suggested in this paper (see aper see . Species(TIF)Click here for additional data file.Table S1List of taxa included with localities, collector’s numbers and Genbank accession numbers.(DOCX)Click here for additional data file.Table S2e) used in the coalescent simulations.Effective population size estimates (N(DOCX)Click here for additional data file.Table S3Assignment of genes to label 1 (L1) or label 2 (L2) in the multilabelled species tree analysis .(DOCX)Click here for additional data file.Methods S1Supplemental methods.(DOCX)Click here for additional data file.