Corvus corax) have admixed mosaic genomes formed by the fusion of non-sister lineages  that diverged ~1.5 million years ago. Phylogenomic analyses and concordant patterns of geographic structuring in mtDNA, genome-wide SNPs and nuclear introns demonstrate long-term admixture and random interbreeding between the non-sister lineages. In contrast, our genomic data support reproductive isolation between Common Ravens and Chihuahuan Ravens (C. cryptoleucus) despite extensive geographic overlap and a sister relationship between Chihuahuan Ravens and the California lineage. These data suggest that the Common Raven genome was formed by secondary lineage fusion and most likely represents a case of ancient speciation reversal that occurred without anthropogenic causes.Many species, including humans, have emerged via complex reticulate processes involving hybridisation. Under certain circumstances, hybridisation can cause distinct lineages to collapse into a single lineage with an admixed mosaic genome. Most known cases of such ‘speciation reversal’ or ‘lineage fusion’ involve recently diverged lineages and anthropogenic perturbation. Here, we show that in western North America, Common Ravens ( Corvus corax) was formed by the ancient fusion of two non-sister lineages of ravens.Speciation reversal is known mainly from recently diverged lineages that have come into secondary contact following anthropogenic disturbance. Here, Kearns et al. use genomic and phylogenomic analyses to show that the Common Raven ( Instead, speciation can follow a complex reticulate process in which hybridisation can create, reinforce or dissolve distinctiveness at all stages of the speciation continuum12. Most empirical examples of reticulate histories involve secondary contact after some amount of reproductive isolation has evolved. In such cases, introgressive hybridisation is often limited to small fragments of the genome, involves only a few individuals or is restricted to narrow hybrid zones11. However, if reproductive isolation fails to evolve in allopatry or erodes upon secondary contact, then hybridisation can result in the collapse of formerly distinct lineages or species into a single hybrid lineage with an admixed mosaic genome17. This reticulate pathway is most often referred to as speciation reversal or lineage fusion, and it can occur at all stages of the speciation continuum, effectively ‘reversing speciation’ or causing ‘despeciation’13. The underlying pathway of reticulation is identical between speciation reversal and lineage fusion; however, the term speciation reversal is most often reserved for situations where the lineages were reproductively isolated prior to lineage collapse, while lineage fusion is used to describe situations where lineage collapse involves divergent lineages that were not reproductively isolated17.Speciation does not always follow a linear bifurcating process where new species emerge after the appearance of barriers to gene flow that drive increasing reproductive isolation16. Few cases of speciation reversal involving deeply diverged lineages have been identified, yet these are crucial for understanding the potential consequences of emerging cases that might be caused by anthropogenic changes in the future. Of particular concern is disentangling why some species maintain or develop reproductive isolation after secondary contact, while others experience rampant hybridisation and ‘despeciate’. Here, we use a natural experiment involving three deeply diverged lineages of ravens in North America—the ‘California’ lineage, ‘Holarctic’ lineage and Chihuahuan Ravens. Previous studies suggest a reticulate history involving two contrasting situations—(1) speciation reversal of phenotypically cryptic, but deeply divergent, California and Holarctic lineages within the Common Raven, and (2) strong reproductive isolation of phenotypically distinctive Common Ravens and Chihuahuan Ravens despite a sister relationship of the California lineage and Chihuahuan Ravens21  and many are linked to recent anthropogenic causes such as climate change and habitat loss21—the Holarctic lineage is found range-wide and the California lineage is restricted to western North America , whereas only one of three microsatellites examined range-wide showed allele frequency differences between California and Holarctic lineages and a high FST value 18. Thus, it remains unclear whether speciation reversal has eroded the phylogenetic signal of previously distinct California and Holarctic lineages in the nuclear genome, or if the nuclear loci examined lack  the necessary phylogenetic power, or alternatively, if mtDNA introgression involving Chihuahuan Ravens could explain mtDNA paraphyly. The latter hypothesis particularly requires testing since maternally inherited mtDNA loci are notoriously poor at reconstructing complex reticulate histories, and can lead to erroneous inferences of speciation history in such cases11. At a finer scale, it is also unclear whether breeding pairs with mismatched mtDNA examined by Webb et al.20 also have mismatched nuclear genomes. Thus, it remains to be tested whether previous inferences about random interbreeding20 and mitonuclear compatibility26 can be applied across the range of Common Ravens or if they are only applicable in the focal population examined by Webb et al.20 in Washington state, United States.Critically, we have not adequately tested the hypothesis of speciation reversal in Common Ravens owing to limited data from the nuclear genome in our previous studiesHere, we combine dense geographic sampling of nuclear genomes and mtDNA to test whether California and Holarctic lineages have undergone speciation reversal, and to determine whether there is genomic evidence for reproductive isolation versus random interbreeding between North American raven lineages. We show that Common Ravens in western North America have extensively admixed genomes formed from the fusion of non-sister California and Holarctic lineages into a single, randomly interbreeding, species. Two dominant phylogenies are present in their genomes—one reflecting extensive introgression and lineage fusion of California and Holarctic lineages, and the other reflecting the original bifurcating speciation history prior to lineage fusion. Genomic evidence supports current reproductive isolation between Common Ravens and Chihuahuan Ravens despite long-term sympatry and a more recent divergence of Chihuahuan Ravens and the California lineage. These lines of evidence offer strong evidence for a conclusion of speciation reversal in Common Ravens. Our study offers one of the best-characterised examples of the genomic consequences of ancient lineage fusion/speciation reversal, and thus advances our understanding of the diversity of evolutionary pathways that lineages can follow through time.n = 441; Chihuahuan Raven n = 28), the ACO1 nuclear intron on the Z sex chromosome , seven autosomal nuclear introns  and 2250 genome-wide SNPs using a double-digest restriction site-associated DNA (ddRAD) protocol  . No other regions/lineages were identified as admixed by three-population tests, including those for Chihuahuan Ravens and either Common Raven lineage .Geographic structuring within Common Ravens in all genomic data sets clearly delimits 1) a region with higher frequency of Holarctic ancestry in Canada, Alaska and the eastern United States, 2) a region with higher frequency of California ancestry in the southwestern United States and (3) a broad region of admixed California/Holarctic ancestry in the western United States. Admixed western United States populations formed a south–north clinal gradient of increasing Holarctic ancestry connecting discrete California and Holarctic clusters in all analyses , whereas in southwestern USA and Mexico, California mtDNA had highest frequency . However, there was substantial variation between states and populations within each state , both autosomal introns and SNPs inferred two clusters delineating pure Holarctic individuals from northern and eastern North America and pure California individuals from the state of California, and place individuals from the western United States in a gradient that connects the pure California and Holarctic clusters Fig.  and 3. I, both auFST = 1.0, n = 5 SNPs) or highly differentiated  between individuals with the purest Holarctic  and purest California ancestry had mixed lineage assignments within individuals from admixed regions in the western United States and Canada—i.e., individuals had a mixture of homozygous California SNPs, homozygous Holarctic SNPs and heterozygous California/Holarctic SNPs  compared to between California and Holarctic lineages  for mtDNA and Z intron, but were more than twice as high between Chihuahuan and Common Raven lineages  compared to between California and Holarctic lineages  for autosomal introns and SNPs. The genomic landscape of divergence was similar between Chihuahuan Ravens compared to either Holarctic or California lineages with generally high FST across the genome and shared peaks of divergences  strongly supported a sister relationship between California and Chihuahuan Ravens . This could signify issues with model fit associated with differentiating absence vs. presence records and the broad range of climatic conditions covered by the localities used, however, it could also indicate that the range of Common Ravens is limited by other variables that were not included in our models . Notably, overlaying separate ENM predictions for Holarctic, western United States and California regions  predicted a slightly more extensive range for Common Ravens than predicted based on occurrence records and field observations Fig. 29. Thisons Fig.  offer a ons Fig. . Nonetheons Fig. . The lowons Fig. . This fiPaleo-ENM predictions from two different climatic models (CCSM and MIROC) for the last glacial maximum  differed substantially for the Holarctic lineage Fig. . CCSM prMtDNA divergences among all North American raven lineages are estimated to have occurred within the past 2 million years  and east into Minnesota, USA  that found random interbreeding between Holarctic and California mtDNA lineages and a lack of selection against hybrids20.The exact timing of lineage fusion remains uncertain, however, most evidence supports ancient secondary contact and long-term random interbreeding between Holarctic and California lineages. Divergence estimates from mtDNA suggest that initial secondary contact could have occurred at least as early as the mid-Pleistocene (140–440 kya) when Holarctic ravens from North America and Eurasia diverged with little-to-no subsequent gene flowtes Fig.  also fit12. Most notably, we find both recent and ancient temporal signatures in mtDNA , heterogeneous vegetation types  and climatic gradients.Our data appear most consistent with California and Holarctic lineages having experienced multiple secondary contact events as they tracked oscillations in Pleistocene climates, rather than a single ancient secondary contact event that initiated lineage fusion8. Each case of speciation reversal documented to date has different strengths of evidence for these different expectations. For example, most empirical examples of speciation reversal  have very narrow geographic scales and thus, in these situations, speciation reversal can result in the complete replacement of both parent lineages with hybrid genomes over a short time frame. This contrasts with the raven situation, where substantial time and gene flow would be required for mosaic genomes comprised of the collapsing California and Holarctic lineages to penetrate across the entire range of Common Ravens . Two lines of evidence suggest that California and Holarctic lineages could have been reproductively isolated prior to secondary contact and lineage fusion. First, the timing of divergence of the Holarctic lineage and the ancestor of the California and Chihuahuan lineages between 0.9 and 2 mya . The challenge now is for taxonomy and conservation legislation to reconsider outdated views of evolution as a strictly bifurcating process and species as ‘end-points’ of evolution in order to better describe earth’s biodiversity and offer protection to naturally formed lineages with mosaic hybrid genomes38.Complex reticulate histories are emerging all across the tree of life—including humans25 . We also provide the most geographically widespread sampling of Mexico (n = 14), populations east of the Sierra-Cascade mountain range in the western United States  and the eastern United States (n = 57). The majority of specimens used in this study were frozen liver or blood tissues obtained as loans from museums and institutions  and California (CAL)25 Fig. . We partn = 218; Chihuahuan Raven n = 6) or published in previous studies by our group 25. We also included previously published mtDNA sequences from Common Ravens from the Old World (n = 35) and Greenland (n = 14) for comparison. We sequenced loci from across the nuclear genome for a subset of specimens that were genotyped for mtDNA—Z sex chromosome intron , seven autosomal nuclear introns , and thousands of SNPs captured using a double-digest restriction site-associated DNA (ddRAD) protocol  , California  and for the Holarctic region . Where possible the same specimen was sampled for mtDNA, intron and ddRAD data sets, however, this was not always feasible owing to variation in DNA quality and sequencing success. For further details of specimen localities, collection year and which individuals are sequenced in each data set, see Supplementary Data We genotyped mtDNA in a total of 441 Common Ravens and 28 Chihuahuan Ravens from North America using mtDNA control region sequences either produced for this study , and from more degraded toe-pad samples in two overlapping fragments using additional internal primers designed for this study from representatives of the California, Holarctic and Chihuahuan mtDNA lineages. Primer details were as follows: Fragment 1 (253 bp)—corII-LGL2/cor-H253 (5′-TGGGATTGAGAATTCATTGGRGT-3′); Fragment 2 (147 bp)—cor-L166 (5′-ACAAGACARGCTTCACCCRAG-3′)/cor-H417. Eight nuclear introns were sequenced from seven different chromosomes (chr) using previously published primers42—chr Z: ACO1 intron 9; chr 1: MYO2/MB intron 2; chr 2: VIM intron 8; chr 4: IRF2 intron 2, CLOCK intron 10; chr 6: PCBD intron 3; chr 8: RPL5 intron 4; chr 12: RHOD intron 1. DNA extraction and PCR of toe-pad samples were performed in a laboratory free of modern bird DNA or PCR amplicons in order to limit the risk of contamination. All sequences derived from historical museum specimens shared haplotypes with individuals sequenced from contemporary tissues, and did not show other evidence of contamination.DNA was extracted from frozen tissues and blood samples using DNeasy Tissue Extraction Kits (QIAGEN) using standard manufacturer protocols, and from toe-pads of museum specimens using a modified protocol to increase the yield obtained from these degraded tissue sources. Specifically, 25 μl of DTT (dithiothreitol) solution and 20 μl of Proteinase K were added at the initial digestion step, and at the final step DNA was eluted with 60 μl of elution buffer instead of 200 μl. PCR and sequencing of modern samples followed standard protocols detailed in previous studies of mtDNA43 , while the phase of length variant heterozygotes was inferred by the subtraction method . Positions with uncertain heterozygous sites (<0.70 posterior probability threshold) were coded with IUPAC symbols. Tests for recombination using the difference of sums-of-squares method were implemented in TOPALi v144 ; these tests found no evidence for recombination in the eight nuclear introns examined in this study.Heterozygous positions in nuclear introns were coded with International Union of Pure and Applied Chemistry (IUPAC) ambiguity symbols. The gametic phase of samples with heterozygous sites were inferred statistically using PHASE v2.145  with minor modifications. Specifically, digestion of the genomic DNA and the ligation of the adaptors were performed in 40 µl in a single step consisting of 1× NEB 4 buffer; 250 µM rATP; 0.5 µM P1 adaptor; 0.5 µM Index A adaptor; 10U SbfI-HF; 10U MspI; 400U T4 ligase; 100–150 ng genomic DNA in a thermal cycler programmed to 37 °C for 1 h; 65 °C for 10 min; slowly cooling to 4 °C . Between eight and twenty samples were then pooled. After each step in the protocol, the libraries were cleaned with 0.9 volumes of Ampure XP beads (Beckman Coulter)  and eluted in 0.1× TE-buffer, in an appropriate volume to be used for the next step. Size selection for 430–530 bp fragment sizes was performed on a Blue Pippin (Sage Science), using 2% agarose cassettes and marker V1. Library amplification was performed in 100 µl reactions, consisting of 10U Q5 HiFi polymerase; 1× Q5 reaction buffer; 200 µM of each dNTP; 0.2 µM of each primer and 14 µl template. The reaction conditions were 98 °C for 30 s;  x 10 to 14 cycles; 72 °C for 2 min; 4 °C hold. Each reaction was split in four tubes during amplification to reduce PCR duplication. Amplified libraries were cleaned twice using 0.65 volumes of Ampure XP beads to get rid of all short DNA fragments that would inhibit sequencing. The molarity of the clean amplified libraries was detected using the DNF-474 or DNF-488-kits on a Fragment Analyzer . Template preparation of the 400 bp libraries was performed using the Ion One Touch 2 or an Ion Chef (Thermo Fisher Scientific) using 12 or 50 pM of library for the respective systems. Single read sequencing runs were performed on 316v2 and 318v2 chips using Hi-Q chemistry on an Ion Torrent PGM 47.Double-digest RAD seq (ddRAD) libraries were prepared following Vivian-Smith et al.http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) identified adaptor contamination and a drop-off in sequence quality at the end of each read. We therefore used the following pipeline to trim, filter and remove adaptor contamination prior to read mapping: (1) remove adaptor sequences using CUTADAPT v1.9.248 with an error rate of 0.1, a minimum overlap length of 10, and discarding any reads shorter than 50 bp, (2) truncate reads up to the first bp at the 3′ that was above Q17 using FastX-toolkit (http://hannon-lab.cshl.edu/fastx_toolkit/), (3) remove stretches of poly A/Ts from the 3′ end that were at least 4 bp long using PRINSEQ-LITE v0.20.449, and finally (4) FastX-toolkit was used to trim the last 20 bp from all reads, truncate reads to a maximum of 300 bp, remove reads shorter than 50 bp and only keep reads where over 95% of bases had quality scores above Q13 and 80% of bases had quality scores above Q17. Of the 63 specimens initially sequenced, we excluded 10 owing to poor sequence quality indicated by raw reads below 190,000. This resulted in a final data set with 6 Chihuahuan Ravens and 47 Common Ravens of which 21 are from purer Holarctic regions , 6 are from purer California regions  and 20 are from admixed regions in the western United States. For details of per individual raw ddRAD reads, see Supplementary Data Raw reads were run through Torrent Suite v4.4 from the ion community to filter barcodes and perform default quality screening. Raw reads were exported as FASTQ files, and reads of individuals sequenced across multiple runs were merged into a single FASTQ file. FASTQC . TMAP optimises a series of established packages  for use with Ion Torrent data and outperforms other read mapping programs for these data owing to its ability to better handle alignments of indels caused by the high rate of homopolymer errors typical of Ion Torrent sequencing technology52. Variant calling and additional filtering were performed in samtools and BCFtools53, which have been shown to perform best for Ion Torrent data, especially when paired with TMAP54. Our variant calling pipeline had the following steps: (1) reintroduce sample IDs into the BAM file headers produced by TMAP using samtools view, sed and samtools reheader, (2) sort and index BAM files using samtools sort and samtools index, (3) index reference using samtools faidx, (4) run samtools mpileup for all individual BAM files against this reference using a homopolymer coefficient of 50 (-h)52, minimum number of 5 reads to call an indel (-m), and applying the -C50 coefficient to better handle short reads with excessive mismatches and (5) call variants using ‘bcftools call’ with the multi-allelic caller and outputting variant sites only. We then performed the following filtering steps using ‘bcftools filter’, SNPSift55 and VCFtools56: (1) exclude all indels and any SNP within 3 bp of an indel and/or with an average quality score less than Q13, (2) only retain SNPs present in at least five individuals with an average quality score above Q20 and a minimum depth per individual of 5 and a maximum average depth of 500, (3) only retain SNPs with a quality score per individual greater than Q20. We then excluded SNPs that were missing from more than 50% of individuals using vcftools–max-missing. To limit the impact of linkage disequilibrium, we first randomly thinned SNPs that were within 50 bp of each other using VCFtools–thin and then we used PLINK v1.0957 to remove SNPs that violated Hardy–Weinberg equilibrium (HWE) and SNPs that were putatively linked based on a squared coefficient of correlation threshold in excess of R2 = 0.8 using a window of 1 kb. We also exported an additional SNP data set with less-stringent filtering in order to explore the genomic landscape of divergence (see below). This data set allowed 30% missing data and did not exclude loci based on linkage disequilibrium (LD), HWE or proximity on scaffolds in the C. brachyrhynchos genome.Filtered reads were mapped to the genome of the American Crow n = 47), this resulted in 1233–2301 SNPs depending on the amount of missing data allowed . The data set containing both Common (n = 47) and Chihuahuan (n = 6) Ravens resulted in 1205–2205 SNPs . Although acknowledging that including more SNPs despite missing data generally increases the power of analyses, we chose to use stringent filters on data sets to be used for population genetic and phylogenetic analyses in order to reduce the impact of known issues with Ion Torrent data54.The final stringently filtered SNP data set was exported from PLINK allowing for SNPs to be missing from either 10, 30 or 50% of individuals, and these were calculated separately for data sets containing all Common Ravens with or without Chihuahuan Ravens. For the data set containing only Common Ravens , and used the more distantly related Nucifraga columbiana as an outgroup. RAXML analyses used the ‘fast ML’ algorithm with 100 bootstrap pseudoreplicates and the GTRCAT substitution model. Phylogeographic structuring within each mtDNA lineage was explored using unrooted parsimony networks estimated in TCS v1.2160 with 95% connection limit from mtDNA alignment that was trimmed to 254 bp and excluded 26 incomplete sequences in order to reduce the impact of missing data. Samples from C. c. tingitanus—a genetically distinct raven subspecies from the Canary Islands off the coast of northwestern Africa24—were omitted from the Holarctic mtDNA network since they did not connect to other Holarctic clade samples under a 95% connection limit.For mtDNA, we estimated a maximum likelihood (ML) phylogeny in RAXML v8.060 with 95% connection limit using phased sequences from each nuclear intron, and then used Bayesian clustering in STRUCTURE 2.3.361 and unrooted Neighbour-Net trees in SplitsTree v4.14.262 to infer geographic structuring and delineate major lineages across all seven autosomal introns. STRUCTURE analyses used unique haplotypes rather than nucleotides in order to avoid biases associated with using multiple linked SNPs within each locus as input data. Preliminary runs for genetic clusters (K) ranging from 1 to 6 showed that the data had little information about population structuring after K3. Final analyses were run ten times for each genetic cluster between 1 and 4 with an initial burnin period of 50,000 generations and 500,000 MCMC generations under an admixture model with correlated allele frequencies among populations. We then used CLUMPAK63 to select the best value of K using the Evanno Delta K method and to summarise the ten independent runs from each K under default parameters. For Neighbour-Net trees, unphased sequences from the seven autosomal introns with heterozygous sites coded with IUPAC symbols were concatenated for each individual. Neighbour-Net tree analyses used uncorrected P distances and considered heterozygous sites as average states.For nuclear introns, we estimated unrooted parsimony networks in TCS v1.2164, Bayesian clustering in STRUCTURE and unrooted Neighbour-Net trees in SplitsTree to infer geographic structuring and delineate major lineages in data sets containing either all ravens  or only Common Ravens  and with differing amounts of missing data . Neighbour-Net and STRUCTURE analyses were performed as per the autosomal introns. Results were near-identical for data sets containing 10% , 30%  and 50%  missing data. We therefore show PCA and STRUCTURE results for the 10 and 50% missing data sets, and Neighbour-Net trees for the 10% missing data set.For genome-wide ddRAD SNPs, we used smartPCA  in the EIGENSOFT packageFST: autosomal introns and SNPs; ϕST: mtDNA and Z intron) and genetic diversity (mtDNA and Z intron: nucleotide diversity (π); autosomal introns and SNPs: Ho/He) were estimated for Chihuahuan Ravens, Common Ravens, California lineage, Holarctic lineage and the admixed western USA region based on the mtDNA, Z intron, autosomal intron and SNP (stringently filtered 10% missing) data sets, and for the seven autosomal introns independently using a locus-by-locus AMOVA in Arlequin v3.5.1.265 with 100 permutations. To explore genomic landscape, we used PLINK v1.0957 to estimate Wright’s FST (Weir and Cockerham’s method) between each lineage at each SNP. We then plotted FST between each pair of lineages for each SNP against their position on scaffolds in the C. brachyrhynchos reference genome using the less-filtered data set  with and without excluding SNPs that violated HWE. Putatively admixed individuals from Canada were not included in the Holarctic lineage for these measures of FST. SNPs within the top 5% of FST values between each pair of lineages in the stringently filtered data set  and SNPs with FST > 0.2 between each pair of lineages in the less-filtered data set  were exported for phylogenetic analyses and to create individual genomic profiles (see below). To limit the impact of potentially linked SNPs on these analyses, we excluded SNPs in the less-filtered data set that were within 5000 bp of each other.Measures of population differentiation . To aid interpretability, these graphs exclude populations from the eastern United States and Mexico. Populations were designated by grouping geographically proximate specimens within a state or province. For Z intron and mtDNA data sets, ancestry proportions represent the observed frequency of California and Holarctic mtDNA lineages or 20, we created genomic profiles for all Common Ravens that were sampled in at least three data sets by scoring each individual as belonging to Holarctic, California or admixed genetic clusters in each data set. For mtDNA, individuals were assigned to Holarctic and California lineages using RAXML. For Z-linked intron, individuals were assigned to California and Holarctic lineages based on the A/G SNP that separated California alleles and a single Holarctic allele in the unrooted network. Heterozygotes for this SNP were assigned as ‘admixed’. For autosomal introns and ddRAD SNPs, individuals were assigned to California and Holarctic lineages based on their STRUCTURE membership coefficients .To explore evidence for mosaic genomes and test the hypothesis of no assortative mating between California and Holarctic lineagesFST = 1, n = 5 SNPs) or almost fixed  between pure Holarctic and California lineages. For each bi-allelic SNP, we designated the allele that was fixed or near-fixed in the pure populations as California or Holarctic and then we scored each individual from the rest of the range as homozygous or heterozygous for these California or Holarctic alleles at each SNP. If mosaic genomes exist and gene flow is unrestricted between California and Holarctic lineages in the admixed western United States populations, we expect to find uncoupling of mtDNA and nuclear assignments  and we expect to see evidence of admixture even in SNPs that are fixed and nearly fixed between pure California and Holarctic regions .To further explore whether Common Ravens in admixed regions show evidence of having substantially admixed nuclear genomes that are a mosaic of California and Holarctic parental lineages, we created SNP-level genomic profiles. We selected all SNPs from the less-filtered data set  that were fixed  failed to converge. Instead, we use SNPs with FST > 0.2 from the less-filtered data set . These high FST SNPs are potentially resistant to admixture between collapsing California and Holarctic lineages, and thus our goal was to test whether phylogenetic analyses of these SNPs support the same topology as mtDNA or whether they support the same topology as the other nuclear data sets. For comparison we also estimated Neighbour-Net trees for SNPs from the less-filtered data set with FST > 0.2 between California and Chihuahuan Ravens  and Holarctic and Chihuahuan Ravens . The stringently filtered and less-filtered SNP data sets produced near-identical patterns of population structure in all four subsets of SNPs examined  seven autosomal introns, (2) all genome-wide SNPs  and (3) SNPs with the highest NPs Fig. .62 and smartPCA in the EIGENSOFT package64 to explore the different phylogenetic signals present in each of data set. In all analyses, admixed individuals from the western United States connected distinct California and Holarctic lineages based on pure individuals alone  and population mean . We applied a strict clock for all nuclear loci and used an exponential prior on the clock rate for all loci (M = 1.0), except for VIM intron 8, which was fixed to 1.0. HKY + I + G was used as the substitution model on all nuclear loci and we used empirical base frequencies, estimated kappa, gamma, shape and proportion of invariants. *BEAST was run twice for 1 × 108 generations, sampling every 5000 generations and with a burnin of 1 × 107 generations. SNAPP species tree analyses on the SNP data sets used a Yule prior for the species tree, a 1/X hyper prior on lamda and default settings for all other priors. We calculated mutation rate U and V for each data set using BEAUti 2, these were fixed in all analyses while coalescent rate was estimated . SNAPP was run twice for 1 × 107 generations, sampling every 1000 generations and with a burnin of 1 × 106 generations. For both *BEAST and SNAPP analyses, we used TRACER v1.6 (http://tree.bio.ed.ac.uk/software/tracer/) to ensure convergence of independent runs , and then used LOGCOMBINER to combine log and tree files from the two independent runs after excluding burnin. TREEANNOTATOR was used to estimate the maximum clade-credibility tree (MCC) using mean heights for each data set. Finally, TreeSetAnalyser was used to calculate the 95% HPD of tree topologies and the percentage of trees within this 95% HPD set that supported a sister relationship between California and Holarctic lineages, California and Chihuahuan lineages or Holarctic and Chihuahuan lineages.Unrooted Neighbour-Net analyses in SplitsTree v4.14.219. We therefore used BEAST v1.7.569 to more robustly estimate the timing of divergence of mtDNA lineages. We collapsed mtDNA sequences from 441 Common Ravens and 28 Chihuahuan Ravens into 143 unique haplotypes and included C. brachyrhynchos MSB20931 as an outgroup. JModeltest270 was used to select the best substitution model for this data set using default parameters, a BioNJ starting tree and the AICc criteria to select the best substitution model. BEAST analyses used a Yule speciation prior on the tree model, HKY + G substitution model as selected by JModeltest2, empirical base frequencies and an uncorrelated lognormal relaxed clock with a lognormal prior with a mean of 0.026 and log standard deviation of 0.12 on the substitution rate. This prior distribution captured the substitution rate inferred for control region from the 95% HPD interval estimated in Lerner et al.71 (0.019–0.035 substitution/site/lineage/mya). BEAST analyses were run twice for 1 × 107 generations sampling every 1000 generations with a burnin of 1 × 106 generations. Independent runs were combined, and final MCC trees calculated using the same approach as per *BEAST and SNAPP species tree analyses for nuclear data sets.Previous divergence estimates for the North American raven lineages were not tree-based, and therefore do not account for possible rate heterogeneity across lineages31 was used to predict ecological niche models (ENMs) using bioclimatic variables with 2.5 arc-min resolution from the WorldClim database72 and geo-referenced occurrence records for Common Ravens and Chihuahuan Ravens obtained from GBIF  combined with geo-referenced localities of genomic samples used in this study , purer Holarctic ancestry  and admixed ancestry  are predicted to have disjunct or overlapping distributions during the Pleistocene. We used the following regional subsets in the absence of mtDNA genotypes for all GBIF occurrence records: (1) separate ENMs for GBIF samples from regions with purer California, purer Holarctic and admixed ancestry, (2) single ENM combining GBIF samples from purer Holarctic and admixed western United States populations, (3) single ENM combining GBIF samples from purer California and admixed western United States populations and (4) separate ENMs using only individuals genotyped as having either Holarctic or California mtDNA.Maximum entropy species distribution modelling in MAXENT v3.3.3.k74. Paleo-ENMs were predicted by projecting these present-day ENMs onto bioclimatic variables predicted under both the community climate system model (CCSM)76 and the model for interdisciplinary research on climate model (MIROC-ESM)77 for climatic conditions during the last glacial maximum 78 and with ‘fade-by-clamping’ selected74. MIROC and CCSM models differ in their estimates of precipitation and temperature for the LGM, and thus their combined use offers a better estimate of putative suitable conditions for the species being modelled69. Model fit and accuracy was evaluated by visually comparing present-day ENMs to species range limits29, and using area under the receiver operating curve measures (AUC). Note that AUC scores for widespread generalist species with extensive presence records, like Common Ravens, are expected to be lower than those with highly restricted ranges, where an AUC of 1.0 is considered to indicate good fit30.The 19 bioclimatic variables were cropped to North America, and then ENMtools was used to test for correlations between bioclimatic variables. Final present-day ENMs were predicted using ten bioclimatic variables that were not strongly correlated  under recommended default settings  and using a random 30% of samples for testing following recommendations in ref. ACO1: MG591008–MG591247; RHOD: MG583407–MG583517; IRF2: MG590382–MG590504; PCBD: MG590505–MG590618; MYO2/MB: MG590619–MG590732; RPL5: MG590733–MG590880; VIM: MG590881–MG591007; CLOCK: MG583286–MG583406. Raw ddRAD reads were deposited in sequence read archive under BioSample accession numbers SAMN08118047–SAMN08118108.British Columbia specimens collected for this study are accessioned at the UBC Beaty Biodiversity Museum-Cowan Tetrapod Collection. Sanger sequences were deposited in GenBank under the following accession numbers—mtDNA control region: MG594926–MG595149; Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1


However, its mechanism had to be clarified.The ideal plant architecture (IPA) includes several important characteristics such as low tiller numbers, few or no unproductive tillers, more grains per panicle, and thick and sturdy stems. We have developed an indica cultivar 9311 as reference, 307 627 SNPs, 57 372 InDels, and 3 096 SVs were identified in the 7302R genome. The 7302R-specific variations were investigated via the synteny analysis of all the SNPs of 7302R with those of the previous sequenced none-IPA-type lines IR24, MH63, and SH527. Moreover, we found 178 168 7302R-specific SNPs across the whole genome and 30 239 SNPs in the predicted mRNA regions, among which 8 517 were Non-syn CDS. In addition, 263 large-effect SNPs that were expected to affect the integrity of encoded proteins were identified from the 7302R-specific SNPs. SNPs of several important previously cloned rice genes were also identified by aligning the 7302R sequence with other sequence lines.We performed re-sequencing and genome-wide variation analysis of 7302R using the Solexa sequencing technology. With the genomic sequence of the Our results provided several candidates account for the IPA phenotype of 7302R. These results therefore lay the groundwork for long-term efforts to uncover important genes and alleles for rice plant architecture construction, also offer useful data resources for future genetic and genomic studies in rice.The online version of this article (doi:10.1186/1939-8433-5-18) contains supplementary material, which is available to authorized users. According to the protocol, a DNA library with an average insertion length of 484 bp was constructed and 6.35 G bases were generated. The alignment of reads was used to build consensus genome sequences for 7302R. Moreover, approximately 4.77 G high-quality raw databases were aligned with the reference sequence of cultivar 9311 using the SOAPaligner . An oveGenome-wide variations were then examined via SOAPsnp11 and SOAPsv using a conservative quality filter pipeline , and 301The frequencies of SNPs, InDels, and SVs for 7302R were plotted at a 100 kb sliding window, with a step size of 50 kb along each chromosome, by comparing them with those of IR24, MH63, and SH527. The SNP/InDel/SV frequency was defined as the corresponding number of SNPs/InDels/SVs divided by the number of nucleotides within the 100 kb interval, excluding the uncovered nucleotides. Each sample was compared with the corresponding interval to identify regions that showed non-random variation frequencies  and 8 517 were non-synonymous coding sequences (Non-syn CDS) , Xa1 , and Xag et al. ). Other u et al. ), Xa26 , rTGA2.r et al. ), and Pin et al. ), have an et al. ). Howeveg et al. ), DEP1 , GS3 , EUI1 , Gn1a  and qSWa et al. ), had rao et al. ) locus ag et al. ), a possd Karlov ), which a et al. ) DNA seqIn the present study, we report variations over the whole genome of a rice cultivar with an IPA phenotype. However, further analysis of more related lines is necessary to better understand the IPA mechanism although useful information have been proposed to account for the IPA phenotype. Several follow-up steps can also be taken to determine candidate genes that may contribute to this phenotype. The large-effect SNPs and known important rice gene-located SNPs should also be strictly selected and considered for functional verifications. Furthermore, we have developed several genetic populations with 7302R for the dissection and mapping of IPA components. The QTL mapping result and variation distributions are expected to make candidate fixing and further functional confirmation easy. The present study therefore lays the groundwork for long-term efforts to uncover genes and alleles important in rice plant architecture construction, also offers useful data resources for future genetic and genomic studies in rice.Data files are generally TXT. For Windows user, “Editplus” or “UltraEdit” is recommended as the browser program. SNPs information of 7302R; Format description(left to right). 1. Chromosome name. 2. Position of locus. 3. Nucleotide at corresponding locus of reference sequence. 4. Genotype of sequencing sample. 5. Quality value. 6. nucleotide with the highest probability(first nucleotide). 7. Quality value of the nucleotide with the highest probability. 8. Number of supported reads that can only be aligned to this locus. 9. Number of all supported reads that can be aligned to this locus. 10. Nucleotide with higher probability. 11. Quality value of nucleotide with higher probability. 12. Number of supported reads that can only be aligned to this locus. 13. Number of all supported reads that can be aligned to this locus. 14. Total number of reads that can be aligned to this locus. 15. Order and quality value. 16. Estimated copy number for this locus. 17. Presence of this locus in the dbSNP database. 1 refers to presence and 0 refers to inexistence. 18. The distance between this locus and another closest SNP. (SNP 19 MB)Additional file 1: InDels information of 7302R; Format description(left to right). 1. Chromosome. 2. Position. 3. Indel type and number. 4. Bases. 5. Strand . 6. Homozygosis or heterozygosis. 7. Average quality. 8. The number of supported read pair. 9. The number of all crossed read pair. (INDEL 2 MB)Additional file 2: SVs information of 7302R; Format description(left to right). 1. Chromosome name. 2. Type of structure variation. 3. Minimal value of start position in cluster. 4. Maximal value of end position in cluster. 5. Estimated start position of this structure variation. 6. Estimated end position of this structure variation. 7. Length of SV. 8. Breakpoint of SV (only for insertion). 9. Unusual matching mode . 10. Number of paired-end read which support this structure variation. (SV 233 KB)Additional file 3: Authors’ original file for figure 1Authors’ original file for figure 2Authors’ original file for figure 3Authors’ original file for figure 4Authors’ original file for figure 5Authors’ original file for figure 6Below are the links to the authors’ original submitted files for images.


Myosin 10 is an actin-based molecular motor that localizes to the tips of filopodia in mammalian cells. To understand how it is targeted to this distinct region of the cell, we have used total internal reflection fluorescence microscopy to study the movement of individual full-length and truncated GFP-tagged molecules. Truncation mutants lacking the motor region failed to localize to filopodial tips but still bound transiently at the plasma membrane. Deletion of the single α-helical and anti-parallel coiled-coil forming regions, which lie between the motor and pleckstrin homology domains, reduced the instantaneous velocity of intrafilopodial movement but did not affect the number of substrate adherent filopodia. Deletion of the anti-parallel coiled-coil forming region, but not the EKR-rich region of the single α-helical domain, restored intrafilopodial trafficking, suggesting this region is important in determining myosin 10 motility. We propose a model by which myosin 10 rapidly targets to the filopodial tip via a sequential reduction in dimensionality. Molecules first undergo rapid diffusion within the three-dimensional volume of the cell body. They then exhibit periods of slower two-dimensional diffusion in the plane of the plasma membrane. Finally, they move in a unidimensional, highly directed manner along the polarized actin filament bundle within the filopodium becoming confined to a single point at the tip. Here we have observed directly each phase of the trafficking process using single molecule fluorescence imaging of live cells and have quantified our observations using single particle tracking, autocorrelation analysis, and kymographs. The besA and B)  .dK = 0.6 μm), which may dimerize when the local M10 concentration is high (i.e. within the filopodium). Weak dimerization has been reported for other myosin families (e.g. myosin 6) where it has been suggested to play a role in regulating motor activity  lipids  lipids , 28–31. rization , 28. Therization –34.M10's distinct localization to the filopodial tip and the fact that it has constitutive motor activity makes it a model system for understanding mechanisms of active protein translocation and targeting. The predominantly polarized organization of actin within cells  in princA and B, left panels). Video imaging revealed individual fluorescent objects of diffraction-limited size and intensity similar to a single eGFP, which moved rapidly at the basal plasma membrane and trafficked within the filopodia (A and C). In addition to filopodial and plasma membrane localization, there was also a diffuse background fluorescence signal within the cell body arising from fluorescent protein present within the cytosol, but not bound to the plasma membrane (A and B). The absence of localization of FL-M10 at stress fibers or other actin-rich regions of the cells is in marked contrast to cells transfected with non-muscle myosin 2b fused to eGFP (NM-M2b) as demonstrated by single-frame and time-averaged TIRF images . NM-M2b appeared as bright fluorescent puncta with a wide range of individual intensities that localized along the stress fibers (D) and did not localize to filopodia.Live cells, which had been transiently transfected with full-length M10 fused to eGFP (termed FL-M10), were visualized by TIRF microscopy. Consistent with earlier studies , the celilopodia , A and Cmembrane , A and Bs fibers D and didE) before they disappeared within a single video frame (i.e. ∼50 ms) due to either photobleaching or detachment from the membrane . The spatial trajectories of the objects were analyzed by plotting the mean squared displacement (MSD) versus time interval (dt). These plots  were curvilinear (concave downwards) for the overwhelming majority of objects. Such behavior is inconsistent with a simple Brownian random walk, which would give rise to a linear straight-line graph. Only very few objects exhibited MSD versus dt plots with either linear or concave upward appearance typical of unrestricted random walk or motorized and persistent motion in a preferred direction (respectively). When such events are rare, as in our data sets, it is difficult to distinguish between instances of true motorized motion and random statistical variation, because short duration Brownian walk trajectories will sometimes appear to be highly directed, just by chance.In addition to the FL-M10 clusters at the filopodial tips, FL-M10 molecules showed a number of behaviors as they moved at other regions of the cell. To analyze the patterns of movement, individual fluorescent objects of diffraction-limited size and intensity similar to that of a single eGFP were identified and tracked by computer . FluoresFig. 2G). The short-range lateral diffusion coefficient (Dlat) determined from the initial gradient of MSD versus dt plots was 0.2 μm·s−1, which is similar to other membrane-associated proteins ).The main conclusion from these results is that the overwhelming majority of FL-M10 molecules diffuse at the plasma membrane in an anomalous fashion, moving less far than expected at longer observation times G. The shproteins , 39. We A, right panels, supplemental Movie 1, and “Experimental Procedures” for further technical details).In addition to the fluorescent objects that diffused relatively slowly at the plasma membrane and could therefore be identified and tracked by computer, visual inspection of the video recordings revealed a fluctuating and speckled fluorescent background signal arising from the rapid motion of cytosolic molecules diffusing within the evanescent field. To map the average rate of diffusion of all fluorescent objects at a particular location in the cell, we performed temporal autocorrelation analysis on the video data. The locally averaged autocorrelation time, τ, of the intensity fluctuations measured at each pixel location was calculated, and the value was converted to a pseudo-color map that reports the average rate of diffusion of all objects moving at a given pixel location. The maps were superimposed over the original fluorescence video data . These regions correspond to areas that contain mainly cytosolic molecules and relatively few membrane-bound molecules. The rapid movement of FL-M10, particularly at the central regions of the cell, demonstrates that a significant fraction of the molecules are cytosolic and do not associate strongly either with the actin cytoskeleton or plasma membrane.i.e. the lower three constructs shown in Fig. 1A). As for FL-M10 (above), the different protein constructs were transiently transfected into live cells that were then viewed by TIRF microscopy. The movement of molecules that bound at the plasma membrane was again assessed by single particle tracking and analysis of MSD versus dt plots (B).To understand how the tail domains of M10 act to target the full-length molecule to different regions of the cell, a panel of eGFP-tagged recombinant proteins in which the motor region had been deleted were tested (dt plots B.versus dt plots were curvilinear (concave downwards) for all of the constructs, as found for FL-M10 (G). All constructs containing PH domains had short-range Dlat values that were similar to FL-M10 (B–D). However, the isolated MyTH4-FERM construct behaved differently and exhibited higher mobility than the other constructs tested . This finding was confirmed by autocorrelation analysis, which also showed that the MyTH4-FERM construct had a higher mobility than either PH123 or FL-M10 . The construct containing both MyTH4-FERM and PH123  had similar mobility to PH123 and FL-M10 indicating that the low mobility conferred by PH domain binding at the plasma membrane dominated the behavior of FL-M10. The low plasma membrane affinity and high mobility of MyTH4-FERM suggested that this region of the molecule is less important for targeting FL-M10 to the plasma membrane.Individual MSD r FL-M10 G. All coo FL-M10 , B–D. Hoboxed region, Fig. 4A) using kymographs  showed a mixture of persistent tip-wise motion, stalling and random back-and-forth motions within the filopodia . The velocity of molecules while undergoing smooth directed movement was ∼1.4 μm·s−1, similar to that measured previously for M10 in vitro at 37 °C . However, when data for all objects were pooled, it was apparent that the trajectories did not show continuous directed motion but nearly all exhibited periods of diffusive motion and stalled behavior. In earlier studies, the characteristic run length of myosin 10 was determined by histogramming the duration and distance moved by individual eGFP-tagged molecules along surface-immobilized actin filaments or filament bundles . The histograms had a peak value centered around zero, with a shoulder on the right-hand side . The histograms were then fitted to the sum of two Gaussian distributions. One population had a net velocity close to zero ; corresponding to periods of either stalled and random back-and-forth motion. The other population had an instantaneous velocity of +0.28 ± 0.69 μm·s−1  for all objects that had been tracked and histogrammed the distribution of velocities D. The hi9 μm·s−1 , correspE). These plots indicate that the individual paths cluster along a central region, ∼100 nm in diameter, consistent with electron micrographs of the filopodial actin core structure (A). Such super-resolution images revealed interesting structural features including small defects in the filopodial shaft structure and thin bridging structures, which link adjacent filopodia. The super-resolution reconstructions show that filopodia project from a root region (∼1 μm across) that rapidly tapers to a shaft diameter of around 116 nm (B and C). Small imperfections (defects) in filopodial ultrastructure were present in about 10% of filopodia. Although movement of FL-M10 in the cell body was chaotic at distances greater than 2–3 μm from the filopodial base (as discussed before), we observed directed motion in the “root region” of filopodia on many occasions .Because the single particle tracking analysis was performed with sub-pixel (∼30 nm) resolution, the paths taken by individual molecules can be plotted over the original fluorescence image to reveal structural features that are smaller than the diffraction limit E. These tructure . This obtructure  by plotttructure A. Such sd 116 nm , B and CA and 6C). Cells transfected with M10-motor-PH produced the same number of substrate-adherent filopodia as cells transfected with FL-M10, and there was clear localization of M10-motor-PH to the filopodial tips (A and B). Furthermore, the mobility and fraction of plasma membrane-bound M10-motor-PH was also indistinguishable from FL-M10 (C and D). Kymographs of M10-motor-PH trafficking were similar to FL-M10 (D), and the velocity measured from the kymographs was again ∼1.4 μm·s−1 as measured for FL-M10. Single molecule tracking additionally showed that the filopodial trafficking for this construct was also similar to FL-M10. The instantaneous velocity distribution analysis again indicated two populations (E), one with a velocity close to zero ; and the other had only the last 86 residues deleted (Δ852–938), removing just the distal part of the SAH domain and the entire CC sequence, termed M10-Δ2.We next determined whether deleting the SAH domain from the FL-M10 construct affected the behavior of M10. Residues 809–932 were originally predicted to form a coiled-coil . A peptiFig. 1B) for cells transfected with M10-Δ1 or M10-Δ2 compared with cells transfected with either FL-M10 or M10-motor-PH constructs. Kymograph analysis of filopodial trafficking for M10-Δ1 and M10-Δ2 also gave similar results to the FL-M10 and M10-motor-PH constructs, and the velocity measured directly from the kymographs was 1.4 μm·s−1 . Intrafilopodial trafficking again indicated two types of behavior . The major population had zero net velocity , and the other showed directed movement toward the filopodial tip  than FL-M10 . However, the mean velocity of the M10-Δ2 moving fraction was increased close to (and not significantly different from) FL-M10 .Analysis of cells transfected with M10-Δ1 and M10-Δ2 expression constructs  showed tFig. 6dial tip . The meaWe have used TIRF-based single molecule imaging to track the fate of individual fluorescently tagged molecules within live mammalian cells to help elucidate how M10 is targeted to the tips of filopodia. Simple visual inspection of the TIRF imaging video data revealed that full-length M10 shows three distinct types of behavior as follows: it diffuses freely within the cytosol, moves more slowly when bound to the plasma membrane, and then in a directed manner within the filopodia. We quantified these observations using a combination of single particle tracking, kymographs, and correlation analysis. By studying different deletion mutants, we have dissected which regions of the M10 molecule are important for each type of behavior. Our study leads to a model for M10 trafficking in which diffusive and active movements combine to rapidly target myosin 10 to the filopodial tip.Dlat, > 5 μm2·s−1), and periodically explores regions of plasma membrane by performing intermittent two-dimensional diffusional movements, with a lateral diffusion coefficient at the membrane, Dlat ∼0.2 μm2·s−1. Then, after locating an appropriate region of membrane at the base of the filopodium, the myosin motor is activated, and the molecule moves in a highly directed manner along the polarized actin filament bundle within the filopodium at a maximum velocity of ∼1.4 μm·s−1. Once at the tip, the M10 molecules are essentially trapped and therefore accumulate as a characteristic cluster or punctum. Even within the filopodium, M10 movement shows discontinuities in which smooth motion is interrupted by periods of stalled and diffusive motion, such that the averaged velocity over a 500-ms time window is much slower (∼0.3 μm·s−1) than for regions of smooth continuous motion seen on the kymographs (∼1.4 μm·s−1).Our data show that M10 moves rapidly through the three-dimensional volume of the cell by diffusion  and replacing these with a parallel coiled-coil did affect filopodia formation. However, none of these studies used constructs that included the tail domains, as we have done here, but only used truncated “HMM-like” constructs. In one study, an M10-HMM-like construct was artificially dimerized using the coiled-coil forming region of myosin 5 or by fusion with the inducible dimerization domain, FKBP, placed at residue 861 (B). This retains the EKR-rich region of the SAH domain but removes the distal region, as well as the anti-parallel CC sequence (B) again did not show strong filopodial localization. A longer construct, in which the tail domains were added after the GCN4 zipper, showed an increased filopodial localization compared with the HMM construct, suggesting that the presence of the tail domains does play a role in filopodial localization. However, neither construct promoted an increase in filopodial number. Neither of these studies investigated any effects on M10 movement within the filopodia.Previous studies have shown that deleting the distal SAH domain and CC region B and repidue 861 B. This rsequence  and all sequence  showed tsequence B again dA drawback of both of these studies is that a parallel coiled-coil is placed next to the EKR-rich region (∼first 40 residues of the SAH domain), deleting downstream SAH and anti-parallel coiled-coil region. In this case, we would expect the EKR-rich region to remain as an SAH domain, as we found this was the case when we tested a myosin 5-SAH chimera . The eff2·s−1), suggesting that M10 is not able to move processively on cortical actin filaments while associated with plasma membrane in central regions of the cell, also it neither binds nor moves on actin stress fibers or cortical actin structures. Truncated constructs containing various combinations of M10 tail regions showed similar behavior and mobility to the full-length molecule, except for the isolated MyTH4 FERM domain, which had increased mobility and lower affinity for plasma membrane. Deletion of the MyTH4-FERM domain from the parent molecule did not affect either mobility at the plasma membrane or speed of trafficking within the filopodia. Neither did it affect the number of surface adherent filopodia, suggesting that this domain is not required for the motile behavior of this myosin. This finding is consistent with earlier studies that also showed the MyTH4-FERM domain is not required for the induction of substrate-attached filopodia in COS-7 cells P3 in both regions of the cell but that the motor is able to move processively only when in the immediate vicinity of the filopodium as suggested by early studies P2). Although that study showed the MyTH4FERM domain also interacted with the motor domain and was additionally implicated in regulating its ATPase activity, it seems that the PH/PtdInsP3 interaction is most important for controlling this regulatory pathway. It has also been suggested that the binding to PtdInsP3 induces formation of dimers of M10 P studies . The req studies . Isolate studies , 44. Mors of M10 , but our3) and then a two-dimensional search for the correct type of actin. Once it reaches the base of the filopodium (versus time spent bound and slowly diffusing at the plasma membrane similar to the ratio of the two respective diffusion coefficients (Dcyto and Dmem). Then, when a molecule binds at the plasma membrane it will explore a region given by g) and undergoes diffusion in the cytosol, it will move a similar distance as shown in f). This would allow a new region of the membrane to be explored each time the molecule rebinds. If we make a rough calculation using realistic values , then for each mean dwell time of 10 s at the membrane and 0.4 s within the cytosol the M10 molecule would explore a region of ∼2 μm2. This may help to explain how M10 translocates rapidly and efficiently from the cytosol to the distant filopodial tips, which would presumably be much slower if targeting relied solely on random diffusion or on directed motion along the randomly oriented network of cytoskeletal actin filaments, stress fibers, and other cortical actin structures.M10 exhibits directed movement only at regions close to the base of the filopodium and shows clearly directed motion only after entering the main filopodial shaft that is composed of fascin-bundled actin. It has been suggested that the tropomyosin isoform TM3 (TmBr3/Tpm1.7), together with Arp2/3, helps promote filopodia formation by increasing the number of short filaments without capping proteins, and these then recruit the bundling protein fascin to generate filopodia . Certainlopodium , it then 0.1 s−1  and assuThe full-length bovine myosin 10 cDNA P79114) together with the tail constructs (PH123-MyTH4-FERM and MyTH4-FERM) were generous gifts from Prof. Richard Cheney (University of North Carolina)  together. A PCR-bThe recombinant M10 constructs were transfected into HEK293 or HeLa cells (no differences were observed in results for these two cell types). Cells were plated onto glass coverslips and transfected using either FuGENE 6  or Gene-juice  following the recommended protocols. 12–16 h later, the cells were either fixed and then stained for actin using Alexa-546 phalloidin  or prepared for live-cell imaging.C).Cells were plated into a single well of a 6-well plate, before transfecting as described above. 24–48 h later, cells were washed with PBS and then scraped into Laemmli buffer. Samples were run on a 7.5% acrylamide gel, transferred to nitrocellulose, blocked in PBS containing 5% milk for 1 h, then incubated with an anti-GFP antibody (Abcam), washed in PBS/Tween, and then incubated with an anti-rabbit horseradish peroxidase antibody before exposing to film C.m HEPES (pH 7.4) and 10% serum, and the coverslip was assembled into a small chamber for viewing the cells on an inverted microscope at 37 °C in a custom-built total internal reflection fluorescence microscopy system (see below). Approximately 10% of the cells had a suitable level of expression, such that single molecules could be visualized as single spots when viewed by TIRF microscopy. This equates to a final concentration of eGFP fusion protein in cells in the nanomolar range .The cell growth medium was replaced with Hanks' balanced salt solution with 20 m−2. The incident laser beam angle was adjusted to 64° to create the evanescent field at the glass-water interface. A digital EMCCD camera  was used to acquire video sequences that were stored directly on a computer hard drive using a computer frame-grabber card and proprietary software. The microscope's image magnification was calibrated using a reticule, giving 100 nm per pixel in both x and y camera axes. Experiments were performed at 37 °C, and video records were collected at either 10, 33, or 50 frames·s−1. The fluorescence intensity of single GFP molecules was characterized using control specimens in which GFP was antibody immobilized at very low surface density (<1 fluorescent spot per 1 μm2) and imaged under identical conditions  to our live cell imaging experiments.The TIRF imaging system has been described elsewhere , 38; howDlat <0.2 μm2·s−1), which was sufficiently slow to allow the path of individual molecules to be tracked between adjacent video frames using a “nearest-neighbor” tracking algorithm . The Dlat values were then color-coded, using a pseudo-color look-up table, to give a visual map that could be overlaid onto the original video data (using ImageJ).Fluorescent objects that moved freely within the cytosol were too fast to be individually tracked because their paths could not be unambiguously determined between adjacent video frames. Instead, we used autocorrelation analysis to estimate the speed of objects moving at each pixel location. The autocorrelation time, τ, depends upon the time it takes a molecule to traverse an optical volume, which is a convolution of the rate of diffusion, the imaging point spread function, evanescent field depth, and camera pixel size. Autocorrelation analysis was conducted at each pixel location, across the entire field of view (up to 512 × 512 pixels), for the duration of the video record. The characteristic autocorrelation time, τ, was then calculated at each pixel position. The τ values were then locally averaged over an 8 × 8 pixel window size (computations were performed using IgorPro). The resulting matrix of values was converted to diffusion coefficient values by performing identical analysis on simulated video data and creating an empirical conversion between τ and e.g.E and To create a physical map of the actin bundle within the filopodium. Particle trajectories  were determined with sub-pixel resolution and plotted on top of one another to create a composite image of the track paths along the axis of the filopodia e.g.E and 5.A).Cells that had been fixed and stained for actin using Alexa-546 phalloidin were imaged either using a Deltavision deconvolution microscope using the ×63 oil objective (for counting filopodia number) or using a Zeiss 880 LSM Airy scan confocal microscope, using a PlanApochromat ×63, 1.4NA oil immersion objective lens . Raw Airy scan images were deconvolved using the Zeiss Zen software to generate the images shown A.To count the number of filopodia, deconvolved images of whole cells taken close to the surface of the coverslip were analyzed in ImageJ. Filopodia were identified on the basis of their actin staining and the presence of Myo10 at the tip of the filopodia. Filopodia numbers, from 11 to 14 cells, were counted (4–7 cells from two separate experiments), and the data were expressed as filopodia number per cell. This approach only considers substrate-attached filopodia and not dorsal filopodia, which are challenging to estimate using a light microscopy approach.M. P. and J. E. M. designed the research; T. G. B., T. A. N., and G. I. M. performed the research; G. I. M., M. P., T. G. B., and J. E. M. contributed new reagents/analytic tools; G. I. M., T. G. B., T. A. N., M. P., and J. E. M. analyzed the data; M. P., G. I. M., T. G. B., and J. E. M. wrote the paper. All authors reviewed the results and approved the final version of the manuscript.


The progress of medicine, science, technology, education, and culture improves, year by year, quality of life and life expectancy of the populace. The modern human has a chance to further improve the quality and duration of his/her life and the lives of his/her loved ones by bringing their lifestyle in line with their sequenced individual genomes. With this in mind, one of genome-based developments at the junction of personalized medicine and bioinformatics will be considered in this work, where we used two Web services: (i) SNP_TATA_Comparator to search for alleles with a single nucleotide polymorphism (SNP) that alters the affinity of TATA-binding protein (TBP) for the TATA boxes of human gene promoters and (ii) PubMed to look for retrospective clinical reviews on changes in physiological indicators of reproductive potential in carriers of these alleles.A total of 126 SNP markers of female reproductive potential, capable of altering the affinity of TBP for gene promoters, were found using the two above-mentioned Web services. For example, 10 candidate SNP markers of thrombosis  can cause overproduction of coagulation inducers. In pregnant women, Hughes syndrome provokes thrombosis with a fatal outcome although this syndrome can be diagnosed and eliminated even at the earliest stages of its development. Thus, in women carrying any of the above SNPs, preventive treatment of this syndrome before a planned pregnancy can reduce the risk of death. Similarly, seven SNP markers predicted here  can elevate the risk of myocardial infarction. In line with Bowles’ lifespan theory, women carrying any of these SNPs may modify their lifestyle to improve their longevity if they can take under advisement that risks of myocardial infarction increase with age of the mother, total number of pregnancies, in multiple pregnancies, pregnancies under the age of 20, hypertension, preeclampsia, menstrual cycle irregularity, and in women smokers.According to Bowles’ lifespan theory—which links reproductive potential, quality of life, and life expectancy—the above information was compiled for those who would like to reduce risks of diseases corresponding to alleles in own sequenced genomes. Candidate SNP markers can focus the clinical analysis of unannotated SNPs, after which they may become useful for people who would like to bring their lifestyle in line with their sequenced individual genomes.The online version of this article (10.1186/s12864-018-4478-3) contains supplementary material, which is available to authorized users. Incessant progress in medical and biological sciences, advancement of technology, and education year in and year out improve quality of life and life expectancy of the population, creating comfortable conditions for active living. Nonetheless, there are numerous factors that adversely affect human health. They can include, for example, different kinds of environmental pollution, an increase in population density, which leads to the rapid spread of infections and parasitoses, and an increase in psychological stress. This situation not only reduces the quality of life and longevity of the individual but also has a deferred, long-term effect on the next generation, by acting as a mutagen . The accThe effects of the above factors limit individual reproductive potential: a concept used in population ecology to assess the evolutionary success of an individual  or a popPredictive-preventive personalized medicine may help to improve individual reproductive success. Its methods include prediction  of the probability of a specific disease, analysis of individual indicators, biomarkers , andIn addition, according to Bowles’ lifespan theory , which lWithin the framework of the biggest modern scientific project “1000 Genomes”, 10545 individual genomes have already been sequenced  using thAs far as human health is concerned, the clinical search for biomedical SNP markers is the only acceptable method. Nevertheless, it is so laborious and expensive that its application to all 8.58 billion potentially possible SNPs  and all −/−) are always inviable since their development cannot proceed past the blastula stage because their maternal supply of TBP is exhausted  regions upstream of transcription start sites for the human genes containing the known biomedical SNP markers that alter TBP’s binding to promoters of these genes (Tables 0 regionsThe humanESR2gene (estrogen receptor β) contains a known SNP marker  dehydrogenase 1, progesterone receptor, and glutathione S-transferase μ3, respectively. Their promoters have the known SNP markers rs201739205, rs10895068, and rs1332018, which elevate risks of breast  region  for both ancestral and minor alleles of the human gene promoter being analyzed. On this basis, using a package R . (PDF 220 kb)Additional file 2:Supplementary method. Keyword search in the PubMed database. (PDF 221 kb)Additional file 3:Table S1. Clinically known dependences between reproductive potential and hereditary diseases whose SNP markers were predicted in this work. (PDF 198 kb)


SNPs), the aim of the study was to identify genetic regions that may be under selection for racing performance. We hypothesized that the selection for racing performance, in combination with unauthorized crossbreeding of Coldblooded trotters and Standardbreds, has created regions in the genome where the Coldblooded trotters and Standardbreds are similar, but differ from the North‐Swedish draught horse. A fixation index (Fst) analysis was performed and sliding window Delta Fst values were calculated across the three breeds. Five windows, where the average Fst between Coldblooded trotters and Standardbreds was low and the average Fst between Coldblooded trotters and North‐Swedish draught horses was high, were selected for further investigation. Associations between the most highly ranked SNPs and harness racing performance were analyzed in 400 raced Coldblooded trotters with race records. One SNP showed a significant association with racing performance, with the CC genotype appearing to be negatively associated. The SNP identified was genotyped in 1915 horses of 18 different breeds. The frequency of the TT genotype was high in breeds typically used for racing and show jumping while the frequency of the CC genotype was high in most pony breeds and draught horses. The closest gene in this region was the Endothelin3 gene (EDN3), a gene mainly involved in melanocyte and enteric neuron development. Both functional genetic and physiological studies are needed to fully understand the possible impacts of the gene on racing performance.The Swedish‐Norwegian Coldblooded trotter and the heavier North‐Swedish draught horse both descend from the North‐Swedish horse, but the Coldblooded trotters have been selected for racing performance while the North‐Swedish draught horse is mainly used for agricultural and forestry work. By comparing the genomes of Coldblooded trotters, North‐Swedish draught horses and Standardbreds for a large number of single‐nucleotide polymorphisms ( Descending from the North‐Swedish horse, the Coldblooded trotter is a unique racehorse breed with a draught horse origin. During the 19th and the first part of the 20th century most North‐Swedish horses were used for forestry and agricultural work. However, horse racing has fascinated humans for hundreds of years and many of the North‐Swedish horses were not only used for work but also for racing. The first official harness race with North‐Swedish horses took place already in the early 1800s. When interest in harness racing grew breeders began to select for faster and more sustainable horses. While many of the horses were used for both working and racing, the intensive selection for racing performance traits started to negatively influence the breeding of good working horses. Therefore, in 1964, the decision was made to divide the North‐Swedish horse into two different breeds: the North‐Swedish draught horse and the lighter Coldblooded trotter  between Coldblooded trotters, North‐Swedish draught horses and Standardbreds, the aim of the study was to identify genetic regions that are under selection for harness racing performance. These regions may for example contain genes influencing energy metabolism, muscle composition or temperament, as these are all examples of traits where the Coldblooded trotters are similar to Standardbreds but different from the North‐Swedish draught horses. Identifying novel genes important for performance is not only of value for the racing industry. Genes that take part in the regulation of energy metabolism and other biological processes that impact racing performance may also shed light on metabolic defects and diseases in horses as well as in other species.The study was divided into three different parts: (1) a Delta fixation index (Fst) analysis, (2) a SNP association analysis, (3) genotyping of the top‐SNP identified in the Delta Fst analysis in a variety of breeds.In total, 42 horses  were included in part i. The two trotting breeds were elite performing horses. They were selected based on Estimated Breeding Value (EBV) and pedigree, to only include horses that had been bred for racing, and to avoid including horses with the same parents or grandparents. The Identical By Descent (IBD) value was calculated in PLINK using the –genome command, and the threshold was set to maximum 0.25 . The DNA samples were genotyped on one of two different Illumina SNP50 Genotyping BeadChips, one that contained 54,602 SNPs  and one that contained 57,165 SNPs . The two datasets were merged and quality control (QC) was performed in PLINK  or a minor allele frequency (maf) <0.00001 . In addition, SNPs that were not in common between the breeds  and X‐chromosome SNPs  were excluded from the analyses. In total, 37,246 SNPs remained and were included in the analyses.DNA was extracted from 350 The statistical analyses were performed in PLINK and the software program for statistical computing R , but the earnings for Norwegian trotters were in Norwegian currency (NOK). In order to set all earnings to Swedish currency an average exchange rate was calculated (μ = 0.95) for the years 2003–2015 and multiplied with the Norwegian earnings  were selected from the Fst analysis. From those, the SNP with the highest single Delta Fst value was selected and its association with racing performance in Coldblooded trotters was investigated. All Coldblooded trotters had previously been genotyped on the 670K Axiom Equine Genotyping array  was included as a covariate due to previous studies having shown a major impact of this gene on harness racing performance , or had previously been genotyped on the 670K Affymetrix Equine Genotyping Array  .The horses were either genotyped for the most significantly associated SNP with the StepOnePlus Real‐Time PCR System (Thermo Fisher) using a custom designed TaqMan SNP Genotyping Assay (Applied Biosystems) , 0.041 (Coldblooded trotters vs. North‐Swedish draught horses) and 0.088 (North‐Swedish draught horses vs. Standardbreds).Endothelin 3 (EDN3) gene   also showed significant associations with racing performance  in horses  genes, which have been reported to have major influence on performance in harness racing breeds and Thoroughbreds (Binns et al. EDN3 for performance and exercise in horses are necessary to understand how this gene may contribute to racing performance. Nevertheless, the results from this study provide important information about the complex genetic regulation of performance with additional studies needed to fully understand the exact impact of the identified region on harness racing performance.Aside from the EDN3 gene, which encodes for a vasoactive peptide that is crucial for the development of melanocytes and enteric neurons. The study provides novel information about the genetics of horse performance, but additional studies are needed to confirm the findings.From the Delta Fst analysis five genomic regions where Coldblooded trotters and Standardbreds were genetically similar but together differed from North‐Swedish draught horses were identified and used for association analysis with harness racing performance. One SNP, g.22:45748491C>T, was significantly associated with racing performance results in 400 Coldblooded trotters. The CC genotype appeared to negatively influence performance results. Interestingly, the TT genotype was at a high frequency in athletic breeds such as Thoroughbreds, Standardbreds, Coldblooded trotters, and Warmbloods while the frequency of the CC genotype was high in pony breeds and draught horses. The closest gene next to the SNP identified was the There are no conflicts of interest to declare.