Retrogenes generally do not contain introns. However, in some instances, retrogenes may recruit internal exonic sequences as introns, which is known as intronization. A retrogene that undergoes intronization is a good model with which to investigate the origin of introns. Nevertheless, previously, only two cases in vertebrates have been reported.Homo sapiens) genome for retrogenes that evolved introns and analyzed their patterns in structure, expression and origin. In total, we identified nine intron-containing retrogenes. Alignment of pairs of retrogenes and their parents indicated that, in addition to intronization (five cases), retrogenes also may have gained introns by insertion of external sequences into the genes (one case) or reversal of the orientation of transcription (three cases). Interestingly, many intronizations were promoted not by base substitutions but by cryptic splice sites, which were silent in the parental genes but active in the retrogenes. We also observed that the majority of introns generated by intronization did not involve frameshifts.In this study, we systematically screened the human (Caenorhabditis elegans. Our results provide novel insights into the evolutionary role of introns.Intron gains in retrogenes are not as rare as previously thought. Furthermore, diverse mechanisms may lead to intron creation in retrogenes. The activation of cryptic splice sites in the intronization of retrogenes may be triggered by the change of gene structure after retroposition. A high percentage of non-frameshift introns in retrogenes may be because non-frameshift introns do not dramatically affect host proteins. Introns generated by intronization in human retrogenes are generally young, which is consistent with previous findings for  Retroposition, or RNA-based duplication, is the process by which reverse-transcribed mRNAs are inserted into new genomic positions, which generates retrocopies . RetrocoArabidopsis and Populus, 29 retrogenes have undergone intronization, which represent about 15.3 % of all known retrogenes [Homo sapiens) genome [Since intronization of retrogenes was first reported , this kitrogenes . In conttrogenes ,13. Prevtrogenes . This fr) genome . How gene-value cutoff (1e-10), coverage cutoff (70 %) and identity cutoff (40 %). Next, we retained candidates with more than three introns absent or only one or two introns absent but with a small Ks (<2) or other RNA-based duplication evidence, for example, a poly(A) track. Finally, as described previously [The human genome data were downloaded from the UCSC Genome Browser database (release hg19) ,19. Thenutoff 1e-, coveragWe wrote a series of PERL programs to look for intron-containing retrogenes on the basis of annotations from ENSEMBL (GRCh37) ,23. We iWe utilized the mRNA and EST annotations from the UCSC Genome Brower Database to search for transcription evidence of intron gain in retrogenes ,19. For Ka), synonymous substitution rate (Ks) and Ka/Ks values between the intronic regions of retrogenes and their parental copies, by implementing the codeml program in the PAML package following the Nei-Gojobori method [Ka/Ks estimation between the exonic regions of retrogenes and their parental copies in the same way.We estimated the non-synonymous substitution rate  were transcribed in the reverse direction relative to that of their parents. For XXyac-R12DG2.2 there are 10 annotated transcription patterns and introns appeared in four of the 10 patterns  of the mRNA. Some part of the retrocopy had changed into an intergenic sequence, and some part acted as a portion of an intron  [Caenorhabditis elegans[In total, eight of the 10 introns created by intronization were non-frameshift introns. This proportion 80 %) is significantly higher than the percentage of frameshift introns generated by chimerization based on intron-gain retrogenes  (0 % is si= 0.017) -29 and P= 0.017) ,31, we fs elegans, our obsPrevious studies showed that most intronizations were caused by base substitutions at the 5′ and 3′ SS ,11. HoweC. elegans, intronization is reported to be a major contributor to intron creation and most introns generated by this mechanism are young [P = 0.023). Furthermore, in our data set, no intronized retrogene (0/5) was retroposed from chromosome X (‘out-of-X’). The retrogenes from chromosome X were mostly old and evolved after the divergence of eutherian mammals (human or mouse) and marsupials (opossum) [. For retrogenes that underwent intron gains by chimerization, the proportion of ‘out-of-X’ retrogenes was 37 %  . For retKa, Ks, and Ka/Ks values between the intronic regions of retrogenes and their parental copies as well as between the exonic regions of retrogenes and their parental copies. The Ka values in the intronic regions were higher than those in the exonic regions . These findings are consistent with the conclusion that introns evolved faster than exons.To evaluate the evolutionary rates of retrogenes, we calculated Ka/Ks values smaller than 1 , which suggested that the corresponding regions were under negative selection. By checking for evidence of expression, we found that three of the five intronized retrogenes showed evidence for expression at the protein level and the additional two retrogenes showed transcription evidence at the RNA level. This result indicated that most intronized retrogenes were functional and should be under negative selection.In addition, the exonic regions of most intronized retrogenes had With regard to the three retrogenes that gained introns after transcription in the opposite orientation compared with the parent, they were annotated to be in the non-coding regions of other genes. We observed that CSMD3 and WBP2NL evolved faster than the other retrogenes Table . This fiC. elegans[In this study, we systematically searched the human genome for retrogenes that underwent intron gain in the coding region and in total identified 15 retrogene introns. These newly generated introns evolved at a faster rate than neighboring exons. In contrast to the findings in plants , we foun. elegans, retroge. elegans,44. Furt. elegans,13,45. CSome questions arise from careful examination of our observations. For example, for the retrogene RPS3AP5, in which the new intron was created by insertion of an external sequence, the process by which the new intron was created is unknown. In addition, in searches of UCSC ,19, EnseFor the eight non-frameshift introns generated by intronization, we examined whether they are under natural selection by checking their genetic variation in different human populations with the 1000 Genomes Browser . HoweverOur results showed that retrogenes may gain introns in three ways: insertion from an external sequence, transcription in the reverse direction compared to that in the parent, and intronization. In addition to base substitution, intronization also may be promoted by cryptic splice sites. For introns generated by intronization, non-frameshift introns might have greater evolutionary success than frameshift introns, because non-frameshift introns have only a small effect on the host proteins or are neutral. Furthermore, intronization tended to occur in young retrogenes.BS, branch site; PPT, polypyrimidine tract; SS, splice site.The authors declare that they have no competing interest.LFK and ZLZ together carried out the identification of intronized retrogenes and data analysis, and performed the statistical analyses. LFK performed the PCR analysis and helped to draft the manuscript. ZLZ conceived the study, participated in its design and analysis, and drafted the manuscript. QZ helped to perform the data analysis and statistical analyses, participated in the design of the study and helped to draft the manuscript. LYC provided the materials for experiments. ZZ participated in the design of the study and helped to draft the manuscript. All authors read and approved the final manuscript.Transcripts uniquely mapped to retrogenes. This file lists transcripts that spanned the introns of their mapped retrogenesClick here for fileEvidence for transcription of retrogene introns (from the UCSC Genome Browser database). This file contains snapshots from the UCSC Genome Browser database that displays the transcription of retrogenes that gained intronsClick here for fileList of human tissues sampled for the experiments. This file lists the human tissues that we used for the experiments to validate the existence of retrogene introns Click here for fileExperimental validation of retrogene introns in TMEM14D and HSP90AA4P. This file shows the experimental results for validating the existence of retrogene intronsClick here for filePhylogenetic tree for vertebrates. A diagram of the phylogenetic tree for vertebratesClick here for fileChromosome and time of origin of intronized retrogenes. This file shows the origination times of intronized retrogenesClick here for fileChromosome and time of origin of retrogenes that gained introns by chimerization. This file shows the origination times of retrogenes that gained introns by chimerizationClick here for fileTranscription annotations (from the UCSC Genome Browser database) of retrogene introns in the parental gene. This file contains snapshots from the UCSC Genome Browser database displaying transcription annotations of retrogene introns in the parental geneClick here for fileSequences of primer pairs used to amplify the retrogenes and their parents. A table that lists primer pairs we used to amplify the retrogenes and their parentsClick here for fileProtein-level alignments of intron-gain retrogenes (“Sbjct”) and their parents (“Query”) by GeneWise. This file contains alignments of intron-gain retrogenes and their parents in protein levelClick here for fileNucleotide-level alignments of retrogene introns  and parental genes . This file contains alignments of intron-gain retrogens and their parents in DNA levelClick here for filePositions of three retrogenes  in the human genome (from the UCSC Genome Browser database). This file contains snapshots from the UCSC Genome Browser Database displaying the positions of three retrogenesClick here for fileComparison of splicing signals (percentile score) in the corresponding region of the new intron in the parental gene and neighboring introns. This file shows the results for the comparison of splicing signals in the corresponding region of the new intron in the parental gene and neighboring intronsClick here for fileGenetic variation of four retrogenes in different human populations. This file displays alignments of genomes of different human populations in the region of four retrogenes.Click here for file


Multilocus phylogenies can be used to infer the species tree of a group of closely related species. In species trees, the nodes represent the actual separation between species, thus providing essential information about their evolutionary history. In addition, multilocus phylogenies can help in analyses of species delimitation, gene flow and genetic differentiation within species. However, few adequate markers are available for such studies.In order to develop nuclear markers that can be useful in multilocus studies of mammals, we analyzed the mammalian genomes of human, chimpanzee, macaque, dog and cow. Rodents were excluded due to their unusual genomic features. Introns were extracted from the mammalian genomes because of their greater genetic variability and ease of amplification from the flanking exons. To an initial set of more than 10,000 one-to-one orthologous introns we applied several filters to select introns that belong to single-copy genes, show neutral evolutionary rates and have an adequate length for their amplification. This analysis led to a final list of 224 intron markers randomly distributed along the genome. To experimentally test their validity, we amplified twelve of these introns in a panel of six mammalian species. The result was that seven of these introns gave rise to a PCR band of the expected size in all species. In addition, we sequenced these bands and analyzed the accumulation of substitutions in these introns in five pairs of closely related species. The results showed that the estimated genetic distances in the five species pairs was quite variable among introns and that this divergence cannot be directly predicted from the overall intron divergence in mammals.We have designed a new set of 224 nuclear introns with optimal features for the phylogeny of closely related mammalian species. A large proportion of the introns tested experimentally showed a perfect amplification and enough variability in most species, indicating that this marker set can be very helpful in multilocus phylogenetics of mammals. Due to the lower variability and stronger stochasticity of nuclear markers with respect to mitochondrial genes, studies should be designed to make use of several markers like the ones designed here. Phylogenetic analyses of closely related species are affected by specific problems that are different from those present in phylogenies of more distant species . The firSystematic efforts to find novel markers for phylogenetic studies have been performed in different groups of organisms -21. MarkIn mammals, introns have been mainly used to resolve deep groupings but also to study more recent phylogenies -33. HoweWe extracted all introns smaller than 50,000 nucleotides from the genomes of human, chimpanzee, macaque, cow and dog, which comprise three mammalian orders . The total number of extracted introns per genome ranged between 153,659 in the cow and 173,320 in the dog. Using information from the ENSEMBL database, we arrived at an initial set of 11,835 one-to-one orthologous introns in the five mammalian species  were discarded because the design of PCR primers could be difficult in them. This filter affected 5.2% of the introns available in the previous step.Most of these introns had an inadequate length for their amplification and were therefore discarded in our initial filtering processes. First, we restricted the intron length in Mammalian genomes have a large number of duplicated genes due to different gene or genome duplication processes . These gNext, we eliminated introns that were too accelerated or decelerated in some lineages . That is, we selected introns whose rates of evolution were similar to the global genomic rate and therefore with a largely neutral molecular clock in mammals. Furthermore, trees with large branches in particular lineages may correspond to hidden paralogues that may have remained undetected up to this point. These paralogues are very problematic for estimations of evolutionary rates and other parameters necessary for their use in phylogenetic analyses. To detect this type of introns, we first constructed a global genomic tree from the concatenation of all introns available at this step Figure . This trThe next step was designed to eliminate the most conserved introns, which could be involved in some function and are therefore not variable enough for the phylogeny of closely related species. To do this, we employed two different measures of divergence. First, we calculated with the Ktreedist software the scaling factor from each intronic phylogenetic tree to the global genomic tree. This measure allowed us to discard the introns that showed the slowest overall evolutionary rates. Second, we calculated another measure of divergence, the primates total branch length, by using the corresponding alignments and trees with only the three primate species . This measure is more accurate and less affected by alignment imperfections possibly generated when comparing sequences that are too divergent. We then selected the introns that had a scaling factor lower than 1.15 and a primates total branch length higher than 0.075 substitutions per position. This rendered a dataset of 225 introns that excluded the most conserved ones.Finally, a visual inspection step was performed to detect any poorly aligned sequences caused by wrong annotations or clearly incorrect orthology assignments. Only one intron had to be eliminated in this step. The resulting final set was thus composed of 224 new phylogenetic markers Figure . In the Homo sapiens of each new marker is represented in Figure The genomic location in Repetitive sequences present in the intron set were analyzed with the RepeatMasker software. Of the 224 introns, 163 were found to contain repetitive elements. The most frequent elements were SINEs, which were present in 70% of the introns that bore any kind of element, followed by LINEs, which were found in 26% of them. Introns with repeats had, on average, 18% of their sequence corresponding to repetitive elements. These types of repetitive sequences normally evolve in a neutral way, mostly by point mutations, and therefore their presence is not a problem for sequence-based analysis methods that assume a normal nucleotide substitution model. If all species in the alignment have the repeat  this fragment can be used normally as any other sequence. When only some species have the repetitive element , we observed that alignment programs do not have problems dealing with these repetitive elements, and therefore the alignments can also be used for further sequence or phylogenetic analyses.More problematic are microsatellites . Since versus 0.0043 for the initial set. Thus, human SNPs also showed a slight increase in the genetic variability of the introns in the final set.In order to test the degree of variability of the 224 final introns with respect to the initial data sets, we estimated the genetic distance between human and chimpanzee for each intron. To do this, we constructed the alignments of the primate species , which could be used without further cleaning. Then, we estimated the corresponding maximum-likelihood trees and measured the patristic distances between human and chimpanzee. We then compared the distances obtained for the final set of 224 introns with the sets corresponding to the different filtering processes Figure , which cb, which is the most popular marker for mammalian phylogenetics [b distance between human and chimpanzee. To do this, we measured the patristic distance in a maximum-likelihood phylogenetic tree similarly reconstructed with human, chimpanzee and macaque cytochrome b sequences obtained from GenBank [b gene. Furthermore, alignments containing the exons flanking the 224 introns were constructed for the three primates, maximum likelihood trees were built as above, and the resulting trees were used to calculate the distance between Homo sapiens and Pan troglodytes for both the upstream and downstream exons of each intron. The resulting mean genetic divergence was 0.006 substitutions/position, which implies that the 224 selected introns have, on average, 2.3 more divergence than their corresponding flanking exons.To compare the mean intronic genetic distance with the divergence of cytochrome genetics , we obta GenBank . The resThe intron filtering processes carried out above were designed to select a set of optimal introns for the phylogeny of closely related mammalian species. However, many unidentified factors may affect the amplification of these introns in different species. To experimentally test the real performance of these introns and the validity of the designed bioinformatic analysis we randomly selected twelve introns among the largest ones in the final data set for sequencing in different mammals.Equus caballus), Sumatran orangutan (Pongo abelii), little brown bat (Myotis lucifugus), European hedgehog (Erinaceus europaeus), domestic cat (Felis catus), Northern tree shrew (Tupaia belangeri), gray mouse lemur (Microcebus murinus), African bush elephant (Loxodonta africana), lesser hedgehog tenrec (Echinops telfairi), nine-banded armadillo (Dasypus novemcinctus) and common shrew (Sorex araneus). These genomes mostly corresponded to low-coverage genome projects but many of them were valid for certain exons. Exonic alignments, containing between 11 and 15 different species, were constructed. These alignments allowed us to design the 3' end of every primer in the most conserved part of the exonic alignment. Degenerate bases (up to a limit of 48 per primer) were used to make the primers suitable for as many different mammalian species as possible.In order to design primers of wide spectrum, the flanking exonic sequences already gathered  were complemented by (whenever possible) a few others from the ENSEMBL database: horse , Bornean orangutan (Pongo pygmaeus), snow leopard (Uncia uncia), tiger (Panthera tigris), least weasel , and European polecat (Mustela putorius). This set included several closely related species in order to analyze their intronic divergence. Table Panthera tigris and CARHSP-1 and PNPO-3 for Myotis escalerai, where more specific primers had to be designed to improve the PCR band. The PCR bands of these introns were subsequently sequenced, and the sequences were compared to the known introns, which confirmed their correct amplification. In addition to the sequence of the PCR band, the exact sequence of one allele for each specimen was obtained by cloning the sequenced PCR product in a plasmid vector.To test these primers, we used genomic DNA extracted from six mammalian species: Iberian mouse-bat , the horse  and the little brown bat (Myotis lucifugus). This extended species set allowed us to assess the variability of our introns in five pairs of closely related mammalian species, which comprised four different mammalian families: Mustelidae, which included Mustela nivalis and M. putorius, with an estimated divergence time of 2.8 million years [Panthera tigris and Uncia uncia, which diverged 2.9 million years ago [Pongo pygmaeus and P. abelii, with 3.8 million years of estimated divergence [Homo sapiens and Pan troglodytes, with 6 million years of estimated divergence [Myotis lucifugus and M. escalerai, which diverged 12.2 million years ago [The sequenced introns were added to the introns already downloaded for human, chimpanzee, macaque, dog and cow, and this set was complemented by three additional genomes that had information for the seven successful introns: the Sumatran orangutan . Among the important criteria used to select these markers were that they belonged to single-copy genes, were not highly conserved, and did not show disparate rates of evolution in different lineages. The experimental validation of these introns showed that, after some optimizations, they could be amplified in different mammalian species, yielding a single PCR band. In addition, an analysis of the genetic distances estimated in several pairs of closely related mammalian species revealed that introns may show different degrees of divergence in different pairs, and that it is not possible to predict which introns will be more variable in each group. In any case, it may be necessary to carry out initial pilot studies with several introns to decide which ones perform best for each species or group of closely related species. In addition, the use of several introns will reduce the stochasticity associated to mutational and coalescence processes, which is particularly large for nuclear sequences. The availability of a large set of introns like the one provided here will greatly facilitate this work.Homo sapiens) version NCBI 36 [Pan troglodytes) version Pan_troglodytes-2.1 [Macaca mulatta) version Mmul_1 [Canis familiaris) version CanFam2.0 [Bos taurus) version Btau_3.1 [Monodelphis domestica [The following mammalian genomes were downloaded from the ENSEMBL database  in GenBa NCBI 36 , chimpanytes-2.1 , rhesus n Mmul_1 , dog  against one region of the genome. Since each genome is subdivided into large fragments, normally chromosomes, we checked that every single hit was also composed of a single subalignment and therefore corresponded to a single exon.To detect duplicated genes, BLAST searches  of the pA reference tree reflecting the global genomic evolution was constructed using all single-copy introns. To do so, the 1344 intron alignments that passed the BLAST filter were concatenated to generate an alignment of 768,745 positions. This alignment was used to build a maximum-likelihood phylogenetic tree using RaxML version 2.4.4, which can handle big alignments . PhylogeHomo sapiens genome was downloaded using the Biomart platform [Repetitive sequences present in the final set of introns were analyzed with the program RepeatMasker version 3.2.8 using the corresponding library for each species . The intplatform , and theMyotis escalerai); the Bornean orangutan (Pongo pygmaeus), obtained from the INPRIMAT Consortium and the Biomedical Primate Research Centre in Rijswijk; the snow leopard (Uncia uncia) and the tiger (Panthera tigris), obtained from the Animal Tissue Bank of Catalunya; and the least weasel  and the European polecat (Mustela putorius), obtained from Collection of Tissues and DNA of the Museo Nacional de Ciencias Naturales.Samples of six mammalian species spanning different orders were obtained. The chosen species were the Iberian mouse-bat (HM147892-HM147933.DNA extractions were performed using the DNEasy Blood and Tissue Kit (QIAGen), following manufacturer's instructions. PCR reactions were carried out in 25 μl of final reaction volume, containing 50 - 100 ng of template DNA, 0.5 - 1 μM of each primer, and 0.75 units of Promega GoTaq. Amplification conditions were as follows: initial denaturation at 95°C for 5 minutes, followed by 28 to 35 cycles comprising 30 seconds of denaturation at 95°C, 30 seconds of annealing at 52-65°C (this temperature turned out to be highly variable among different species and introns and had to be adjusted for each particular case) and extension at 72°C for 30-60 seconds. The resulting PCR products were loaded in 1% agarose gels stained with SYBR Safe DNA Gel Stain (Invitrogen). Bands were manually excised from the gel and subsequently purified using Illustra GFX PCR and DNA Gel Band Purification Kit . The resulting products were then sequenced using different sequencing services. Both heterozygous point mutations and indels that caused difficulties in assigning a clear sequence to each allele of the individuals were observed in several sequences. Therefore, we cloned each PCR product into the pstBlue-1 vector (Invitrogen) to be able to isolate one of the alleles. The plasmid containing one of the alleles was isolated from the bacterial cultures, purified using the GenElute Plasmid Miniprep Kit (Sigma Aldrich), and subsequently sequenced. Resulting sequences were analyzed using Geneious version 4.5.5 [For each successfully amplified and sequenced intron, a new multiple sequence alignment was created containing the six newly obtained sequences plus further sequences from ENSEMBL. As above, MAFFT was used to build these alignments, which were then cleaned using Gblocks with the same parameters as above. We constructed the phylogenetic trees using PhyML with the GTR model of evolution and four substitution rate categories. In addition, for each of these introns we analyzed the divergence for five pairs of closely related species. To do this, we constructed new pairwise intronic alignments for each species pair using MAFFT as above, but without applying Gblocks, and estimated maximum-likelihood distance using PAUP  with theJC and JI conceived and designed the experiments. JI did the experimental work and the main analyses. JJ contributed to the original design of the problem, participated in discussions and collaborated with samples. JC and JI wrote the paper. All authors reviewed and approved the final manuscript.Final set of 224 introns selected for the phylogeny of closely related mammalian species. The first page of each intron lists the following information: description (function of the gene to which the intron belongs), gene name according to the HUGO Gene Nomenclature Committee (in parenthesis), intron number, chromosome where it is located in Homo sapiens, intron start (the location of the first base of the intron in the corresponding human chromosome), human intron length, intron alignment length , flanking exons length (the size of both the upstream and the downstream exons that flank this intron in Homo sapiens), SNP density (the number of single nucleotide polymorphisms described for human in this intron divided by its length), K tree score , scaling factor , human-chimpanzee distance in substitutions per position (the maximum-likelihood distance between the introns of Homo sapiens and Pan troglodytes measured in the primates phylogenetic tree), and total primate branch length in substitutions per position . For the representation of the exon and intron alignments, a few wrong definitions of intron ends and starts that we found were manually corrected. The five-species alignments of both upstream and downstream exons are represented, but only the 160 bases closest to the intron are displayed. To help assess sequence conservation, positions with more than 50% identities are highlighted. The maximum-likelihood phylogenetic tree of the intron for the five species is also shown. The root was placed at 7% of the branch separating primates and laurasiatherians . The second page of each intron shows the five-species alignment of the intron. Introns are ordered in this document by total primate branch length.Click here for file


Similarly, large-scale analysis of intron dynamics in a few vertebrate genomes has identified only intron losses and no gains, indicating that intron gain is an extremely rare event in vertebrate evolution. These studies suggest that the intron-rich genomes of vertebrates do not allow intron gain. The aim of this study was to search for evidence of de novo introns gained in the domesticated genes during the evolution of mammals and chordates has been analyzed. A significant amount of intron gain was found only in domesticated genes of placental mammals, where more than 70 cases were identified. De novo gained introns show clear positional bias, since they are distributed mainly in 5' UTR and coding regions, while 3' UTR introns are very rare. In the coding regions of some domesticated genes up to 8 de novo gained introns have been found. Intron densities in Eutheria-specific domesticated genes and in older domesticated genes that originated early in vertebrates are lower than those for normal mammalian and vertebrate genes. Surprisingly, the majority of intron gains have occurred in the ancestor of placentals.A phylogenomic approach has been used to analyse all domesticated genes in mammals and chordates that originated from the coding parts of transposable elements. Gain of introns in domesticated genes has been reconstructed on well established mammalian, vertebrate and chordate phylogenies, and examined as to where and when the gain events occurred. The locations, sizes and amounts of This study provides the first evidence for numerous intron gains in the ancestor of placental mammals and demonstrates that adequate taxon sampling is crucial for reconstructing intron evolution. The findings of this comprehensive study slightly challenge the current view on the evolutionary stasis in intron dynamics during the last 100 - 200 My. Domesticated genes could constitute an excellent system on which to analyse the mechanisms of intron gain in placental mammals.Reviewers: this article was reviewed by Dan Graur, Eugene V. Koonin and Jürgen Brosius. Spliceosomal introns are one of the major eukaryote-specific genome components, and their evolution has been studied extensively during the last decade [for recent reviews see -11]. TheMaximum likelihood reconstruction of intron gain and loss in eukaryotes has revealed a significant excess of losses and a nonuniform distribution of gains and losses ,5,12-14.In some large taxonomic groups, such as in vertebrates and especially in mammals, no intron gain was observed in the genome-wide comparisons ,22,23. LSuch absence of intron gain in »recent« evolutionary history might be real, but could also be artefactual, the consequence of inadequate taxon sampling or inadequate comparisons, since only the »old« orthologous genes have been compared. To test the claims on the absence of intron gain in some taxonomic groups such as mammals ,22 and ide novo gained intron. The prerequisite for recognizing the origin, extent and timing of de novo gained introns is reliable and wide taxon sampling [Domesticated genes -31, origsampling . In the sampling ,35. By ude novo intron gain in domesticated genes of mammals from an analysis of their exon/intron structures. The location, size and amount of de novo gained introns in domesticated genes of mammals and chordates have been analyzed. Surprisingly, a burst of intron gain was found only in domesticated genes of placental mammals. De novo gained introns shows clear positional bias since they are distributed mainly in 5' UTR and coding regions, and rarely in 3' UTRs. Up to 8 de novo gained introns have been found in the coding regions of some domesticated genes, with intron density of 4 introns per kb. Surprisingly, the majority of intron gains have taken place in the ancestor of placentals. None of these cases of intron gain have been observed in any of the previous genome-wide analyses [In this study a phylogenomic approach  has beenanalyses ,22 as a analyses -39.de novo, either in the coding or untranslated regions of the gene. An extensive phylogenomic analysis of all domesticated genes in chordate and mammalian genomes has therefore been made. The exon/intron structures of domesticated genes were studied in detail throughout mammals, vertebrates and chordates. The most important part of this study was to find the transition point where and when TEs were transformed into domesticated genes, allowing de novo gain of introns to be precisely pinpointed in these genes.In the majority of previous genome-wide studies of the intron gain there was a remarkable degree of uncertainty concerning the ancestral intron states. Single copy domesticated genes originated from multicopy TEs, their parts or from their remains -31. SincThe rich collection of numerous mammalian genomes, belonging to all three major extant mammalian lineages, Eutheria , Metatheria  and Prototheria (monotremes), is a major advantage in studying the origin and evolution of domesticated genes. In the placental mammals, genomes of four superorders  are well represented. In addition to the mammalian genomes, all other available vertebrate and chordate genomes were analyzed in order to find the transition point from TEs to domesticated genes. Until now only a few studies of TE-derived domesticated genes have been published -31, but By the phylogenomic analysis of all available domesticated genes in mammalian, vertebrate and chordate genomes, unequivocal data about their origins (when and in which taxonomic group they originated) and numerous gene-related data  have been obtained. Crucial information about the transition points from TEs to domesticated genes has been obtained in this study. Even more importantly, the gene structures of domesticated genes has provided direct evidence for extensive intron gain in placental mammals. An extensive phylogenomic analysis of these genes will be presented elsewhere .The analysis of all known domesticated genes in chordates and mammals  from the entire DNA transposon, b) by a complete DNA transposon being fused to the »normal« gene in the form of a single long exon that is 3' end located, and c) the most prevalent case, by gene fusion or exon shuffling of DNA binding domains (DBD) of DNA transposons with »normal« genes (where the exonization is necessary before the gene fusion). Therefore, in the case of DNA transposon-derived genes, intron gain can be recognized easily only in the first case, while the second and a third cases are much more difficult for inferring intron gain in these genes. In the majority of the cases of fused entire transposases or just the DBDs, the newly recruited exons remain intact as very long or relatively short exons, but they are mostly without any intron. Therefore, in the case of DNA transposon-derived genes, these fused transposases and DBDs have been excluded from the analysis of intron gain. Progenitors of novel domesticated genes were from diverse DNA transposon superfamilies, such as from Tc1/piggyBac ,31.Of the 36 analyzed orthologous DNA transposon-derived genes, only 11 have been used for the inference and timing of intron gain  . 34 orthologous retroelement-derived genes were analyzed and intron gain was found in 27 genes , there was a large scale gene origination in the ancestor of placentals (evolutionarily young genes), at least in some classes of transcription factors e.g. in C2H2 ZNFs). To test the extent of intron gain in some other placental-specific gene families I analyzed the presence of intron gain in KRAB and SCAN ZNF genes ,44, espeH2 ZNFs. de novo gained introns in domesticated genes vary greatly. In the case of DNA transposon-derived genes   I have cn humans . Since mron gain ,7, howevThe availability of RefSeq genes ,35 and nde novo gained introns were evolutionarily conserved in mammals. The intron sequences of all human domesticated genes were compared to sequences obtained from WGS genomic sequences from the representatives of all placental superorders. Surprisingly, intron sequences were highly conserved between human and Afrotheria in only 9 out of 19 retroelement-derived genes, showing an overall conservation of 70 - 75% between the representatives of all four superorders of placental mammals analyzed  has shown that numerous intron gains occurred in the ancestor of placentals and that intron gain is still ongoing in mammals. At least 50 - 70 cases of intron gain can be documented from the analysis of >30 domesticated genes and retrogenes, and a few more cases can be documented also for placental-specific transcription factors . Up to 100 cases of intron gain are recognized from the analysis of ~200 orthologous genes. These intron gains have occurred at different time points of placental evolution, the vast majority of them in the ancestor of placentals and the others in diverse lineages or species of placental mammals.de novo intron gain. The availability of numerous mammalian and vertebrate genomes was crucial for tracing the origin of these genes, the origin of specific exon/intron structures and for determining the extent of intron gain.This study provides clear evidence for extensive intron gain in the ancestor of placental mammals. Vertebrate, and especially mammalian, genomes contain a number of genes that have originated from transposable elements (TEs) or their remains -31. SincIt has been shown that intron losses outnumber intron gains in eukaryotic orthologous genes ,14. Genode novo gained introns is similar to that reported in diverse eukaryotic lineages [Rates of intron gain in the past tens to hundreds of million years in diverse eukaryotes have been very low ,12-14. Slineages ,17,20. Tde novo gain of introns in these genes to be pinpointed precisely. The number of gained introns in these genes varies greatly, from 1 to 8. Domesticated genes in placentals and chordates accumulated a large number of introns, such that their density in these genes has become close to that in »normal« genes [An extensive phylogenomic analysis of all the domesticated genes in chordate and mammalian genomes has provided crucial information as to where and when TEs were transformed into domesticated genes, allowing l« genes . The avel« genes ,14. The l« genes . The selde novo gained introns in domesticated genes are the 5' UTRs and coding regions, while 3' UTR locations are very rare. These preferred intron locations are similar to those of the »normal« chordate genes [de novo gained introns in domesticated genes differ from the recently reported case of mammalian retrogenes, where newly gained introns are preferentially located in the 5' UTRs [The sizes of the gained introns in domesticated genes are highly variable, ranging from a few hundred to a few thousand base pairs. DNA transposon-derived genes contain longer introns than retroelement-derived genes, just as evolutionarily older domesticated genes contain much longer introns than evolutionarily younger domesticated genes. Surprisingly, the longest introns exist in the gag-derived ZCCHC16 gene, and the second intron in the mouse (~410 kb long) is also the longest intron in the chordate domesticated genes. This gene resembles mammalian retrogenes with very long introns . The prete genes . However 5' UTRs .The extensive information on intron position conservation collected from the genomic alignments for all placental-specific intron gains has shown that the great majority of intron positions in placental-specific domesticated genes are highly conserved. The great majority of gained introns in domesticated genes have been fixed in the eutherian ancestor, as demonstrated by their presence and sequence conservation in all eutherian superorders. From the genomic alignments we can readily trace the genes and their exons and introns compared at the nucleotide level over quite large evolutionary distances (at least 80 - 100 Myr) and, what is surprising, see remarkable level of conservation at the nucleotide level.de novo gained introns has been analyzed, and a striking conservation of the intron sequences in their entire length was found in 9 out of 19 retroelement-derived domesticated genes, showing 70 - 75% nucleotide identity between humans and Afrotheria, Xenarthra and Laurasiatheria. Comparison of the entire domesticated genes between human and the representatives of all placental superorders also shows ~75% nucleotide identity between humans and Afrotheria, Xenarthra and Laurasiatheria. Conservation of intronic sequences has been observed in some other Boreoeutheria genes [The rate of sequence divergence in introns is very high, therefore many introns are less conserved in sequence between organisms than their associated exons ,52. The ia genes ,53. It hia genes , therefoia genes . The mutia genes ,55. The ia genes ,53.Alu SINEs that can change the mode of splicing of the flanking exons [The analysis of domesticated genes in ASTD database has shown the presence of alternative splicing. Up to 14 alternative splicing events can be seen per domesticated gene. It is interesting that more alternative splicing events can be seen in human than in mouse orthologous genes. Alternative splicing in domesticated genes may have originated by mutations in splicing sites (evolution of weaker splice sites), by sequence changes in the intronic and exonic splicing silencers or enhancers (generating lower or higher densities) or by accumulation of ng exons ,48. Compng exons ,57. By sng exons ,48.de novo gained introns in the ancestor of placental mammals probably being much higher. This study pointed to the serious problems arising from comparison of orthologous introns in coding regions only and from sparse taxon sampling in the genome-wide analyses of intron gain [The presence of ~100 intron gains in placental mammals is remarkable, and clearly represents just the tip of the iceberg, the number of ron gain ,22,23. Nron gain  or distaron gain ,22 introTo obtain a genome-wide insight into the extent of intron gain in placental mammals, highly accurate exon/intron structures  of genes from marsupials and platypus are needed, as well as from the key Afrotheria  and Xenarthra  species. The major problem in comparative genomics of mammals is the quality of sequence data for just 2× genome covered species . Therefode novo intron gain from the analysis of their exon/intron structures. De novo gained introns show clear positional bias, since they are distributed mainly in 5' UTR and coding regions, while 3' UTR located introns are very rare. Large numbers of de novo gained introns have been found in the coding regions of some domesticated genes, reaching as many as 8 introns per gene. Surprisingly, the majority of intron gains have occurred in the ancestor of placentals, more than 160 Mya. None of these cases of intron gain were observed in earlier genome-wide analyses of limited numbers of evolutionarily younger placental species. All previous claims for the absence of intron gain in mammals, or in the last 100 - 200 My of eukaryotic evolution, were the consequence of inadequate taxon sampling and the comparison of only the »old« orthologous genes. Thus, as this study has demonstrated, adequate taxon sampling is crucial for the reconstruction of intron dynamics and evolution.The first evidence for intron gain in the ancestor of placental mammals has been provided. A phylogenomic analysis of domesticated genes in mammals and chordates has provided evidence for http://www.ensembl.org and the nonredundant (NR), EST, GSS, HTGS, WGS, as well as the diverse taxon-specific  genome databases at the National Center for Biotechnology Information (NCBI) http://www.ncbi.nlm.nih.gov. Comparisons were performed using the diverse BLAST tools [-5 and other parameters to default settings. Domesticated genes, as well as diverse gag, integrase and transposase domains, have been used as queries. DNA sequences were translated using the Translate program http://www.expasy.org/tools/dna.html. The reference set of human representatives of the domesticated genes is available in Additional file The databases analyzed were the Ensembl ST tools  with thede novo gained introns in domesticated genes of mammals and chordates have been analyzed. The availability of RefSeq genes [The locations, sizes and numbers of eq genes ,35 and nAll the nonredundant representatives of the gag-, integrase- and DNA-transposon-derived domesticated genes have been included in the analyses. Protein or nucleotide sequences were aligned using Clustal W2 . All theThe extensive information on intron position conservation has been collected from the Ensembl genomic alignments for all placental-specific intron gains. By this approach the genes and their exons and introns can be compared at the nucleotide level over quite large evolutionary distances (at least 80 - 100 Myr) and a remarkable level of conservation at the nucleotide level can be demonstrated. The human orthologous gene was always aligned with at least one representative of the placental superorders Afrotheria, Xenarthra, Laurasiatheria and Euarchontoglires. The intron density of a gene is given as the number of introns per kb of coding sequence (introns in CDS) or as number of introns per kb of 5' UTR sequence (5' UTR introns).All domesticated genes have been checked in the ASTD database (Alternative Splicing and Transcript Diversity 1.1)  for the http://www.repeatmasker.org[TE content was analyzed using the RepeatMasker web site asker.org.DBD: DNA binding domain; LCA: last common ancestor; Mya: million years ago; My(r): million year; TE: transposable element; UTR: untranslated region; ZNF: zinc finger.The author declares that they have no competing interests.DK designed and executed the analyses, and wrote the manuscript.Dan Graur, University of Houston, United States of AmericaNo comments. An interesting idea.Eugene V. Koonin, NCBI, NLM, NIH, United States of AmericaKordiš reports apparent gain of a large number of introns in "young" vertebrate genes, primarily in genes that evolved from domesticated transposable elements. A burst of intron insertion is mapped to the origin of placental mammals. Clearly, this is a very interesting and important finding that runs against the numerous published observations of the (near) lack of intron gain throughout the evolution of mammals or even the evolution of vertebrates.Author's response: I agree with the above statement.However, the "refutation" of the intron stasis scenario should be taken in perspective. This entire study involves approximately 200 "young" genes, and not all of them have gained many introns. However interesting, this finding pertains to a small fraction of genes in any given animal genome, so quantitatively this is a small correction to the stasis picture. There is no need to over-dramatize the revision of the existing views. This is not to deny the importance of the result. To me, the emerging pattern of evolution of eukaryote gene architecture is simple: whenever an intron-less gene is added to an intron-rich genome via any route, it is rather quickly saturated by introns until intron density close to that in "old" genes is reached. In this respect the results of this work extend and generalize the previous observations on chloroplast-derived genes in plants (Ref. 26) The selective and/or neutral factors affecting this saturating insertion of introns remain unknown and seem to be of great interest, and so are the molecular mechanisms of intron insertion. One has to agree with the author of the article that domesticated genes are an excellent model to study these fascinating and fundamental problems.Author's response: I really don't like to refute your intron stasis scenario and to overdramatize the revision of the existing views about the extent of intron gain. As this study pointed out, in the previous genome-wide analyses of intron gain  the serious problem was the comparison of orthologous introns in coding regions only and the sparse taxon sampling. None of the cases reported here were observed in the previous studies of closely related  or distantly related species . In the closely related mammalian species analysed, intron gains occurred before those species originated. In comparisons of distantly related vertebrate species only »old« orthologous genes have been compared, and evolutionary novelties were excluded from such analyses, however the neglected intron gains occurred after the analysed species originated. Therefore, as this study shows, the overall extent of intron gain could be much higher than reported in previous studies. As this study demonstrated, the solution to the above problems is to analyse the highly neglected evolutionary gene novelties at particular time points . This study therefore provides a further cautionary example in using only closely or distantly related species and sophisticated statistical methods in directionalizing intron loss/gain events, and underscores the importance of using appropriately selected taxa and evolutionary gene novelties for accurate inferences of genome evolution.The article as it stands now presents many omissions and lost opportunities some of which seem to be a must for revision whereas others are extensions of the work done that perhaps could be postponed until subsequent publications. Here are several specific areas where the submitted manuscript appears to be amiss. The information on intron position conservation probably should be much more detailed and specific and should be presented in the main paper not in an additional file, probably in the form of a table or figure.Author's response: As requested I have added the extensive information on intron position conservation in a large supplementary file that contains genomic alignments for all placental-specific intron gains. This has the advantage over presentation in a table or figure, since one can readily see the genes and their exons and introns compared at the nucleotide level over quite large evolutionary distances (at least 80 -100 million years) and, what is surprising, see the remarkable level of conservation at the nucleotide level. The human gene is always aligned with at least one representative of the placental superorders Afrotheria, Xenarthra, Laurasiatheria and Euarchontoglires. In this way a direct insight into the extent of conserved intron positions can be seen, as well as the several cases of highly conserved introns. It should be noted that most of the mammalian genomes are now available as low-coverage (~2×) assemblies. Sequencing errors in some of these genomes can still have surprising effects for apparent lineage-specific insertions of introns. A careful reinspection of such a data is always needed to exclude sequencing errors.The inferred number of gained introns should be given specifically, claiming a "remarkable number" is not enough.Author's response: This suggestion is accepted and the inferred numbers of gained introns are now given specifically.In the same vein, I believe that specific numbers should be presented on intron densities in the domesticated genes (per kilobase not per gene) and compared to the intron densities in other genes.Author's response: I calculated intron densities for all Eutheria-specific intron gains as well as for some cases of older intron gains that occurred during the evolution of vertebrates. I also compared these data with the intron densities in other genes. The requested figure showing the histograms of intron densities in gained introns compared to other genes has been added to the MS.Further, it is unclear to me why not performing a formal, maximum-likelihood reconstruction of the history of intron gains and losses in the analyzed genes; this can be easily done using the COUNT software :1910-2). Such a reconstruction would yield a more robust and more informative evolutionary scenario.Author's response: I have added genomic alignments for all placental-specific intron gains. These data clearly demonstrate that the massive intron gain has occurred only at the particular time point, namely in the ancestor of placental mammals. The numbers of additional intron gains or losses are indeed very small and are restricted to the particular species or to the small taxonomic groups. Therefore maximum likelihood analysis was not practical because of the small number of potential intron gains and losses.This suggestion also brings up the issue of illustration. The current manuscript contains no figures which is strange considering the subject. For example, it is easy to imagine a figure showing the phylogenetic tree of vertebrates with the ML estimates of intron gains and losses in the analyzed genes mapped to specific branches.Author's response: I know that it is strange that the current version of manuscript contained no figures. As explained above the requested phylogenetic tree of vertebrates with the ML estimates of intron gains and losses in the analyzed genes mapped to specific branches was not practical because of the small number of potential intron gains and losses. This MS clearly demonstrates that the only case of massive intron gain in the domesticated genes occurred only at one particular time point, namely in the ancestor of placental mammals. However, outside this evolutionary time point the additional intron gains or losses are very small and are restricted to particular species or to small taxonomic groups.Other figures are easy to come up with as well, for example, histograms of intron densities. Some interesting possibilities are not pursued at all, e.g. comparing the structures of the splice sites for the new and older introns.Author's response: The requested figure showing the histograms of intron densities in gained introns compared to those in other genes has been added to the MS. Intron densities are indeed not as high as in the typical mammalian genes, but still they are remarkable with 4 introns/kb of exonic sequence. I checked also the structure of the splice sites for the new and older introns but no differences in the conservation of splicing signals can be found, as already reported for some other cases of intron gains. All gained introns use canonical splice sites without any deviations.Also, do any of these genes undergo alternative splicing? Knowing this could provide a clue to the evolutionary factors behind intron saturation.Author's response: In the ASTD database (Alternative Splicing and Transcript Diversity 1.1) I checked all domesticated genes for the presence of alternative splicing and found several examples of alternative splicing. It is interesting that larger amounts of alternative splicing are observed in DNA transposon-derived genes than in the retroelement-derived genes. In some genes (e.g. human PNMA2 gene) up to 14 alternative splicing events have been documented in the ASTD database. It is interesting that the majority of these alternative splicing events can be found only in humans but not in mouse. Alternative splicing events can be traced only in a limited number of mammals  - no data are available for any other placental lineage. There are numerous possible causes of alternative splicing events, from the intronic silencers or enhancers, exonic silencers or enhancers, to the diverse repetitive elements. In humans, it has been demonstrated that Alu SINEs can generate alternative splicing. Since most of the alternative splicing events are limited to humans the involvement of Alu SINEs is among the most interesting possibilities. The presence of alternative splicing events exclusively in humans indicates that these events are indeed quite recent, but the absence of comparative data cannot provide their precise timing. However, since human and mouse belong to the same eutherian superorder Euarchontoglires, the timing of these events can be more restricted to the primates or to the younger lineages within primates (e.g. to Hominoidea). Alternative splicing has also been implied in the intronization model of intron gain, however, I believe that this model cannot explain the observed situation. As is evident, all (or the vast majority) of gained introns in domesticated genes have been fixed in the eutherian ancestor, as demonstrated by their presence and sequence conservation in all eutherian superorders. But the alternative splicing events can be found, in the majority of cases, only in humans. This may indicate very recent, probably regulatory, adaptation in the human lineage.Finally, I find the conservation of the actual sequences of introns illustrated in Additional File Author's response: The requested discussion of the conservation of the intron sequences has been added to the MS.Jürgen Brosius, University of Muenster, GermanyThis is an interesting approach towards detection of relatively novel events of intron generation. The author is using genes that were derived from, by nature intronless, transposable DNA elements or transposed retroelements. This has the advantage that events that occurred over the past 100 to 200 million years could be detected. My main problem with the current version is the terminology of intron gain. Looking at the events, it turns out that in many cases no novel sequences actually were gained but existing sequences converted to introns. Perhaps the term intronization would fit better and I would recommend the author to search PubMed using that term. There are 10 matches and the first use of the term intronization is from Gromoll et al. (2007). The paper merely describes the disuse of one of the exons in the LH receptor type I and type II genes. A second early and relevant paper describes an Alu element induced intronization and could be cited as well .Author's response: From the literature data we can only see a few cases of intronized sequence in the normal gene that already contained introns . All such introns were quite short (~50 bp) in both Caenorhabditis  and Cryptococcus (Roy 2009). For me it is highly unlikely that several quite long introns in domesticated genes have been generated by the intronization of coding sequences. The main reasons for this are the sizes of the ancestral gag and integrase domains, which are in the range of 250-400 amino acids. The sizes of the domesticated genes are in the range from ~100 (contraction) to ~2000 (expansion) amino acids. Therefore it is highly unlikely that existing sequences have been converted to introns in domesticated genes. It was estimated recently (Roy 2009) that intronization may not be a major overall contributor to modern day gene structures and that intronization is unlikely to explain a large fraction of modern introns. I think that nonsense-mediated decay (NMD) is the more likely mechanism to enable intron gain.In several parts of the manuscript the author mentions a "significant amount of intron gain" or the "remarkable number of de novo gained introns etc. This is relative and it would be better to provide at least approximate numbers instead.Author's response: This suggestion is accepted and most cases were changed into the approximate numbers.At the end of the background section the author mentions studies restricted to the superorder Boreoeutheria. The original publication concerning the classification is Springer et al. (2001).Author's response: This suggestion is accepted and the original citation has been added to the MS.How can the author rule out that young genes with introns in fact encode non-protein coding RNAs that acquired introns or simply feature insertions that are not being removed by splicing? It would be useful to provide information on how many of the transcripts are supported by bona fide messenger RNAs, or at least ESTs, or even better by protein sequence data. In additional file Author's response: All these domesticated genes are protein coding genes and a few may also encode non-protein coding RNAs. Domesticated genes are also highly expressed in particular tissues . Rich collections of EST/expression profiles for all these genes are present in the literature as well as in the NCBI Genes section, at the Gene Expression Atlas and in many additional transcriptomic databases. In any case, for the annotation of the well covered taxa (such as human and mouse) numerous ESTs and cDNAs exist and helped before in establishing their exon/intron structures at the Ensembl and NCBI Genes as well as for the recognition of some cases of alternative splicing events.The frequently mentioned fact that novel introns can be quite large is not surprising. Once an intron had been generated, the floodgates are open for constant bombardment with transposed elements  potentially increasing the sizes of such introns to a great extent over the past 100 to 200 million years.Author's response: I agree with this statement. Some of the gained introns are indeed large or even huge. I checked the TE repertoires of some of these introns in different species by RepeatMasker. Up to 50% of the intron size can be occupied by diverse TEs. Different species of placental mammals  shows unique lineage- or species-specific TE content in these introns. This finding indicates that introns in each species are indeed under constant bombardment with TEs.In general, it would be interesting to obtain information as to the origin of the sequences that now are being used as introns. My guess is that most of the sequences will be derived from pre-existing sequences at the corresponding loci previously serving as exons or intergenic regions. As mentioned above, one would expect additional TEs that were inserted after the initial intronization. Should the initial intronic sequence be derived from sequences outside the genes' loci, one might obtain clues about events including mobile element insertions that contributed to such novel introns.Author's response: Despite a rigorous search I could not identify homologous parental origin for any novel intron elsewhere within the placental and marsupial genomes, consistent with the other studies about the intron gain. Homology searching has shown just orthologous introns in placental mammals, but the sources of these sequences are still unknown. Searching for TEs in the long introns with RepeatMasker has shown the presence of species-specific repeats in the analysed species. This means that these TEs has been inserted in the particular lineages . Regarding the current division of Eutheria into three sister groups Afrotheria, Xenarthra and Boreoeutheria it is evident that the majority of intron origination events in domesticated genes has occurred in the eutherian ancestor, but they were later independently bombarded with lineage-specific TEs in all three eutherian sister groups and also inside Boreoeutheria in Laurasiatheria and in Euarchontoglires (as evident from the big differences in TE repertoires in these introns between rodents and primates). I agree that it is possible that some intergenic regions might also serve as a source of introns. This will be more extensively explained in a separate MS  by the analysis of conserved synteny in Eutheria-specific domesticated genes. It should be noted that by homology searching with introns of domesticated genes in three marsupial genomes no homologous sequences can be found. Since these genes originated in the LCA of placental mammals no orthologs (except a few cases: e.g. PEG10) can be found in marsupials. Knowing the limits of nucleotide homology-searching for unconstrained sequences it is impossible to infer the origin of intron sequences in domesticated genes.In any event, as mentioned before, the terminology "intron gain" leads to the expectation that there is perhaps a general mechanism in placental mammals leading to the de novo generation of intron sequences. A more thorough investigation will probably show that most events will be due to "bricolage" or tinkering  via random mutations including small or large indels leading to jury-rigged outcomes including intronization.Author's response: It has been demonstrated recently by the Lynch group  and others  that the cellular surveillance systems have played a crucial role in the endogenous origin of introns throughout the eukaryotes. I think that these explanations are more appropriate than those suggested by the reviewer.In the paragraph titled "ongoing gain and loss of introns in domesticated genes: evidence from comparative genomics in mammals" the author refers to the great diversity in the numbers and positions of introns in younger orthologous placental specific domesticated genes and states that this indicates that some introns have not been fixed yet. This would agree with phylogenetic studies using transposed element insertions showing that during periods of rapid speciation of placental mammals such markers can be polymorphic (unfixed) and phylogenetically unreliable due to incomplete lineage sorting .Author's response: I agree with this comment.Often it is mentioned that the majority of intron gains have occurred in the ancestor of placental mammals  a fossil of a Jurassic eutherian mammal (Juramaia sinensis) was found. This fossil is 160 My old and provides new estimates for the divergence of marsupials and placentals. It extends the first appearance of the eutherian-placental clade by about 35 Myr from the previous record, it also has reduced and resolved a discrepancy between the previous fossil record and the molecular estimate for the placental-marsupial divergence. Because Juramaia is unambiguously placed on the placental side of the marsupial-placental divergence, the marsupial-placental divergence must have occurred before Juramaia. Therefore this new fossil serves to reset the minimal age at 160 Myr for the basal-most diversification of marsupials and placentals. This new eutherian fossil age is now similar to the age of placentals at 160 Myr with 95% posterior distribution from 143 to 178 Myr by the latest molecular estimate.The tables in the main section need further explanation: for example, what is the significance of a range for intron numbers such as 5 to 13 for the POGZ gene etc., Table Author's response: Intron numbers are variable in some genes or in some species, this is connected to the loss or additional gain of introns. »5' UTR or coding«: precise location is always clear, either in 5' UTR or in coding region. These genes in human or mouse have been really correctly annotated (at the Ensembl or at the NCBI Genes), but in some other low coverage genomes erroneous annotations can still be found. Explanations:-5' UTR/coding: introns are present both in 5' UTR and in coding region (for example: Homo RGAG1 gene contains 2 introns in 5' UTR and one intron in coding region),-5' UTR or coding: introns are present either in 5' UTR or in coding region (for example ZCCHC12 gene: in humans three introns are present in 5' UTR while in chimp 5 introns in coding region were reported (erroneous annotation that was corrected very recently),-5' UTR or 3' UTR: introns are present in one species in 5' UTR and in the other species in 3' UTR .The fact that introns are readily being generated in the 5' flanking regions of retrogenes has been discussed as early as 1992 by Brosius and Gould. This could be cited.Author's response: This reference has been added to the MS.Where the author mentions that "the highest rate of recent intron gain yet observed in genome-wide ortholog comparisons was in ascomycetous fungi" he could add the publication of Denoeud et al. (2010) on massive intron gain in the tunicate Oikopleura dioica.Author's response: The citation about the massive intron gain in Oikopleura genome has been added to the MS.Also, recent work on the SCAN domain, which is derived from the C-terminal portion of the gag capsid (CA) protein from the Gmr1-like family of Gypsy/Ty3-like retrotransposons could be cited . This paper and others  show that parts of retrogenes contribute to novel protein domains to existing genes, giving an alternative explanation to some of the "intronization" events.Author's response: The requested reference has been added to the MS.The last part of the last sentence of the first paragraph in the results section is unclear and should be modified.Author's response: The unclear sentence has been clarified.References that the author could cite:Baertsch R, Diekhans M, Kent WJ, Haussler D, Brosius J. Retrocopy contributions to the evolution of the human genome. BMC Genomics. 2008 Oct 8;9:466.Brosius J, Gould SJ. On "genomenclature": a comprehensive (and respectful) taxonomy forpseudogenes and other "junk DNA". Proc Natl Acad Sci U S A. 1992 Nov 15;89(22):10706-10.Brosius J. Genomes were forged by massive bombardments with retroelements and retrosequences. Genetica. 1999;107(1-3):209-38.Churakov G, Kriegs JO, Baertsch R, Zemann A, Brosius J, Schmitz J. Mosaic retroposon insertion patterns in placental mammals. Genome Res. 2009 May;19(5):868-75.Emerson RO, Thomas JH. Gypsy and the birth of the SCAN domain. J Virol. 2011 Aug 24.Denoeud F, et al. Plasticity of animal genome architecture unmasked by rapid evolution of a pelagic tunicate. Science. 2010 Dec 3;330(6009):1381-5.Gromoll J, Lahrmann L, Godmann M, Müller T, Michel C, Stamm S, Simoni M. Genomic checkpoints for exon 10 usage in the luteinizing hormone receptor type 1 and type 2. Mol Endocrinol. 2007 Aug;21(8):1984-96.Jacob F: The Possible and the Actual .Sela N, Mersch B, Gal-Mark N, Lev-Maor G, Hotz-Wagenblatt A, Ast G. Comparative analysis of transposed element insertion within human and mouse genomes reveals Alu's unique role in shaping the human transcriptome. Genome Biol. 2007;8(6):R127.Springer MS, de Jong WW. Phylogenetics. Which mammalian supertree to bark up? Science. 2001 Mar 2;291(5509):1709-11.Warren WC et al. Genome analysis of the platypus reveals unique signatures of evolution. Nature. 2008 May 8;453(7192):175-83.Author's response: Some of these references have been added to the MS.Reference set of human domesticated genes.Click here for fileIntron gain in Eutheria-specific KRAB-ZNF, SCAN-ZNF and KRAB-SCAN-ZNF genes. Intron numbers are shown for the well annotated human genes. The ancestral state for KRAB-ZNF genes is 1 to 2 introns in the coding region and 1 intron in the 5' UTR. The ancestral state for SCAN-ZNF genes is 1 intron in the coding region and 1 intron in the 5' UTR. The ancestral state for SCAN-KRAB-ZNF genes is 2 to 3 introns in the coding region and 1 intron in the 5' UTR.Click here for fileIntron sizes in human domesticated genes.Click here for fileConservation of intron positions in domesticated genes.Click here for fileAlternative splicing in PNMA2.Click here for fileOngoing gain and loss of introns in domesticated genes.Click here for fileHighly conserved introns in domesticated genes of placental mammals.Click here for fileLineage-specific accumulation of TEs in orthologous introns in four placental superorders.Click here for file


Hereford population , estimating effects of imposed selection within L1 based on allele frequencies at 50 K SNP loci, and examining loci-specific effects of heterozygosity on the selection criterion. Animals were genotyped using the Illumina BovineSNP50 Beadchip, and SNP were mapped to UMD3.0 assembly of the bovine genome sequence. Average linkage disequilibrium (LD), measured by square of Pearson correlation, of adjacent SNP was 0.36 and 0.16 in L1 and AHA, respectively. Difference in LD between L1 and AHA decreased as SNP spacing increased. Persistence of phase between L1 and AHA decreased from 0.45 to 0.14 as SNP spacing increased from 50 to 5,000 kb. Extended haplotype homozygosity was greater in L1 than in AHA for 95.6% of the SNP. Knowledge of selection applied to L1 facilitated a novel approach to QTL discovery. Minor allele frequency was (FDR < 0.01) affected by cumulative selection differential at 191 out of 25,901 SNP. With the FDR relaxed to 0.05, 13 regions on BTA2, 5, 6, 9, 11, 14, 15, 18, 23, and 26 are co-located with previously identified QTL for growth. After adjustment of postweaning gain phenotypes for fixed effects and direct additive genetic effects, regression of residuals on genome-wide heterozygosity was −235.3 ± 91.6 kg. However, no SNP-specific loci where heterozygotes were significantly superior to the average of homozygotes were revealed (FDR ≥ 0.17). In conclusion, genome-wide SNP genotypes clarified effects of selection and inbreeding within L1 and differences in genomic architecture between the population segment L1 and the AHA population.Hereford is a major beef breed in the USA, and a sub-population, known as Line 1 (L1), was established in 1934 using two paternal half-sib bulls and 50 unrelated females. L1 has since been maintained as a closed population and selected for growth to 1 year of age. Objectives were to characterize the molecular genetic architecture of L1 ( L1) was established in 1934 by joining two paternal half-sib bulls with 50 unrelated Hereford females. Thereafter, L1 has been selected primarily for growth to 1 year of age and continuously maintained as a closed population  may identify non-random associations of linked loci and may indicate population divergence ; second, by estimating effects of the imposed selection on allele frequencies and; finally by examining loci-specific effects of heterozygosity on the selection criterion.http://www.ars.usda.gov/Main/docs.htm?docid=6238), while the remaining bulls (N = 255) were chosen by the American Hereford Association as being generally representative of the Hereford breed. A pedigree consisting of 21,284 Herefords, including 9,419 L1 Hereford cattle was used herein.Two Hereford populations were used in this research: L1 located at Fort Keogh Livestock and Range Research Laboratory, Miles City, MT and registered Herefords sampled from breeders across the US. The L1 population was founded by two paternal half-sib bulls and 50 cows in 1934 and has been selected primarily for growth to 1 year of age since that time. MacNeil  reviewedr2 > 0.99, details in later text) had one SNP removed from the pair. Numbers of SNP used in different analyses are shown in Table All animals were genotyped using the Illumina BovineSNP50 Beadchip , with genotypes coded as 2, 1, or 0 to represent the number of copies of the minor allele. LD was calculated for SNP pairs in the same chromosome that had spacing between 50 to 10,000 kb. More than 91 million SNP pairs that had different spacing were used to estimate LD for L1 and AHA. Chromosome-wide LD was also estimated by using average r2 of adjacent SNP. Genome-wide average distance between adjacent SNP was 238 kb.To characterize L1 and AHA, LD was quantified as the square of the Pearson correlation between SNP genotypes  for postweaning gain in L1 were calculated following MacNeil et al. (Iσe2). Two sets of putative QTL are presented from this analysis. In the first analysis, false discovery rate (FDR) with a critical threshold of 0.01 was employed to adjust for multiple testing  that can detect the non-additive proportion of genetic variance was employed here by modifying the method of Gulisija et al.  and pruning SNP pairs in complete LD , L1 had 13,850 fewer remaining SNP than AHA.r2 of two adjacent SNP differed in different chromosomes, particularly in L1  affected by CSD at 191 out of 25,901 SNP. When the FDR was relaxed to 0.05 there were 1,081 significant SNP clustered into 186 candidate regions . As shown in Table The regression of EBLUP residuals for postweaning gain on inbreeding coefficients  was −349.2 ± 68.2 kg. This is consistent with previous results from L1 where the regression was estimated simultaneously with the other fixed effects . L1 had greater average LD than AHA, indicated by higher correlation of SNP genotypes within L1 and fewer haplotypes spanning the genome. The difference in LD between L1 and AHA decreased as SNP spacing increased, indicating their relatively recent divergence. Persistence of phase indicated that the large LD generated by the bottleneck which occurred at the formation of L1 has eroded. Knowledge of selection applied to L1 since its inception provided an opportunity for a novel approach to QTL discovery. Coupled with previous reports of QTL for growth traits in beef cattle, this approach provided 13 candidate regions for further investigation. These data also reveal no loci affecting postweaning growth where the heterozygotes were significantly superior to the average of the homozygotes.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.http://www.frontiersin.org/Livestock_Genomics/10.3389/fgene.2012.00285/abstractfgene.2012.00285/abstractThe Supplementary Material for this article can be found online at Supplementary Table S1Genomic regions wherein the regression (b ± SE) of number of minor alleles on cumulative selection differential for postweaning gain was significant (FDR < 0.05) in L1.Click here for additional data file.


Z2 topological insulator. To illustrate this idea, we consider both Kane-Mele (graphene-based) and Bernevig-Hughes-Zhang models: the latter is proposed for HgTe/CdTe 2D quantum well. Much focus is on the role of valley degrees of freedom, especially, on how they are projected onto and determine the 1D edge spectrum in different edge geometries.A graphene nano-ribbon in the zigzag edge geometry exhibits a specific type of gapless edge modes with a partly flat band dispersion. We argue that the appearance of such edge modes are naturally understood by regarding graphene as the gapless limit of a  K- and K'-valleys--in the first Brillouin zone [π," manifesting as the absence of backward scattering [pn-junction, or Klein tunneling [π in the interference of electronic wave functions. Another feature characterizing the electronic property of graphene lies in the appearance of partly flat band edge modes in a ribbon geometry [Z2 topological insulator (Z2TI). A Z2TI is known to possess a pair of gapless helical edge modes protected by time reversal symmetry. Similar to the gapless chiral edge mode of quantum Hall systems, responsible for the quantization of (charge) Hall conductance [helical edge modes ensure the quantization of spin Hall conductance. The Kane-Mele model [topological mass term, induced by an intrinsic spin-orbit coupling) is a prototype of such Z2TI constructed on a honey-comb lattice. Edge modes of graphene and of the Kane-Mele model show contrasting behaviors in the zigzag and armchair ribbon geometries [Z2 topological order in the Kane-Mele model. To illustrate this idea and clarify the role of valleys, we deal with the Kane-Mele and the Bernevig-Hughes-Zhang (BHZ) models [Graphene has a unique band structure with two Dirac points, uin zone ,2. Its tuin zone ,4. The fattering . A direcunneling ,7, whereunneling -10, is ageometry -13. It hgeometry . The Dirductance , the helle model ,17 (= grometries . In this) models  in paral) models .t1 is the strength of hopping between nearest-neighbor (NN) sites, i and j, on the hexagonal lattice. The tight-binding Hamiltonian H1 has two gap closing points, H1, the former being also purely imaginary: z sis the z-component of Pauli matrices associated with the real spin, and ij νis a sign factor introduced in [it2 induces a mass gap of size Let us consider a minimal tight-binding model for graphene: le model , hoppingduced in . The orit2/t1 ratios are chosen as t2/t1 = 0 and t2/t1 = 0.05 in the above two cases, respectively (t1 is fixed at unity). Dotted lines represent projection of t2) the existence of a pair of gapless helical edge modes is ensured by bulk-edge correspondence [Tight-binding implementation allows for giving a precise meaning to two representative edge geometries on hexagonal lattice: armchair and zigzag edges . Different geometries correspond to different ways of projecting the bulk band structure to 1D edge axis. In the armchair edge, the two Dirac points pondence .t2 → 0, however, the edge modes survive only in the zigzag edge geometry, as a result of different ways in which t2 are absorbed in the bulk Dirac spectrum in this limit. In the zigzag edge, on the contrary, the helical modes connecting t2 → 0. Notice that Z2 topological insulator.Therefore, they appear both in armchair and zigzag edges. In the graphene limit: L can be employed to determine the presence (or absence) of a topological gap with the precision of 1/L. The behavior of such gapless surface states that exist on the topologically non-trivial side is continuous, up to and at the gap closing. They also evolve continuously into gapped surface states on the trivial side. The flat edge modes appear at the gap closing when they do.Note here that such surface phenomena as fiat and helical edge states are characteristics of a system of a finite size, and the evolution of such gapless surface states is continuous, free from discontinuities characterizing a conventional phase transition as described by the Landau theory of symmetry breaking. The study of a system of a finite size Inspired by the contrasting behaviors of edge modes of graphene and of the Kane-Mele model in the zigzag and armchair ribbon geometries, let us consider here the BHZ model in different edge geometries . The BHZx and Γy are 2 × 2 hopping matrices:where Γσ =  is another set of Pauli matrices different from s = , and represents an orbital pseudo spin. Note also that Equation 1 describes only the  spin up part. To find the total time-reversal symmetric Hamiltonian, Equation 1 must be compensated by its Kramers partner [X1 = , X2 =  and M = . The gap closing at M occurs at Δ = 8B, whereas the gap closings at X1 and X2 occur simultaneously when Δ = 4B. Thus, at Δ = 4B, "valley" degrees of freedom appear as a consequence of the square lattice regularization. In contrast to K- and K'-valleys in graphene; however, the valleys here, X1 and X2, are two of the four time-reversal invariant momenta in the 2D Brillouin zone. Away from the gap closing, the spectrum acquires a mass gap. The sign of such a mass gap, together with the chirality χ, determines their contribution to the spin Hall conductance: e2/h is shown. The symmetry of the valence orbital is indicated in the parentheses, which is either, s  or p (inverted gap), corresponding, respectively, to the parity eigenvalue: s δ= +1 or p δ= -1. The latter is related to the Z2 index ν as (-1)ν = ∏DP δDP [Z2 non-trivial phase is characterized by ν = 1, and corresponds to the range of parameters: 0 < Δ/B < 4 and 4 < Δ/B < 8. Note that in the ν = 1 phase, contributions from Γ and M to X1 and X2 reinforce each other. In this sense, the role of X1 and X2 are analpogous to that of K and K' in the Z2 non-trivial phase of the Kane-Mele model. partner . The lat ∏DP δDP . The Z2 K- and K'-points onto the edge in armchair and zigzag edge geometries, notice that here in the straight edge, Γ and X2 are superposed on the xk-axis. Similarly, X1 and M are projected onto the same point. In the zigzag edge of BHZ model, Γ and M are superposed, whereas X1 and X2 reduce to an equivalent point at the zone boundary.Figures E(xk) = ± A sin xk. As is clear from the expression, the spectrum does not depend on Δ/B, which is very peculiar to the straight edge case. Only the range of the existence of edge modes changes as a function of Δ/B . We have seen that a flat edge spectrum appears in the two cases, whereas in the latter case, the flat band edge modes connect the two valleys that have emerged because of the (square) lattice regularization. The appearance of flat band edge modes in the zigzag graphene nano-ribbon was naturally understood from such a point of view.BHZ: Bernevig-Hughes-Zhang.The authors declare that they have no competing interests. KI carried out much of the analytical and numerical studies, and wrote the manuscript. SM made a significant contribution to the analytic part. AY contributed mainly to the numerical part. YK supervised the project.


Animal travel between habitat patches affects populations, communities and ecosystems. There are three levels of organization of edge properties, and each of these can affect animals. At the lowest level are the different habitats on each side of an edge, then there is the edge itself, and finally, at the highest level of organization, is the geometry or structure of the edge. This study used computer simulations to (1) find out whether effects of edge shapes on animal behavior can arise as emergent properties solely due to reactions to edges in general, without the animals reacting to the shapes of the edges, and to (2) generate predictions to allow field and experimental studies to test mechanisms of edge shape response. Individual animals were modeled traveling inside a habitat patch that had different kinds of edge shapes . When animals responded edges of patches, this created an emergent property of responding to the shape of the edge. The response was mostly to absolute width of the shapes, and not the narrowness of them. When animals were attracted to edges, then they tended to collect in convexities and disperse from concavities, and the opposite happened when animals avoided edges. Most of the responses occurred within a distance of 40% of the perceptual range from the tip of the shapes. Predictions were produced for directionality at various locations and combinations of treatments, to be used for testing edge behavior mechanisms. These results suggest that edge shapes tend to either concentrate or disperse animals, simply because the animals are either attracted to or avoid edges, with an effect as great as 3 times the normal density. Thus edge shape could affect processes like pollination, seed predation and dispersal and predator abundance. Animal travel between habitat patches affects populations, communities and ecosystems. On a large scale this affects metapopulation dynamics, where extinction rates of subpopulations are crucially dependent on dispersal rates There are three levels of organization of edge properties, and each of these can affect animals. At the lowest level are the different habitats on each side of an edge, then at a higher level there is the edge itself, and finally, at the highest level of organization, is the geometry or structure of the edge - for example, perimeter∶area ratio, corridors and edge shapes (convexities vs. concavities). This paper will focus on the highest level of edge organization, edge geometry.One fundamental and widespread type of edge geometry is edge shape. The idea that animals might be affected by edge shape was first suggested by Hardt and Forman Eurema nicippe) butterflies selectively travel through habitat corridors. However this can be explained solely by how they respond to edges; the butterflies' attraction to edges results in them concentrating in corridors, without having to recognize that these are specific types of edge structures Edge properties are important in two ways: ecologically, what is important is whether the edge property affects the animal's movement, but mechanistically, what is important is whether the animal responds behaviorally to that edge property. And these might not be the same, because an animal's behavior to an edge property at a lower organizational level might cause an emergence effect at a higher level. For example, at a lower level, most animals respond differently to different habitats. If an animal avoids a habitat, then the edge will affect that animal's movement, without the animal recognizing the edge itself. Similarly, at a higher level, sleepy orange (Thus the objectives of this study are to use computer simulations to: (1) find out whether effects of edge shapes on animal behavior can arise as emergent properties solely due to reactions to edges in general, without the animals reacting to the shapes of the edges; and (2) generate predictions to allow field and experimental studies to test mechanisms of edge shape response.The general approach was to model individual animals traveling inside a habitat patch that had different kinds of edge shapes. Sizes and shapes of edge types was varied, as was the behavior of the animal to the edge . I then compared densities and directions of animals at various places near the edge, to see how these were affected by edge shape and animal edge behavior. Simulations were carried out in Mathematical 7.0 Specifically, the habitat patch was 30×30 units in size Figure 1I tested all combinations of 4 edge shape widths ×3 animal edge behaviors. Edge shape widths were 4, 7, 10, 15 units, and were measured at the width of the base. Animal edge behaviors were avoidance, neutral and attraction. Each run of the simulation used one type of avoidance behavior and was simulated by a run of 2,000,000 steps.The animal movement was simulated using a correlated random walk with independent turning angles distributed with a circular normal distribution When animals intersected an edge, then their reflecting angles were affected by their edge behavior, with the following step angles, φ: Neutral, φ = mirror reflection angle; Attraction, φ = mirror reflection angle - 0.75 * (angle between mirror reflection and edge); Avoidance, φ = mirror reflection angle - 0.40* (angle between mirror reflection and perpendicular to edge). Note that after intersecting the edge, there was no random component to the new angle. The specific parameters were chosen so that attraction and avoidance would have a similar proportional effect on density - a 5x effect on density adjacent to the edge vs. inside Figure 3Animal responses were measured at various positions along the edge and inside from the edge. Response statistics measured were density of locations and three measures of directionality. Densities were standardized by dividing by the mean density in the central part of the patch - thus a density of 1 means no edge effect.I used three independent measures of directionality. The first measured edge-following - i.e. how parallel vs. perpendicular the trails were to the edge of the patch . Edge-following was estimated by |cos(θ)|−|sin(θ)|, where θ = the angle between the animal and the edge. Edge-following was −1 when the trail was perpendicular to the edge, 0 at 45°, and 1 when it was parallel to the edge.The second one measured directionality towards the tip of the treatment, along the edge . To-tip was estimated by sin(θ), where θ = the angle between the animal and the edge, oriented such that θ = 0° when pointing towards the edge. To-tip was 1 when the trail pointed towards the center of the treatment and -1 when it pointed away from the center. Thus 0 meant no directionality. To-tip was undefined for the Straight treatment.The final one measured directionality towards the inside of the patch, perpendicular to the edge . To-inside was estimated by cos(θ), where θ = the angle between the animal and the edge, oriented such that θ = 90° when the animal pointed towards the inside. To-inside was 1 when the trail pointed towards the mowed area and 1 when it pointed towards the outside. Thus 0 meant no directionality.Animal densities were obviously affected by edge shape e.g. Figure 4In order to compare the relative importance of absolute width vs. narrowness of the edge shapes, I combined all results into one plot Figure 5Comparing types of shapes, we see that animals were much more affected by the Out than the In shapes. For example, densities in the Out shape varied by a range of 100-fold when comparing Attraction to Avoidance  Sialia sialis) The general structure has two parts to it - the reaction upon detecting the edge, and then after actually encountering it. In the first part, when my modeled animals detected an edge, they changed mean turning angle either towards or away from the edge by a specific bias value. A bias of 0 means a zero mean turning angle, a bias of 1 means the animal always turns directly towards the edge, and a bias of -1 means the animal always turns directly away from the edge. This type of behavior structure has been shown in the only two studies that have specifically measured turning angles near edges. Fender's blue butterflies (Poecile atricapillus) tend to travel parallel to edges In the second part of my edge reaction model, animals reflected from the edge with a variable angle. Here a bias of 0 means complete reflection, a bias of 1 means always parallel, and a bias of −1 means always perpendicular. No study has measured specific angles of reflection at edges, however, some have reported general agreement: chickadees  and monarch (Danaus plexippus L.) butterflies turning away from habitat edges for 4 types of habitat edges. Six out of those eight combinations show a smaller edge avoidance than I observed (Parnassius smintheus) butterflies to fly towards vs. away from forest edges at different distances from the edge. My measure of this tendency  showed an overall directionality towards the edge, averaged over all distances, which is reached by parnassian butterflies only within 0.3 units of the edge (The specific parameters in my model for avoidance vs. attraction were chosen so that (1) attraction and avoidance gave proportionally similar, but opposite directions, responses to each other e.g. Figure 3the edge . Thus thAlthough my model only predicts edge shape effects when animals just respond to edges, in some situations we can use these predictions to test whether animals do recognize edge shapes. For example, Hardt and Forman An alternate hypothesis, suggested by my model, is that herbivore behaviour at edge shapes is governed just by their response just to edges, not to edge shapes. First we need to determine their response to edges. Since the herbivores, that normally live in the forest, do graze on the grass in the open areas, this suggests that they are attracted to the edges. Then my model predicts that if they are attracted to edges, they would concentrate at convexities, not concavities. Thus we would expect that seed dispersal of the woody colonizers, and thus their density, would be greater at convexities.Since my model's predictions are the opposite of Hardt and Forman's Another aspect of predictions is the scale of the edge shapes. The units used in this model are biologically relevant and thus useful for predictions in other systems. In this model, “steps” do not represent physical steps of animals - rather, they represent decision points These results also generate predictions for the effects of edge tortuousity. It has been long noted that a higher perimeter:area ratio increases emigration My results showed that simply reacting to edges causes an emergent property of responding to edge geometry. This considerably broadens the scope of edge shape effects because animals need a certain level of cognition to recognize edge geometry; one would expect many more species to recognize and react to edges  Alces alces) Ursus arctos) Puma concolor coryi) Ursus americanus) near urban areas Leopardus pardalis) in United States Another potential effect of edge shape that more directly affects humans, is on the behavior of large ungulates. Collisions between vehicles and large wildlife (e.g. white-tailed deer (This is a new area of research and my model has only been able to answer a few questions. It would be useful to test these model predictions with experimental and field work, and also to explore general questions. How do edge shapes affect animal movement, edge permeability and emigration? Which types of animals respond most to edge shape? Which properties of animals could be used to predict animal response. These types of studies have been difficult in the past because it has been difficult to track animals with a high enough resolution to identify edge behavior, and with enough data points to have enough edge shape encounters. However in the last ten years GPS devices for remotely tracking animals have increased in capability. For example GPS collars now are as light as 150 grams