We also found that ectopic expression of Tbx5 is associated with foot feathers in chickens, suggesting similar molecular pathways underlie phenotypic convergence between these two species. These results show how changes in expression of regional patterning genes can generate localized changes in organ fate and morphology, and provide viable molecular mechanisms for diversity in hindlimb scale and feather distribution.Birds display remarkable diversity in the distribution and morphology of scales and feathers on their feet, yet the genetic and developmental mechanisms governing this diversity remain unknown. Domestic pigeons have striking variation in foot feathering within a single species, providing a tractable model to investigate the molecular basis of skin appendage differences. We found that feathered feet in pigeons result from a partial transformation from hindlimb to forelimb identity mediated by DOI:http://dx.doi.org/10.7554/eLife.12115.001 Animals ranging from fish to birds display dramatic diversity within and among species; yet remarkably little is known about the genetic and developmental mechanisms that underlie this variation. In birds and their extinct dinosaur relatives, the distribution of scales and feathers on the feet is a highly variable trait.Different breeds of domestic pigeon all belong to the same species but have feet that can be feathery or scaly to different extents. Classical genetics experiments suggested that only a few genes are involved in the transition from scaled to feathered skin on the feet of pigeons. However, the molecular basis for this transition was unknown.Pitx1, which is typically considered a hindlimb gene, is expressed at lower levels in the developing legs of breeds with feathered feet than in a breed with scaled feet. The experiments also showed that Tbx5, a gene that is expressed in the forelimbs of many animals, is expressed abnormally in the embryonic hindlimbs of breeds of pigeon and chicken with feathery feet.Domyan et al. set out to identify the genes involved in the transition from scaled to feathered feet by mating different breeds of pigeon in the laboratory and then sequencing the birds’ DNA. They also surveyed the entire DNA sequences of many additional pigeon breeds with and without feathered feet. This combined approach showed that two regions of the pigeon genome have a profound effect on the number and size of feathers on the feet of domestic pigeons. These regions contain genes that are known to play key roles in controlling the growth of a limb and whether it develops into a leg or a wing. In developing pigeon embryos, Domyan et al. found that a gene called Pitx1 and Tbx5 in feather-footed breeds, and whether the same genes control the foot feathers of other species of birds.Together, these findings suggest that the hindlimbs of domestic birds with feathery feet are more like wings at the molecular level, which results in them being covered in feathers rather than scales. Future work will now aim to discover the specific DNA sequences that alter the expression of DOI:http://dx.doi.org/10.7554/eLife.12115.002 Likewise, paravians (birds and their close non-avian theropod dinosaur relatives) and other dinosaurs evolved diverse feather coverings on their legs and feet that sometimes resemble flight-like feathers  and feathers elsewhere. Exceptions to this pattern can provide insights into the evolutionary and developmental basis of skin appendage diversity. Some raptors and boreal birds evolved foot feathers instead of scales  exhibit stunning variation within a single extant species (grouse (gr) mutants, and by larger feather 'muffs' in birds that also carry mutant alleles at the Slipper (Sl) locus  locus   locus . In muffariation . TherefoHedgehog  and scaled (Scandaroon) breeds . Using quantitative trait locus (QTL) mapping with 130 F2 offspring genotyped at 3803 polymorphic markers (10 odds ratio (LOD) > 4.6; To identify chromosome regions that contribute to feathered feet, we generated an F) breeds . F1 hybr markers , we idenacross breeds, the two most-highly differentiated pFst signals implicate the same genomic regions as the QTL study: genomic scaffold 79 is located on LG11 , and scaffolds 70 and 95 are adjacent to one another on LG20 (p=9.81 x 10–13)  10.7554/eThe peak pFst region on scaffold 79 contained a 44-kb deletion (from 6.719–6.763 Mb) that was homozygous in 10, and heterozygous in 2 of the 15 feather-footed birds . Birds hgr locus), and point to scaffold 70 as the major-effect locus for longer toe feathers in birds with muffs (Sl locus)  showed signatures of selection and shared similar haplotypes on scaffold 70  , 3. Thusl locus) .Pitx1, a gene that encodes a homeobox-containing transcription factor that is normally expressed in the vertebrate hindlimb but not the forelimb  and hindlimb (Pitx1) identity and development  plays a role in determining limb-type identity , Indian fantail , and English trumpeter (large-muffed) – at Hamburger-Hamilton stage 25  and was more severely reduced in the large-muffed English trumpeter (p=0.002) , and was higher in the large-muffed English trumpeter (p=0.002)  . Conversp=0.002) . Forelimhindlimb .10.7554/Tbx5 paralog Tbx3 was also differentially expressed in both feather-footed pigeon breeds relative to racing homer (Tbx3 is a target of Tbx5 (cis-regulatory changes do not drive this expression difference (see below). The hindlimb-specific transcription factor Tbx4 is not contained in the candidate regions defined by our QTL mapping and genome-wide association studies, but this gene is a downstream transcriptional target of Pitx1  (Pitx1), and the novel expression of a key forelimb-specific gene (Tbx5).We examined expression of additional genes within the two candidate regions at HH25, and found that the ng homer . This co of Tbx5 , and addof Pitx1 . We ther 6 each) . Thus, tTbx5 and Pitx1 expression in scaled and muffed pigeon embryos at HH25. Interestingly, ectopic hindlimb expression of Tbx5 in muffed embryos was markedly different than its normal forelimb pattern in wild-type pigeons and other vertebrates  to a fairy swallow (muffed), and measured parent-of-origin allele expression in the hybrid embryonic hindlimb at HH25 , and expression of the feathered-foot allele of Tbx5 was nearly 1600% higher than the scaled-foot allele    . In contembryos) . These rTbx5 (log10 likelihood ratio = 10.3)  . The oven breeds  and betwn breeds  is also Pitx1 and Tbx5 expression can result in muscular and skeletal abnormalities. Experiments in both chick and mouse consistently demonstrate that Pitx1 plays a necessary (but not sufficient) role in determining hindlimb-type morphology of the skeleton, muscles, and tendons  to that of the English trumpeter  and the Pomeranian pouter . We found consistent soft-tissue patterning defects in both feather-footed pigeon breeds: the fibularis longus (FL) tendon inserts on the dorsal rather than ventral surface of the proximal tarsometatarsus, the flexor perforans et perforatus (FPP3) muscle adopts a longitudinal rather than pennate fiber orientation, and a slip of the FPP3 fuses with the FL tendon    . Ectopic at HH25  and, as embryos) . Hence, ears ago .Pitx1 was implicated in foot feathering in silkie chickens  and feathered-foot (silkie and Cochin) chicken embryos at HH25    . This aputations . Also ines each) . In all,Extensive classical breeding experiments in pigeons demonstrate that complex derived traits can often be parsed into component parts , 2012. Tgrouse (gr) and Slipper (Sl) – are responsible for most of the variation in foot feathering  and subtly different roles for this transcription factor in limb outgrowth and identity might have evolved in these lineages  are subtler and largely restricted to lateral structures in the adult. Therefore, we propose that different aspects of fore- and hindlimb morphology could have different dosage- and/or stage-dependent requirements for exposure to identity cues. Our ongoing analyses of musculoskeletal phenotypes in our Flineages .Pitx1 and Tbx5 misregulation, and how this ultimately results in the transformation of scaled into feathered epidermis. Mutations at these genes can cause congenital limb deformities in humans, including clubfoot and Liebenberg syndrome  have extensive foot feathering. However, recent paleontological evidence suggests that feathers – not scales – might be the ancestral hindlimb skin appendages in birds and some of their close non-avian dinosaur relatives . 114 F2 offspring survived to 6 months of age, at which time they were euthanized and phenotypic measurements taken. Proportion of the tarsus was measured by dividing the length of the dorsal tarsus that was feathered by the total length of the tarsus , and averaged between the two tarsi. Toe feathers were counted on each toe, and summed across all 8 toes. The length of each of the longest three toe feathers on digit 3 , which bore the longest toe feathers on each foot, was measured to the nearest 1 mm and averaged for each bird.Animals were housed in accordance with the University of Utah Institutional Animal Care and Use Committees of University of Utah (protocols 10–05007 and 13–04012). 130 F2 offspring, 50 ng of DNA was digested with ApeKI, ligated to barcoded adapters, and then 10 ng of each barcoded sample was pooled in batches of 26 individuals and purified (Qiagen PCR Purification Kit). DNA fragments 550–650 bp in size were selected using Pippin Prep , and amplified by 10–12 cycles of PCR using custom indexed primers. Libraries were purified with Ampure beads  and sequenced using 100- or 125-bp, paired-end sequencing on the Illumina HiSeq2000 platform at the University of Utah Genomics Core Facility. Reads were trimmed to 90 bp, filtered for quality, and de-multiplexed using Stacks  , identified as closely-spaced double-crossover events, were manually corrected on these linkage groups. Subsequently, the stepwiseqtl function was used to identify additional QTL, and the fitqtl function used to account for the effect of one linkage group while calculating the LOD scores and percent variance explained (PVE) of the other. Significance thresholds of α = 0.05 were calculated with 1000 permutations of each phenotype across all linkage groups. The peak markers for each phenotype were used to test for the effect of each QTL.Genetic map construction and QTL mapping was performed using R/qtl (qtl.org) . MarkersBAM files generated previously for a whole-genome resequencing panel  were comIndividual birds from different breeds were binned into the following phenotypic classifications:Groused: Berlin long-faced tumbler, Lahore, Oriental frill, Shaksharli.Muffed: English long-faced muffed tumbler, English pouter, English trumpeter, frillback, ice pigeon, Indian fantail , Pomeranian pouter , Saxon monk, Saxon pouter. The English pouter is an unusual breed that is sometimes classified by breeders as slipper only. Its foot feathering is far more extensive than groused breeds, which led us to include it in the muffed group for the purposes of the genomic analyses.Scale-footed: African owl, archangel, Birmingham roller, carneau, Chinese owl, cumulet, Egyptian swift, English carrier, fantail, feral , Iranian tumbler, Jacobin, king, Lebanon, Marchenero pouter, mookee, Oriental roller, parlor roller, runt, Scandaroon, Spanish barb, starling, Syrian dewlap, Thai laugher.https://github.com/vcflib/vcflib for software updates).pFst, a modified likelihood ratio test that accounts for genotype uncertainty, extended haplotype homozygosity (EHH), and haplotype network analyses were implemented using the GPAT++ software library : Berlin long-faced tumbler, Berlin short-faced tumbler, Bokhara trumpeter, classic Oriental frill, crested Saxon field color, English trumpeter, fairy swallow, frillback, German double-crested trumpeter, ice pigeon, Indian fantail, Lahore, Mindian fantail, Oriental frill, Persian roller, Pomeranian pouter, Russian tumbler, saint, Schmalkaldner moorhead, Uzbeck tumbler, West of England.Scale-footed : African owl, Altenburg trumpeter, American flying tumbler, American giant homer, American mini crest, American show racer, archangel, Bohemian pouter, Brunner cropper, Budapest tumbler, Cauchois, Chinese owl, cumulet, Danzig highflier, domestic show flight, dragoon, English baldhead long-faced clean-legged tumbler, English carrier, English magpie, English short-faced tumbler, exhibition homer, fantail, Franconian trumpeter, French mondaine, giant runt, Holle cropper, horseman pouter, Italian owl, Jacobin, Jiennesse pouter, king, Lebanon, Spanish little friar tumbler, Maltese, medium-faced crested helmet, Modena, mookee, Norwich cropper, nun, Old Dutch Capuchine, Old German owl, Oriental roller, parlor roller, Portuguese tumbler, Scandaroon, showtype racing homer, Spanish barb, starling, Syrian Baghdad, Texas pioneer, Thai laugher, Thuringer clean leg, Vienna medium-faced tumbler, Voorburg shield cropper, zitterhals.Forelimb and hindlimb buds from HH25 racing homer and Indian fantail embryos were collected and placed in 1% formaldehyde for 20 min at room temperature, then washed 3x in ice-cold PBS and stored at -80°C until chromatin extraction. ChIP was performed on 200 micrograms of chromatin isolated from embryonic pigeon limbs. Control libraries were prepared using 100 ng of input chromatin. A total of 16 libraries were created (8 ChIP and 8 input controls for each breed and limb combination). A validated monoclonal antibody against H3K27ac  was used to perform ChIP, and sequencing libraries were prepared using NEBNext Ultra DNA Library Prep Kit for Illumina with NEBNext Multiplex Oligos for Illumina . All libraries were size selected using SPRI magnetic beads to eliminate adapter dimers. All 8 ChIP libraries showed enrichment for a positive control site relative to input libraries (tested by qPCR). Single-end, 50-bp read sequencing was performed on Illumina HiSeq2000 platform at the University of Utah Genomics Core Facility.bdgdiff in MACS2 (https://pypi.python.org/pypi/MACS2/2.0.10.20130522). Regions with a log10 likelihood ratio ≥ 3 were considered to have differential enrichment between the two groups.Fold-enrichment plots were generated using MACS  and visuTo assay gene expression, limb buds from HH25 embryos were harvested and placed in RNAlater  at 4°C overnight. Total RNA was extracted, cleaned and DNase-treated (Qiagen RNeasy Kit). mRNA was reverse-transcribed to cDNA using oligo-dT and M-MLV RT  according to the manufacturer’s protocol.cDNA was amplified using intron-spanning primers for each target using a CFX96 qPCR instrument and iTaq Universal Sybr Green Supermix . Results were compared by Mann-Whitney U test. Two technical replicates of each sample were performed, and the mean value determined. Differences were considered statistically significant if p<(0.05 / # genes assayed) to control for multiple-testing. Each experiment was performed three times, and the results presented are from one representative experiment. Primers used for each assay are listed in Pitx1 and Tbx5 transcripts were identified by Sanger sequencing in the parents of a cross between an Old Dutch Capuchine  and a fairy swallow (muffed) that was homozygous for the 44-kb deletion upstream of Pitx1, and PyroMark Custom Assays (Qiagen) for each SNP were designed using the manufacturer’s software. Pyrosequencing was performed on cDNA and gDNA derived from HH25 limb buds using a PyroMark Q24 instrument (Qiagen). The signal intensity ratio of feathered allele to scaled allele from cDNA samples was normalized to ratios obtained from gDNA samples from the same embryos to control for allele-specific amplification bias. Normalized ratios were analyzed by Mann-Whitney U test, and considered significant if p<(0.05 / # genes assayed) to control for multiple-testing. Each experiment was performed twice, and the results presented are representative. Primers used for each assay are listed in SNPs in Linear templates for probe synthesis were amplified from cDNA by PCR using primers listed in Embryos used for RNA in situ hybridization were dissected from eggs, and fixed overnight in 4% paraformaldehyde at 4°C on a shaking table, then dehydrated into 100% MeOH and stored at -20°C. RNA in situ hybridization was performed as described . Hybridi In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor, Marianne Bronner, and Detlef Weigel as the Senior Editor.Thank you for submitting your work entitled "A molecular shift in limb identity underlies the convergent evolution and development of feathered feet" for consideration by The reviewers have discussed the reviews with one another and the Reviewing editor has drafted this decision to help you prepare a revised submission.This work represents an elegant use of pigeon genetics to address an important question of evolutionary morphology: the origin of feathered feet. A molecular understanding of this issue would open the door to advances in our knowledge of the scale to feather transition, the origin of feathered appendages, and other problems of significance. Using the tractable pigeon system, with a bevy of tools available for genetic and anatomical analysis, the authors reveal a correlation between feathered feet and a shift in expression of two regulatory factors, Pitx1 and Tbx5. The novelty is slightly dampened by the fact that the genes they found have been known to alter limb identify for more than a decade, but the work still represents a logical approach and valuable documentation, showing us how different approaches can lead scientists to the same important genes. While the genetic approach in pigeon is strong, the chicken variant part needs improvement as detailed below. Importantly, some of the discussion regarding the evolutionary implications seems over-interpreted and needs to be toned down.1) A major problem that must be clarified and rephrased is the relationship to dinosaur feather/scale evolution. While this is certainly an interesting topic, the "four wing dinosaurs" found by Xing Xu and his associates clearly have leg skeleton in the hind feet. Thus, a "feathered limb" can have wing or leg skeleton inside. Consistent with this, 2) "Convergent Evolution" used in the title is too strong and misleading. It should be rephrased in the title, Abstract and Introduction.3) It is claimed that misexpression of a Pitx1-Enr construct disrupts scale formation and that this is consistent with phenotypes obtained following Pitx1 misexpression in the wing. What is shown in the two panels is a severely truncated and abnormal limb. It is not possible to assess any effect on scale formation from this part of the data. It seems to go too far to say that Pitx1-Enr is dominant-negative, when the luciferase assay merely indicates that the construct can repress transcription in a heterologous system. It is incorrect to state that “[…]verify that Pitx1EnR inhibited wild-type Pitx1 activity".4) While the figure title states that "Tbx5 alters development of the foot epidermal appendages" the main text goes much further suggesting "misexpression of Tbx5[…] induced ectopic development of foot feathers". This is not at all clear from the panels in Figure 2—figure supplement 4B. Normal developing feathers are clear in other parts of the embryos at these stages and the structures highlighted do not look similar. The authors should be much more cautious about exactly what has occurred in these embryos.5) An important issue is the localization of the cells that are expressing Tbx5 in the hindlimb, since it is epidermal derivatives that are being analysed largely and these structures are known to be patterned by signals from the mesoderm. It is stated that "ectopic Tbx5 expression was largely localized to the mesoderm of the proximal and posterior hindlimb" based on whole mount in situ hybridization. It would be clearer to show this in a section. It is further stated that this domain "shows a striking correlation with regions of epidermal transformation". This is not clear at all. Also, not too much can/should be read into the expression domain of Pitx1 shown for two breeds of pigeon. Are you certain that these are the correct stages to be analyzing the expression domains of these genes with respect to any possible action they may have on patterning epidermal structures? The same argument applies for the analysis of ectopic Tbx5.7) The data in Additional issues with this section are:The section starts with a misrepresentation of the literature suggesting Tbx5 can result in muscular and skeletal mispatterning.Any potential contribution of reduced Pitx1 expression to the morphological differences described is not considered.Is "ulna" in subheading “Muffed pigeon breeds incur musculoskeletal patterning changes” a typo?Is "lateral" hindlimb really intended. Is this really the primary site of ectopic Tbx5. This is described as 'posterior' in the earlier section.8) In the Discussion, while it is mentioned that additional mutations also contribute to the muff phenotype it is assumed that these must all converge on the Tbx5 and Pitx1 pathways. This train of thought is continued in the suggestion that "a relatively small number of genetic changes are sufficient for a surprisingly large transformation of epidermal appendage morphology and distribution.” I don't believe the data make the case for 'sufficiency' in their argument since the misexpression data are not convincing and the birds could be harbouring a large number of additional mutations that contribute/are responsible for the feathering phenotype.9) There is surprisingly little discussion of work on Tbx5 and Pitx1 function in the mouse. The authors must be aware that much of what they show and conclude is inconsistent with analyses of various mouse mutants of Tbx5 and Pitx1. This has been ignored largely, even though it would be exciting, if indeed true. The current interpretations of the results, without reference to the prior work in other systems, could further muddy an already messy literature on the roles of genes in determining the differences between fore limbs and hind limbs. It is therefore essential to clearly understand the functional significance of the QTL and expression analyses they have carried out. It would be interesting to test if elevating the levels of Pitx1 in a feathered breed would be sufficient to reduce feathers/replace with scales and reduce Tbx5 expression in the leg.10) Have the authors considered whether the specific QTLs identified relate to expression of Tbx5 in the wing or other regions of the embryo or adult? What attempts have been made to demonstrate that the QTL are causative to the expression domain in the leg? Only one possible explanation  appear to have been explored and the final conclusion hangs on what appear to me to be unconvincing functional data.11) Ptilopody refers to the presence of feathers in the shank. There are feathers on proximal parts of pigeon legs  that do not correlate with Tbx5 expression or reduction in Pitx1 expression. This is not mentioned or discussed. Is the assumption that the capacity of Tbx5 is localized to distal regions of the limb? There are of course feathers in many other regions of the bird where Tbx5 is not expressed also. Chicken breeds also show variation in feathering in the hindlimb-cochin and silkie chicken are analyzed to some extent. In silkie and some other chicken breeds (silky-feather) the basis of the mutation is known and/or causes other defects such as ploydactyly in Silkies. These do not correlate with the results and conclusions derived from this work in pigeon. These have not been discussed and are relevant to the significance of the proposed mechanisms in bird/dinosaur evolution.12) The shared genotype for feathered feet between pigeon and chicken may be partial since some chicken with feathered feet have Tbx5 in the leg bud, but no difference in Pitx1, which is on chromosome 13 where the chicken ptilopody locus is located. Can the authors analyze different chicken ptilopody variants to qualify their convergent evolution statement?eLife. Your revised article has been favorably evaluated by Detlef Weigel (Senior editor), a Reviewing editor (Marianne Broner), and two reviewers. The manuscript has been improved but there are some remaining, relatively minor issues that need to be addressed before acceptance, as outlined below. We look forward to receiving a revised version that addresses these points:Thank you for resubmitting your work entitled "Molecular shifts in limb identity underlie development of feathered feet in two domestic avian species" for further consideration at Point 1: The point was to distinguish that there can be changes in feather and scale dermis patterns that are distinct from the other mesoderm derivatives (e.g. skeleton) rather than anything about dosage and/or stage requirements.Point 7: Although the text has been changed, the authors still seem to be implying that the longer fibula is a partial hindlimb to forelimb transformation which it need not be Please avoid the term “limb-specific”.Subheading “Evolution of epidermal appendage distribution”: avialan to avian This work represents an elegant use of pigeon genetics to address an important question of evolutionary morphology: the origin of feathered feet. A molecular understanding of this issue would open the door to advances in our knowledge of the scale to feather transition, the origin of feathered appendages, and other problems of significance. Using the tractable pigeon system, with a bevy of tools available for genetic and anatomical analysis, the authors reveal a correlation between feathered feet and a shift in expression of two regulatory factors, Pitx1 and Tbx5. The novelty is slightly dampened by the fact that the genes they found have been known to alter limb identify for more than a decade, but the work still represents a logical approach and valuable documentation, showing us how different approaches can lead scientists to the same important genes. While the genetic approach in pigeon is strong, the chicken variant part needs improvement as detailed below. Importantly, some of the discussion regarding the evolutionary implications seems over-interpreted and needs to be toned down.We appreciate the positive feedback about our approach and results. We address our results in chicken directly in our responses to specific comments below. We also understand the concern about over-interpretation of the evolutionary implications of our results, and we have toned down our language, especially in the Abstract, Introduction, and Results. We still feel that our work provides exciting new insights about evolutionary changes in epidermal appendage identity and distribution, but we have made this less of a central theme of the paper.1) A major problem that must be clarified and rephrased is the relationship to dinosaur feather / scale evolution. While this is certainly an interesting topic, the "four wing dinosaurs" found by Xing Xu and his associates clearly have leg skeleton in the hind feet. Thus, a "feathered limb" can have wing or leg skeleton inside. Consistent with this, We agree with this assessment, although we wish to point out a couple of items of potential confusion. First, the leg skeletons of muffed pigeons are clearly still legs, and we did not state otherwise in the original manuscript. To clarify this point, we now state in the Discussion:“Collectively, these findings point to an alteration of the identity of the developing hind limb, rather than localized changes to individual epidermal placodes. […] Instead, different aspects of hind and forelimb morphology appear to have different dosage- and/or stage-dependent requirements.”On this same theme, we do not claim that muscle mis-patterning in the hind limb directly resembles the normal forelimb. To emphasize this point, we added the following statement in the Discussion:“These changes are aberrations of normal patterning, although they are not necessarily clear transformations to a more forelimb-like configuration.”2) "Convergent Evolution" used in the title is too strong and misleading. It should be rephrased in the title, Abstract and Introduction.This comment raises an interesting issue. Descent with modification under domestication is widely accepted as evolution, albeit not in a “natural” setting, but we realize that the language we used could lead to confusion. Our interpretation of convergence also seems to have no alternative – inheritance of feathered feet from the most recent common ancestor of pigeons and chickens is rejected by every recent phylogenetic hypothesis of avian evolution. We have altered our verbiage to the more descriptive “genetic mechanisms” and similar terms to avoid confusion and detract from the main messages of our study.3) It is claimed that misexpression of a Pitx1-Enr construct disrupts scale formation and that this is consistent with phenotypes obtained following Pitx1 misexpression in the wing. What is shown in the two panels  is a severely truncated and abnormal limb. It is not possible to assess any effect on scale formation from this part of the data. It seems to go too far to say that Pitx1-Enr is dominant-negative, when the luciferase assay merely indicates that the construct can repress transcription in a heterologous system. It is incorrect to state that“[…]verify that Pitx1EnR inhibited wild-type Pitx1 activity".After further reflection, we agree that the direct implications of these functional experiments are difficult to assess. As a result, we chose to remove them from the revised manuscript.4) While the figure title states that "Tbx5 alters development of the foot epidermal appendages" the main text goes much further suggesting "misexpression of Tbx5[…] induced ectopic development of foot feathers". This is not at all clear from the panels in Figure 2—figure supplement 4B. Normal developing feathers are clear in other parts of the embryos at these stages and the structures highlighted do not look similar. The authors should be much more cautious about exactly what has occurred in these embryos.As mentioned above, we have removed these experiments from the manuscript.5) An important issue is the localization of the cells that are expressing Tbx5 in the hindlimb, since it is epidermal derivatives that are being analysed largely and these structures are known to be patterned by signals from the mesoderm. It is stated that "ectopic Tbx5 expression was largely localized to the mesoderm of the proximal and posterior hindlimb" based on whole mount in situ hybridization. It would be clearer to show this in a section.This is an excellent suggestion. We now include images of sections from each of the three pigeon phenotypes studied  in It is further stated that this domain "shows a striking correlation with regions of epidermal transformation". This is not clear at all.Also, not too much can/should be read into the expression domain of Pitx1 shown for two breeds of pigeon. Are you certain that these are the correct stages to be analyzing the expression domains of these genes with respect to any possible action they may have on patterning epidermal structures? The same argument applies for the analysis of ectopic Tbx5.We agree that the stages we chose to analyze might not represent differences (or similarities) that persist throughout development. To make this point clear, we have specified Hamburger-Hamilton stages in the revised manuscript. Further developmental biology work is needed to fully understand the specific time points at which the actions of these two genes are most important for specific aspects of epidermal, muscular, and skeletal development. However, our genetic and genomic results provide clear motivation for studying these two genes, and our allele-specific expression assays demonstrate that cis-regulatory differences specifically at these two loci affect their expression levels.7) The data in Thank you for highlighting these points of potential confusion. We agree that the fibula of muffed pigeons is still a fibula in both morphology and position, and we suspect that a typo (pointed out below) contributed to the impression that we were mistakenly identifying the fibula as an ulna. On the contrary, our intent was to convey that the fibula of the English trumpeter in “We also found that the fibula, which is normally reduced in pigeons relative to the homologous forelimb structure (ulna), was enlarged[…]”Further confusion might have originated from the typo in the following statement:“Ectopic expression of Tbx5 in the hind limbs of chick embryos produces a similar enlargement of the ulna.”In the above sentence, “ulna” should have been “fibula.” We have made this change in the revised manuscript.Additional issues with this section are:The section starts with a misrepresentation of the literature suggesting Tbx5 can result in muscular and skeletal mispatterning.Thank you for this comment. Our effort to be economical with space unfortunately resulted in the following confusing statement:“In mouse and chicken embryos, experimental manipulation of Pitx1 and Tbx5 expression can also result in muscular and skeletal mispatterning .”We have now expanded the Discussion at the beginning of this section. We start with a similar overview statement, and provide additional commentary to de-convolute the known roles of these two genes on muscular and skeletal patterning in different vertebrates.“In mouse and chicken embryos, experimental manipulation of Pitx1 and Tbx5 expression can result in muscular and skeletal abnormalities. […] Given the dramatic musculoskeletal defects observed in other organisms with experimentally altered Pitx1 and Tbx5 expression, we compared the hind limb morphology of adult feral pigeons[…]”Any potential contribution of reduced Pitx1 expression to the morphological differences described is not considered.As mentioned above, and as detailed in the Discussion (“Roles of Pitx1 and Tbx5 in diversity and disease” subheading), we now discuss the impact of Pitx1 misexpression and mutant alleles in a bird, two mammals, and a fish. These examples illustrate that decreases in Pitx1 expression can result in skeletal changes, although we are not presently able to determine the specific effects of Pitx1 versus Tbx5 on musculoskeletal morphology in muffed pigeons. Follow-up work will entail detailed phenotyping of the musculoskeletal system of our F2 cross by dissection and CT analysis, and these phenotypes will allow us to separate the effects of these two loci on musculoskeletal changes. To emphasize this point, we now state in the aforementioned subsection of the Discussion:“Our ongoing analyses of musculoskeletal phenotypes in our F2 cross, which includes individuals with different combinations of feathered-foot alleles of Pitx1 and Tbx5, will allow us to understand the separate and epistatic effects of these loci on musculoskeletal anatomy.”Is "ulna" in subheading “Muffed pigeon breeds incur musculoskeletal patterning changes”a typo?Yes, thank you for identifying this error. “Ulna” has been changed to “fibula” in the revised manuscript.Is "lateral" hindlimb really intended. Is this really the primary site of ectopic Tbx5. This is described as 'posterior' in the earlier section.Thank you for pointing out this discrepancy. The posterior limb bud of the embryo becomes the lateral side of the adult limb. To clarify this point, we have altered the original statement to read:“Notably, all of the modified structures of ptilopodous pigeons develop in the posterior  and dorsal hind limb, which are the primary sites of ectopic Tbx5 expression.”8) In the Discussion, while it is mentioned that additional mutations also contribute to the muff phenotype it is assumed that these must all converge on the Tbx5 and Pitx1 pathways. This train of thought is continued in the suggestion that "a relatively small number of genetic changes are sufficient for a surprisingly large transformation of epidermal appendage morphology and distribution.” I don't believe the data make the case for 'sufficiency' in their argument since the misexpression data are not convincing and the birds could be harbouring a large number of additional mutations that contribute/are responsible for the feathering phenotype.We agree that other genes are probably involved in the foot feathering phenotype. However, multiple lines of evidence demonstrate that only two loci have major effects on the phenotype. First, classical genetic studies implicate major effects of the grouse and slipper loci, the molecular identities of which were previously unknown. Second, our QTL mapping study implicated just two loci of major effect on the phenotype. Importantly, our F2 intercross led to the recombining of muffed and scaled genomes, thereby minimizing the effects of the parental genetic backgrounds on phenotypic variation in the F2 offspring. If other loci had significant effects on the phenotype, we would have observed additional significant QTL. As the size of our F2 population increases, we might have enough power to map additional loci that make small contributions to the phenotype. Some of these loci might converge directly on the Tbx5 and Pitx1 pathways, but we see no reason to assume that they will. However, this does not diminish the result that the two QTL we mapped in this study do indeed control a large proportion of phenotypic variance in the cross. Third, our genome scans comparing scale-footed and feather-footed breeds independently detected the same two chromosome regions as our QTL mapping study. If other loci exerted major effects on the phenotype or were selected repeatedly across breeds, we would have detected them in our selection scans. It remains possible that other loci have minor effects on the phenotype, and we might not have the power to detect them in our whole-genome scans of scale-footed and feather-footed breeds. Our results also do not exclude the possibility of allelic heterogeneity at modifier loci, which would make them harder to detect. However, these possibilities do not diminish the two highly significant associations we detected with our current data set. We agree that our misexpression data do not make a clear case for developmental sufficiency, but our results robustly support genetic sufficiency of these loci for foot feathering.9) There is surprisingly little discussion of work on Tbx5 and Pitx1 function in the mouse. The authors must be aware that much of what they show and conclude is inconsistent with analyses of various mouse mutants of Tbx5 and Pitx1. This has been ignored largely, even though it would be exciting, if indeed true. The current interpretations of the results, without reference to the prior work in other systems, could further muddy an already messy literature on the roles of genes in determining the differences between fore limbs and hind limbs. It is therefore essential to clearly understand the functional significance of the QTL and expression analyses they have carried out. It would be interesting to test if elevating the levels of Pitx1 in a feathered breed would be sufficient to reduce feathers/replace with scales and reduce Tbx5 expression in the leg.We agree that this is an important topic in the evolution and development of fore- and hind limb identity. In our response to comment 7, we provide a more in-depth discussion of previous work in both the mouse and chicken experimental systems. In short, we find that regulatory alterations to Tbx5 in pigeon and chicken breeds align nicely with previous functional data in chickens. We believe this work doesn't muddy the waters, but rather bolsters confidence in the original Tbx5 overexpression experiments in chickens. Pitx1 and Tbx5 undoubtedly retain similar functions among tetrapods (and other vertebrates), but we find no reason to presume that gene regulation will work identically in avians and mammals. Moreover, the common ancestor of mammals and birds lived more than 300 million years ago and the ectodermal structures that are the focus of this study (feathers) are not even present in mammals. Horton et al.  speculate about an FGF-mediated mechanism that could explain part of the difference in the conflicting data for the role of Tbx5 in forelimb identity, and we now cite this paper in the manuscript. For example, in the Discussion, we now state:“We note that, although genetic manipulations indicate that Tbx5 does not specify forelimb identity in mice, the divergence time between mammals and birds is deep (>300 million years) and subtly different roles for this transcription factor in limb outgrowth and identity might have evolved in these lineages .”We also agree that the suggested Pitx1 misexpression experiment is worthwhile. We hope to conduct this experiment soon.10) Have the authors considered whether the specific QTLs identified relate to expression of Tbx5 in the wing or other regions of the embryo or adult? What attempts have been made to demonstrate that the QTL are causative to the expression domain in the leg? Only one possible explanation  appear to have been explored and the final conclusion hangs on what appear to me to be unconvincing functional data.Thank you for pointing out these important issues that need clarification. It is indeed possible that expression of Tbx5 is altered in other regions of the embryo; however, the embryonic wing is not one of these regions, as demonstrated by the expression assays reported in 11) Ptilopody refers to the presence of feathers in the shank. There are feathers on proximal parts of pigeon legs  that do not correlate with Tbx5 expression or reduction in Pitx1 expression. This is not mentioned or discussed. Is the assumption that the capacity of Tbx5 is localized to distal regions of the limb? There are of course feathers in many other regions of the bird where Tbx5 is not expressed also.We believe that the presence of feathers throughout the body of a pigeon  the basis of the mutation is known and/or causes other defects such as ploydactyly in Silkies. These do not correlate with the results and conclusions derived from this work in pigeon. These have not been discussed and are relevant to the significance of the proposed mechanisms in bird/dinosaur evolution.We agree that our results on foot feathering in pigeons conflict somewhat with previous results in chicken models. However, to our knowledge, the specific mutation(s) leading to foot feathering in silkies and other breeds are not known. Indeed, we believe that the identification of specific candidate genes contributes to the novelty of our work in pigeons. A previous genetic mapping study implicated a chromosomal region around Pitx1 in ptilopody in silkies, but specific genes and mutations were not identified. In fact, the Pitx1 gene was not even mentioned as a candidate gene in that study . Polydactyly in silkies is a different genetic issue than ptilopody (these two loci are unlinked), and we do not address this issue in the manuscript. Ptilopodous silkies can be polydactyl, but not all are. Silky feathering throughout the body is also unlinked to ptilopody. We agree that the disparity between chickens and pigeons is important and we address it in some detail in the last paragraph of the Results section:“Classical genetic studies implicate at least two loci in heavy foot feathering in chickens , although the molecular genetic origins of the trait remain unknown. […] Furthermore, different populations of breeds such as silkies appear to have different constellations of ptilopody loci and alleles, and it is possible that we used strains that do not have Pitx1 mutations .”12) The shared genotype for feathered feet between pigeon and chicken may be partial since some chicken with feathered feet have Tbx5 in the leg bud, but no difference in Pitx1, which is on chromosome 13 where the chicken ptilopody locus is located. Can the authors analyze different chicken ptilopody variants to qualify their convergent evolution statement?The common ancestor of chickens and pigeons did not have feathered feet, so we believe that the feathered feet of these two species is best described as a case of phenotypic convergence. If the reviewer is referring to breed “variants,” we already examined gene expression in two ptilopodous breeds, the Cochin and silke. None of our gene expression data point to a role for Pitx1 in ptilopody in silkies or Cochins, but as we state in the manuscript, we could be examining the wrong stage to implicate this gene. However, we demonstrate differences in Tbx5 expression between the hind limb buds of wild-type and ptilopodous breeds. We also generated silkie x white leghorn hybrid embryos and showed that ectopic expression of the feathered-foot Tbx5 allele is due (at least in part) to cis-regulatory changes, but we do not have genetic mapping or enough genomic resequencing data from chickens to directly test for involvement of specific sequence variants.To emphasize that our results might account for some, but not all, of the ptilopody phenotype in chickens, we have added “in part” to the following statement:“Hence, Tbx5-related developmental mechanisms may, in part, underlie the evolution of foot feathering in two species that last shared a common ancestor over 80 million years ago.”Point 1: The point was to distinguish that there can be changes in feather and scale dermis patterns that are distinct from the other mesoderm derivatives (e.g. skeleton) rather than anything about dosage and/or stage requirements.Thank you for this clarification. We have addressed this comment in the revised manuscript, and the paragraph in question now reads:“Collectively, these findings point to a partial alteration of the identity of the developing hindlimb, rather than localized changes to individual epidermal placodes. […] Therefore, we propose that different aspects of fore- and hindlimb morphology could have different dosage- and/or stage-dependent requirements for exposure to identity cues.”We chose to retain a modified version of the last sentence in the revised manuscript because we believe it proposes an important and testable research hypothesis for future work.Point 7: Although the text has been changed, the authors still seem to be implying that the longer fibula is a partial hindlimb to forelimb transformation which it need not be We have altered this passage to highlight a previous interpretation about the enlargement of the fibula in experimentally induced hindlimb expression of Tbx5. We agree that the longer fibula of pigeons need not necessarily be due to a partial hindlimb to forelimb transformation, but given the strong evidence that epidermal identity is transformed, we believe we are justified in raising this issue and would be downplaying the significance of important experimental interpretations from others’ work by not doing so. We realize that not everyone will agree with these interpretations, but we feel that it is our responsibility to credit these experimental and interpretive precedents. The revised passage reads as follows:“We also found that the fibula, which is normally splint-like and shorter than the tibia in pigeons, was enlarged  and two To tone down our argument further, we replaced “transformation” with “changes” in the following concluding sentence:“Thus, the morphological changes to the hindlimbs of feather-footed pigeon breeds are considerably more than skin deep.”We also deleted from the caption for Regarding the minor issue, we cannot find any description in the comparative anatomy literature that supports the statement about a lack of articulation between the ulna and wrist in avians, nor does our own experience with skeletal preparations of embryonic or adult pigeon material support this statement.Please avoid the term limb-specificWithout guidance about the reason to modify this term, we are somewhat confused about how best to address this request. “Limb-specific” (including “forelimb-specific” and “hindlimb-specific”) is commonly used throughout the limb development and morphology literature to describe gene expression patterns that are specific to one or another set of limbs. Many of the instances of “limb-specific” in the previous version of the manuscript resulted from an earlier request to change “hindlimb”  to “hind limb”; therefore, “hindlimb-specific” (a term in widespread use) became “hind limb-specific,” which is comparatively unusual and awkward. We note that even the first and second round of reviewer comments on this manuscript use “hindlimb” in every case , with the exception of the comment in the first review to make the change from “hindlimb” to “hind limb.” Therefore, with clarity and convention in mind, we elected to change “hind limb” to “hindlimb” in the revised manuscript.To further clarify what is meant by limb specificity, we have expanded the descriptions of Pitx1 and Tbx5 limb expression domains when these genes are first mentioned:“The highest pFst peak on scaffold 79 – corresponding to the major-effect QTL on LG 11 for the proportion of tarsometatarsal feathering – was approximately 200 kb upstream of Pitx1, a gene that encodes a homeobox-containing transcription factor that is normally expressed in the vertebrate hindlimb but not the forelimb . The higWe made several other changes to cut down on the number of times “fore/hindlimb- specific” appears in the text, but we believe this terminology is usually the most meaningful, accurate, and clear way to convey information. We welcome suggestions for alternative terminology.Subheading “Evolution of epidermal appendage distribution” avialan to avianThis change would alter the meaning of the sentence, so we elected to delete the word entirely. Doing so actually broadens the scope of the question we are asking, which we see as a positive outcome:“How might pigeons help us understand the evolution of epidermal appendage distribution and limb morphology in other species?”We also use “avialan” later in the same paragraph to convey an interpretation by Zheng et al. 2013. Changing this term to “avian” would be incorrect and change the meaning of the statement.


Furthermore, chemical doping is susceptible to degradation and can adversely affect intrinsic graphene’s properties. Here we demonstrate strong (1.33 × 1013e/cm2), robust, and spontaneous graphene n-doping on a soda-lime-glass substrate via surface-transfer doping from Na without any external chemical, high-temperature, or vacuum processes. Remarkably, the n-doping reaches 2.11 × 1013e/cm2 when graphene is transferred onto a p-type copper indium gallium diselenide (CIGS) semiconductor that itself has been deposited onto soda-lime-glass, via surface-transfer doping from Na atoms that diffuse to the CIGS surface. Using this effect, we demonstrate an n-graphene/p-semiconductor Schottky junction with ideality factor of 1.21 and strong photo-response. The ability to achieve strong and persistent graphene n-doping on low-cost, industry-standard materials paves the way toward an entirely new class of graphene-based devices such as photodetectors, photovoltaics, sensors, batteries, and supercapacitors.Scalable and low-cost doping of graphene could improve technologies in a wide range of fields such as microelectronics, optoelectronics, and energy storage. While achieving strong p-doping is relatively straightforward, non-electrostatic approaches to n-dope graphene, such as chemical doping, have yielded electron densities of 9.5 × 10 Photoelectron spectroscopy and secondary ion mass spectrometry (SIMS) of Na in the SLG (Na) of ρNa = 1.15 ± 0.05 × 1014 cm−2 at the surface adjacent to graphene; similar densities of alkali metals on graphene are known to induce strong n-doping via electron transfer from the metal atoms to graphene16Na = 7.6 × 1014 cm−2–close to the measured surface-density of Na on SLG), show n-doping with ΔEF = + 474 meV (F = + 426 meV). The measured charge-transfer rate of 0.11e per-Na-atom is also in agreement with literature values of 0.1e–0.2e132O) borosilicate-glass (BSG) substrate shows a Dirac point at VG ~0 V  with multi-layer graphene (GR) transferred onto SLG (8% Naage (VG) . The min the SLG  indicate 474 meV , in clos V inset , indicat2CO3, NaSeO3, or NaOH at the CIGS/air surface18Na = 4.18 × 1013 cm−2 at the surface. In contrast, the CIGS/air surface ρNa in CIGS/Mo/BSG is nearly two orders of magnitude lower (ρNa = 6.16 × 1011 cm−2).Next, to test if graphene/p-type semiconductor/SLG substrates result in n-doping of graphene via Na diffusion through the semiconductor, and the corresponding formation of a p-n junction, we deposited p-type CIGS onto the Mo-side of a Na-rich SLG substrate coated with 330 nm of Mo, and then transferred graphene onto the CIGS surface. CIGS/Mo/SLG was chosen because it is a standard substrate used in the solar industry, and so our measurements will help determine the feasibility and versatility of this doping method in real-world applications. SLG substrates are commonly used because Na in CIGS is known to improve solar cell performance18−1, along with the graphene G peak at 1585 cm−1 and the 2D peak−1; the graphene D peak−1 is negligible, indicating minimal damage during transfer to the rough CIGS surface . For GR/CIGS/Mo/SLG (red-curve), the conductance minimum VG (Dirac point) exceeds the measurement limit and is estimated via extrapolation to be VG = −106 V (13e/cm−2) with ΔEF = +536 meV, surpassing the ΔEF for graphene on neat SLG (green-curve). This strong n-doping results from the high Na density that diffuses through CIGS and concentrates at the CIGS/air surface  (+698 meV) due to the Na-ion dipole interaction on graphene, as calculated using Helmholtz equation , is higher than in GR/SLG (0.11e per-Na-atom), which we attribute to ionization-rate differences of the Na species on the CIGS and SLG surfaces.The doping effects and junction properties of graphene on CIGS/Mo/SLG (and on CIGS/Mo/BSG) are investigated using the four-terminal device in = −106 V , confirm011 cm−2  is not d−2 white light (solid), with (red) and without (blue) the Al2O3 gate-dielectric  with Al2O3 is reduced by 104 to 0.36 nA/cm2 (2/p-Si FET without the Al2O3 dielectric (dashed blue curve) has a Dirac point beyond VG = +100 V, indicating strong p-doping; a GR/SiO2/p-Si FET with the Al2O3 dielectric  and built-in field.electric . These m6 nA/cm2 , the phoG, where J0 is reduced as VG is increased from −100 V to 50 V. Plotting ln(J0) vs. (|VG + ΔVF|)1/2 , we obtain Φb = 0.29 eV  is modeled assuming Landauer transportb = 0.13 eV. Assuming ideal Schottky-diode behavior, ln(J0/T2) vs. 1/T  vs. (|VG + ΔVF|)1/2  is lower than expected, which we believe is due to surface defects and surface sodium doping of CIGS that lowers the surface ionization potential of CIGS relative to the bulk.The behavior of Jinset to  yields ΦΔVF|)1/2  yields Φb) is equal to the ionization potential of the CIGS semiconductor (IPCIGS) minus the work function of graphene (ΦG), and represents the barrier that a hole in the valence band of CIGS must overcome to reach the graphene interface and recombine with an electron there. The activation energy (Ea), on the other hand, represents the characteristic energy that governs the rate of minority carrier (electron) excitation into the conduction band of the p-type semiconductor CIGS. In an intrinsic semiconductor, Ea is equal to half the bandgap, and for a doped p-type semiconductor like CIGS, its value should be close to the bandgap energy, since in this case the Fermi level is close to the valence band.The barrier height  and Ea, which will indicate whether the recombination is predominantly bulk or interfacial. The ideality factor n is expected to become temperature dependent in the presence of tunneling. The measured I–V data is used to determine n as a function of temperature and this is directly incorporated into Eq. (S11), which allows us to separate the effects of Ea and n on the reverse saturation current density J0. nln(J0) vs. 1/T , yielding an activation energy (Ea) of 0.96 eV, which indicates dominant interfacial recombination since it is less than the CIGS bandgap of 1.15 eV  of the diode is measured to be 190 nm using C-V measurements  is much less than the best-fit value of Ea (0.96 eV), both models yield good fits to the same J0vs. T data due to the inclusion of the temperature-dependent n in the activation-energy model.While fitting the  eV Ref. . We haveo 0.49 V . The spaurements . The app eV ref. ), and gr eV ref.  modified13e/cm−2, corresponding to a Fermi energy shift of +426 meV.), robust, and spontaneous n-doping of graphene on the surface of a low-cost industrial-grade soda-lime-glass substrate via surface-transfer doping from the Na. By leveraging the Na diffusion through a p-type CIGS semiconductor deposited onto the soda-lime glass, we applied this method to the formation of a graphene(n)/semiconductor(p) Schottky diode with even stronger graphene n-doping  than was achieved on bare glass. This method of n-doping does not require any high-temperature annealing steps, and should be compatible with a wide range of semiconductor/substrate systems. The junction properties, such as Schottky barrier height and interfacial recombination rate, can be controlled by tuning the doping strength via the thickness of a few-nm dielectric layer such as TiO2 or Al2O3. Advantages of this technique include the lack of external chemicals whose doping strength decays over time, the ability to achieve strong and persistent n-doping of graphene that is placed on top of a p-doped semiconductor, the ability to n-dope graphene on a wide range of p-doped semiconductors via the use of a Na host that is in direct contact with the graphene layer, and the ability to control the strength of the doping via the use of a spacer layer  between the Na host and the graphene layer. Disadvantages include the possible restriction to p-doped semiconductors that are not too strongly affected by the Na diffusion from the Na host to the graphene layer, in the case where the semiconductor lies between the Na host and the graphene layer.In conclusion, we have demonstrated strong  substrates via plasma-enhanced chemical vapor deposition (PECVD) at 160 °C at 1.6 nm/s rate. Next, 1 × 1 μm2–500 × 500 μm2 regions were patterned on the PECVD SiO2 either using optical lithography (or ebeam lithography) techniques depending on the feature size. E-beam was performed using the E-beam lithography JEOL JBX-6300FS system on E-beam resist positive resist ZEP520A  by exposing with a dose of 400 μC/cm2 at 100 keV and developed with hexylacetate for 90 sec. In optical lithography, the tool MA6 Mask aligner was used with positive optical resist S1811spun at 4000 rpm for 45 sec annealed at 110 °C for 1 minute, and developed with MIF 312 3:2 with DI water for 1 minute. These patterned regions were then etched via Reactive Ion Etching (RIE)  using a mixture of (CHF3 and Ar) at 15 nm/min, until the CIGS was exposed. Commercially obtained CVD graphene on Cu foil (Graphene Platform) was then transferred from the Cu substrates to the SiO2/CIGS/Mo glass substrates. The graphene transfer was done by coating the graphene side of the graphene/Cu foils with PMMA , oxygen plasma etching  the opposite side, and etching the Cu using ammonium persulfate (0.1 M) solution overnight. As the Cu is etched away, the graphene/PMMA film floats on the etchant and it is washed in de-ionized water (>18 MΩ resistivity using a Millipore DI system) and it then transferred, graphene-side down, onto the pattered CIGS/Mo/SLG or other control substrates such as neat SLG or BSG substrate. Afterwards, graphene transferred substrates are annealed at 100 °C for 30 minutes in a vacuum oven to remove water, and are subsequently annealed at 200 °C for 15 minutes to soften the PMMA and promote conformal adhesion onto substrates. It is found that the 200 °C annealing step is extremely critical in getting highly uniform, wrinkle- and damage-free graphene films on the rough surfaces of CIGS and SiO2 substrates. Afterwards, the PMMA is removed from the graphene by immersing in acetone overnight and the substrate is further annealed in a Rapid Thermal Annealer at 375 °C in Ar (96%): H2(4%) forming gas for 15 minutes for complete PMMA removal. Next, the graphene is etched following optical (ebeam) lithographic patterning using oxygen plasma etch.  using a negative tone resist mask . After etching the graphene, source-drain electrical contacts (Au (30 nm)/Cr (5 nm)) are deposited using ebeam evaporation after optical (ebeam) lithography patterning. Next, a 200 nm top gate-dielectric layer (Al2O3) is blanket deposited on GR/CIGS/Mo/SLG(BSG) or GR/SLG(BSG) substrates via Atomic Layer Deposition at 1 Ǻ/cycle using (Tri Methyl Aluminum) TMA/Water precursor at 250 °C. On top of the Al2O3, a semi-transparent top-gate (10 nm of Au) is deposited via ebeam evaporation following optical (ebeam) lithography patterning. Next, the source and drain electrodes are exposed through the dielectric layer by RIE etching of Al2O3 using BCl3 by Oxford Plasmalab 100 ICP etcher, on a mask pattern using optical (ebeam) lithography.Given in How to cite this article: Dissanayake, D. M. N. M. et al. Spontaneous and strong multi-layer graphene n-doping on soda-lime glass and its application in graphene-semiconductor junctions. Sci. Rep.6, 21070; doi: 10.1038/srep21070 (2016).


The van der Waals heterostructures of two-dimensional (2D) atomic crystals constitute a new paradigm in nanoscience. Hybrid devices of graphene with insulating 2D hexagonal boron nitride (h-BN) have emerged as promising nanoelectronic architectures through demonstrations of ultrahigh electron mobilities and charge-based tunnel transistors. Here, we expand the functional horizon of such 2D materials demonstrating the quantum tunneling of spin polarized electrons through atomic planes of CVD grown h-BN. We report excellent tunneling behavior of h-BN layers together with tunnel spin injection and transport in graphene using ferromagnet/h-BN contacts. Employing h-BN tunnel contacts, we observe enhancements in both spin signal amplitude and lifetime by an order of magnitude. We demonstrate spin transport and precession over micrometer-scale distances with spin lifetime up to 0.46 nanosecond. Our results and complementary magnetoresistance calculations illustrate that CVD h-BN tunnel barrier provides a reliable, reproducible and alternative approach to address the conductivity mismatch problem for spin injection into graphene. In recent years, there has been a growing interest in van der Waals heterostructures of 2D crystals such as graphene with h-BN. In fact, van der Waals heterostructures can lead to the observation of many interesting phenomena such as tunnel transistors, metal-insulator transition, itinerant magnetism, Wigner crystallization2223272223Recent theoretical studies by Karpan et al. showed that the tunnel contacts with tailored resistance area product (RA) and large spin polarization values can be achieved by engineering the number of h-BN layers at the junction31In this letter, we show reliable and reproducible tunneling behaviour of large area CVD grown h-BN and demonstrate efficient spin injection, transport and precession in graphene. Furthermore, we report an enhancement of the spin signal and spin lifetime by using resistive h-BN tunnel barriers, which circumvents the conductivity mismatch problem. Our observations supported by calculations elucidate that h-BN tunnel barrier resistances can be tailored to achieve desirable magnetoresistance values. While the present work focuses on the graphene spintronic devices, our results are generic for a large class of h-BN based charge and spin tunnel devices.We illustrate the underlying concept and spin injection device structure in 2 substrate by exfoliation from HOPG using the scotch tape method2 chips. The h-BN was then patterned to regions well covering the graphene flakes (see −1 by the supplier (graphene supermarket), which matches well with the thickness based studies on exfoliated h-BN2235We fabricated graphene spin transport devices using h-BN as tunnel barrier and ferromagnetic Co electrodes for spin injection and detection. We prepared graphene flakes on Si/SiOakes see . The posWe fabricated devices with and without h-BN tunnel barriers. In the following description, we refer h-BN0 as the device without any tunnel barrier, and h-BN1, h-BN2, h-BN3 and h-BN4 as devices with h-BN tunnel barriers in the order of increasing RA. The details of the device fabrication are described in the 2. The IV characteristics fit well . In spite of the challenges associated with the growth and transfer of CVD h-BN, we have been able to observe the tunneling behavior in most of the contacts over a large scale, over a wide range of temperature. The graphene channel resistivity in our devices was found to be in the range of 2–6 kΩ with linear I–V behavior. We observed characteristic gate dependent Dirac curves for the graphene channels of our devices with a mobility of 2000–3000 cm2 V−1 s−1. The details of the graphene characterization are presented in We performed the electrical characterization of the devices in different measurement geometries  to ascerNL = ΔVNL/I). RNL changes it's sign for parallel and anti-parallel configuration of the ferromagnetic electrodes in accordance with the changing spin density (majority to minority and vice-versa) at the detector electrodeHaving established the tunneling behavior of the Co/h-BN contacts, we focus on spin dependent transport measurements carried out on our graphene devices. The measurements were performed in a nonlocal (NL) spin valve geometry, where the charge current path is isolated from the spin diffusion signal measured by the voltage probes, as shown in the inset of NL with contact resistance RA for different devices is shown in NL is quantitatively related to the tunnel barrier resistance.The room temperature nonlocal spin valve measurements for devices with a h-BN tunnel barrier (h-BN1 and h-BN2) and without a tunnel barrier (h-BN0) are shown in Lω) while diffusing towards the nonlocal detector contact. In 2, we observed a nonlocal Hanle resistance with ΔRNL ~ 8.5 Ω at 100 K. This would correspond to a nonlocal spin valve signal of 17 Ω. To remove possible non-spin related effects, we have symmetrized our raw Hanle data by taking the average of the negative and the positive magnetic field sweepsNL) due to Larmor precession of the spins diffusing towards the detector from the injector is given by P is the spin polarization of the h-BN/Co contacts (assumed to be the same for both the injector and the detector), and B⊥. L is the effective channel length between the injector and the detector (2 µm in our devices), with a width W and square conductivity sσ of the graphene strip. The diffusing spin polarized electrons have a lifetime sτ and diffusion constant D with a spin diffusion length In order to evaluate the spin lifetime of the electrons in graphene devices with h-BN tunnel barrier, we performed nonlocal Hanle spin precession measurements by sweeping the magnetic field perpendicular to the device geometry while the magnetization axes of the injector and detector electrodes are being kept parallel. The injected spin polarized electrons precess about the perpendicular magnetic field with Larmor frequency  and h-BN3 (sτ ≈ 0.46 ns with RA = 12.6 kΩ μm2). The maximum spin signal and spin lifetime were obtained for the h-BN3 device, giving rise to a spin diffusion length sfλ of 1.4 μm with a spin polarization P of 14% and sτ ~0.46 ns. The diffusion constant obtained from the Hanle fitting is D = 0.00416 m2/s. The spin lifetime sτ is found to be independent of the bias current for our devices as shown in s) is primarily a measure of spin flip scattering (τsf−1) inside the graphene channel. In the case of spin injection from a ferromagnet to graphene, the spin escape rate (τesc−1), a rate by which the spins diffuse back into the ferromagnet and get relaxed, greatly influences the spin lifetime (esc →∞). In addition to this, the spin accumulation at the interface suffers from further relaxation due to spin flip scattering at the ferromagnet-graphene interface17The spin lifetime in graphene devices with h-BN tunnel barrier  are found to be much higher than the device without tunnel barrier (h-BN0). When compared to h-BN0 having T) following the drift-diffusion based analytical formulation by Takahashi and MaekawaiR ~ dR, i→injector, d→detector). The value of the nonlocal spin valve signal ΔRNL for h-BN3  is marked by a red dot along with the theoretical curve. The close agreement between the calculated spin signal (which is dependent on tunnel resistance) and the observed spin valve signal implies that the values of obtained polarization and spin lifetime are reliable. The plot also reveals that the tunnel resistance idR in our device h-BN3 lies close to an optimum value for the observation of the maximum spin signal. Beyond the contact induced relaxation regime, any further increase in tunnel barrier resistance will not only saturate the spin signal but will also result in higher power dissipation in the device. In addition to the saturation of the spin signal, an enhancement in the tunnel barrier resistance inevitably increases the dwell time (nτ) of spins inside the channel. This leads to a down turn (as nτ > sτ) in magnetoresistance (MR) as observed in spin valves. In idR of h-BN3 being closer to maximum. In the calculation, the low MR for small contact resistances is due to the conductivity mismatch, preventing spin injection from the Co contact into graphene. The decay of the MR for large contact resistances is due to relaxation of the spins during the time spent in the graphene channel (dwell time), a time that becomes very long for large contact resistances. Note that the values of spin parameters such as the spin polarization might also depend on the tunnel barrier resistance (TR) which was not taken into account in these theoretical models. In spite of the fact that this might lead to slight deviation on either side of TR ~ idR, the constant values that we employed here are quite reliable in the regime where TR ~ idR as evident from the match in the non-local calculation. This analysis elucidates that CVD grown h-BN is a reliable solution to the conductivity mismatch problem without requiring the deposition of any oxide based tunneling barriers on graphene. It should be noted that the optimum tunnel resistance (chR ∝ sfλ) and thus the quality of the channel. This implies that channels supporting larger spin diffusion length or a higher spin lifetime can overcome the dwell time limit set by the tunnel barrier resistance. With epitaxial graphene channels, Dlubak et al.To understand the effect of barrier resistance on the overall spin signal, we calculate the nonlocal spin signal as a function of the tunnel contact resistance  such as graphene itself. Opportunities are growing with the proposal by Karpan et al.45In addition to the quality of the tunnel barrier, the spin lifetime and polarization depend to a great extent also on the intrinsic scattering mechanisms inside the graphene medium. The observed spin lifetime and diffusion length are significant for our graphene flakes on SiOIn conclusion, we have demonstrated tunnel spin injection into graphene using atomically thin CVD hexagonal boron nitride (h-BN) at room temperature. The CVD h-BN showed reproducible tunneling characteristics with weak temperature dependence of the tunnel resistance over a large scale. Taking the advantage of the variation in the thickness of CVD h-BN, we have been able to investigate the evolution of the spin transport with resistance of the h-BN tunnel contacts. With resistive h-BN tunnel contacts, we observed an enhancement in spin transport with spin lifetimes up to 0.46 ns, yielding a spin polarization of 14%. In light of the magnetoresistance calculations, we show that the CVD h-BN is a reliable and unique way of creating tunnel resistances, which overcomes the conductivity mismatch problem. The integration of CVD h-BN to graphene opens up new avenues to explore the role of h-BN atomic planes for spin injection into other 2D advanced materials350152 (285 nm), highly doped silicon substrate with predefined Ti/Au markers. We used graphene exfoliated from highly oriented pyrolytic graphite (obtained from Advanced Ceramics) using the conventional scotch tape method. Graphene flakes were identified using a combination of optical and atomic-force microscopy. In our experiments, we used graphene flakes with widths around 1.5 µm.The graphene flakes were prepared on a clean SiO3 solution for etching the Cu substrate. After complete removal of Cu, the floating layer of PMMA/h-BN film was washed several times with deionized (DI) water. To remove possible chemical residues, the film was also treated with 10% HCl for 20 min, followed by neutralization with DI water. The PMMA/h-BN film was then transferred onto graphene flakes prepared on a SiO2/Si chip. The chip containing the PMMA/h-BN/graphene heterostructure was dried and baked at 150°C for 5 minutes to improve the adhesion between the h-BN and the graphene. Finally, the PMMA layer was removed using hot acetone and the h-BN/graphene on the chip was annealed at 200°C in an H2/Ar atmosphere for 20 minutes. Careful transfer processes with thicker PMMA and the subsequent optimized baking procedure ensured a uniform and ripple-free h-BN layer on the graphene. The h-BN layer was patterned to shapes covering the graphene flakes using electron-beam lithography and argon plasma etching . During CVD growth, h-BN grows on both sides of the copper foil (h-BN/Cu/h-BN). The h-BN on the top surface was first protected with thick PMMA (A11 e-beam resist), and the h-BN on the lower surface was removed by argon plasma etching. Then the PMMA/h-BN/Cu structure was kept in FeClshown in .Ferromagnetic electrodes were fabricated on h-BN/graphene samples by electron-beam lithography and lift-off techniques. Cobalt thin film contacts of 65 nm thickness with a cap-layer of 15 nm gold were prepared using electron-beam evaporation. Ferromagnetic electrodes of different widths  for the spin injector and detector were fabricated, ensuring different switching fields to achieve a parallel or antiparallel configuration through in-plane magnetic field sweeps. Most of the data presented here correspond to contacts with 130 nm, 250 nm as the injector and detector electrodes.D~10–20 V and mobility ~ 2000–3000 cm2 V−1 s−1.Electrical characterization of the contacts and the device were performed using multiterminal measurement geometry. The contact resistances were found to be in the range of 10–100 kΩ, showing reasonable scaling with area, thus demonstrating the homogeneity of the h-BN tunnel barrier. The high contact resistance and its low temperature dependence shown in  confirm Spin transport and spin precession measurements were performed in nonlocal geometry where the charge current path is separated from the voltage contacts measuring spin signal, in order to exclude spurious signals such as anisotropic magnetoresistance and stray Hall voltage. The measurements were performed using direct currents (DC) of 1–5 µA, and the nonlocal voltage was measured using a nanovoltmeter, under vacuum in a variable temperature cryostat (from room temperature 300 K to 1.5 K) with a superconducting magnet.M.V.K., A.D., J.B. fabricated and measured the devices. T.I., S.P.D. participated in fabrication and measurements. M.V.K. analyzed the data and performed the magnetoresistance calculations. M.V.K. and S.P.D. wrote the manuscript with input from all co-authors. S.P.D. and M.V.K. conceived the idea, and S.P.D. supervised the research.Supplementary Information


The combination of several materials into heterostructures is a powerful method for controlling material properties. The integration of graphene (G) with hexagonal boron nitride (BN) in particular has been heralded as a way to engineer the graphene band structure and implement spin- and valleytronics in 2D materials. Despite recent efforts, fabrication methods for well-defined G-BN structures on a large scale are still lacking. We report on a new method for producing atomically well-defined G-BN structures on an unprecedented length scale by exploiting the interaction of G and BN edges with a Ni(111) surface as well as each other. With the ever-growing family of two dimensional 2D) materials, now comprised of the semi-metal graphene (G)1D materia5781011121314161820Two main approaches have been used to fabricate G-BN heterostructures thus far: The pattern re-growth technique uses photolithographic patterning of graphene or BN layers, generally grown on metal foils1617232425Experiments in the past few years have focused on well-established surfaces for graphene growth such as Ir(111)2517232930Ni(111) is a strongly interacting crystal facet with a lattice constant closely matched to that of both graphene and BN. As a result of the strong interaction, both materials form 1 × 1 structures on Ni(111)32333435−8 mbar range displaces nickel atoms from Ni(111) terraces to form highly symmetric triangular islands embedded into the first metal layer. This growth mode has also been observed for graphene on the Ni(111) surface−7 mbar of ethylene or less) on the other hand results in a growth front proceeding parallel to the ZZ edge of the BN seed, forming narrow ZZ terminated graphene strips. The width of these strips can be tuned through the deposition time. By carefully controlling the preparation conditions, we have produced well-defined graphene strips between 4 nm  surface exceeds that for graphene growth by several hundred degrees . As a result, an atomically perfect ZZ interface between BN and graphene is readily obtained. Graphene attached to BN domains remains embedded in the surface and forms a remarkably straight interface with the Ni(111) surface along the ZZ direction see . This suSome defects in the BN islands are found on all samples investigated in this study. This is not of immediate concern since the functionality of G/BN nanostructures lies with the graphene areas, but a closer investigation to assess the potential impact of these defects on the G-BN interface is warranted. While the mechanism by which these imperfections are formed cannot be conclusively identified based on our experiments, the etching of the BN sheet by hydrogen abundant from the decomposition of the precursors used during the sample preparation and the substitution of B or N atoms with C are likely candidates. The defect density in BN decreases as one approaches the interface with graphene. Atomically resolved STM images of a defective area in BN, shown in ca. 50 nm in length with only few defects. Even at these length scales, the interface is atomically sharp along the entire section shown. Finally, i.e. only by the nucleation density of BN on the substrate.We now turn to the atomic structure of the G-BN interfaces. A close-up view of a G-BN junction in atomic resolution is shown in It is clear from earlier work1115ca. 1 nm wide transition region is observed where graphene and BN seem to intermix. This behaviour suggests that the formation of either C-B or C-N bonds is strongly preferred on the Ni(111) surface and the system attempts to minimize its energy by re-organising unfavourable interfaces.et al.404142433839To confirm this interpretation, we performed DFT calculations  surface. BN islands embedded in nickel terraces with atomically sharp ZZ edges serve as seeds for graphene growth. The surrounding nickel terrace seems to play a vital role in this process by stabilising the original BN seed as well as the emerging graphene structure. The size and shape of the graphene domains can be tuned by carefully controlling the preparation conditions. The total length of such interfaces is only limited by the size of the BN seed and atomically sharp interfaces in excess of 150 nm length are demonstrated. A combination of STM and DFT suggests that the interfaces are most likely of ZZ C-B type.The growth process described here may be readily generalised to nickel foils or epitaxial thin films similar to those used in other G-BN synthesis schemes. Transfer techniques for graphene from nickel foils are well documented in the scientific literature47483N3H6) and graphene from ethylene on a Ni(111) single crystal in ultra-high vacuum (UHV). The crystal was cleaned by repeated cycles of sputtering with neon ions followed by annealing at 1270 K. After the final sputtering cycle and before annealing, the surface was exposed to 30 L of oxygen at room temperature. The oxygen treatment removes any remaining carbon contamination from the crystal surface during annealing−9 to low 10−8 mbar region for up to three minutes. After the desired exposure had been reached, the borazine supply was cut and the sample cooled to the graphene growth temperature immediately. Graphene growth was performed at temperatures between 830 K and 850 K by exposing the BN samples to ethylene at pressures in the low 10−7 mbar range. To obtain jagged graphene domains, the pressure was raised to the mid 10−6 range for a ca. 1 second at the start of the growth interval. After the desired exposure time, the ethylene supply was shut off and samples were annealed at the growth temperature for 15 to 30 minutes to improve the graphene quality. Further details can be found in the T = 77 K. The STM images were post-processed using the Gwyddion (http://gwyddion.net/) and WSxM (http://www.nanotec.es) scanning probe microscopy softwares50Samples were prepared by sequential CVD growth of BN from borazine (B−6 eV. The atomic structures were fully relaxed until the forces present were less than 10−3 eV/Å. The Ni(111) substrate was modeled as three layers below h-BN and graphene. The two lowest layers were fixed to bulk positions with lattice constant 2.488 Å. As nickel is a ferromagnetic material, the calculations allowed spin polarization, and the initial spin moments were chosen to closely match the bulk values. Moreover, we enabled the available relativistic treatment, the scaled zero-order regular approximation, in order to treat the heavier nickel atoms in a proper way. Further computational details specific to each model system can be found in the The all-electron FHI-aims packageHow to cite this article: Drost, R. et al. Synthesis of Extended Atomically Perfect Zigzag Graphene - Boron Nitride Interfaces. Sci. Rep.5, 16741; doi: 10.1038/srep16741 (2015).


Here we demonstrate a solution for fabricating van der Waals heterostructures. Graphene/hexagonal boron nitride (h-BN) heterostructures were synthesized on cobalt substrates by using molecular beam epitaxy. Various characterizations were carried out to evaluate the heterostructures. Wafer-scale heterostructures consisting of single-layer/bilayer graphene and multilayer h-BN were achieved. The mismatch angle between graphene and h-BN is below 1°.Van der Waals materials have received a great deal of attention for their exceptional layered structures and exotic properties, which can open up various device applications in nanoelectronics. However,  Such heterostructures provide a platform to investigate novel phenomenon in fundamental physics, and there are reports indicating superior properties for device applications12131416Van der Waals (vdW) materials are layered structures bonded with vdW forces. This group of materials have received high interest recently for their novel properties and high potential in various device applications12in situ layer-by-layer characterization. As a matter of fact, vdW epitaxy was first demonstrated using MBE process24262728To obtain such heterostructures, mechanical exfoliation and chemical vapor deposition (CVD) growth were widely used20212 layer of 300 nm were used as substrates. They were transferred to an E-beam evaporator system for the deposition of a Co film of 400 nm. The wafers were subsequently loaded on to standard 3″ wafer holders and transferred to an MBE system for growth. A Knudsen effusion cell filled with B2O3 powder  was used as B source. A thermocracker was used to crack acetylene gas  as C source. The C source was tuned by either acetylene gas flow or cracker temperature. An electron cyclotron resonance (ECR) system was used to form nitrogen gas plasma  as N atomic source. The N source was tuned by either nitrogen gas flow or ECR magnetron current.Thermally oxidized Si wafers with a SiOFor a typical growth, the substrate is firstly annealed at 800 ~ 850 °C under a hydrogen flow of 10 sccm for a duration of 30 minutes. At the end of substrate surface treatment, the hydrogen gas flow is stopped and substrate temperature is further ramped up to 870 ~ 900 °C for graphene/h-BN heterostructure growth. The thermal cracker temperature is ramped to 1200 °C and 3 sccm acetylene is introduced into the chamber for graphene growth. The growth lasts from 10 s to 1 minute. Subsequently, h-BN growth follows with minimal time gap. B cell temperature is precisely ramped to 900 ~ 1100 °C right before h-BN layer growth. Nitrogen flow rate is 10 sccm and the growth lasts 10 ~ 15 minutes. At the end of h-BN growth, the substrate temperature is slowly cooled down towards room temperature at a rate of 10 °C/min. The slow substrate cooling process suggests that the epitaxy undergoes layer-by-layer growth mode, which is in contrast to fast cooling procedure with much higher cooling rates in the growth of graphene by precipitation of carbon atoms from the metal substrate in some CVD process303 solution to etch away the Co metal layer, which often takes several days. The film was then transferred onto carbon coated Cu TEM grid and treated with acetone and DI water to remove PMMA. Cross sectional TEM sample was prepared using focused ion beam technique. The graphene/h-BN thin film was covered by an Ir layer and further protected by electron-beam and ion-beam deposited Pt layers.Raman characterizations were performed using a HORIBA LabRam system equipped with a 50-mW 514-nm green laser. Scanning electron microscopy (SEM) images were acquired using a Philips XL30-FEG system. X-ray photoelectron spectroscopy (XPS) was carried out using a Kratos AXIS ULTRA XPS system equipped with an Al Kα monochromatic X-ray source and a 165 mm mean radius electron energy hemispherical analyzer. Transmission electron microscopy (TEM) images and electron diffraction patterns were acquired using a FEI/Philips CM-30 TEM. Plan-view TEM sample was prepared using direct transfer method. After spin coated with PMMA, the sample was submerged in FeClAs grown vdW thin films are transparent and thus not visible for bare eye or through optical microscope . As the In order to achieve continuous wafer-scale h-BN single domain instead of discrete flakes, fine-tuning of the sample growth condition was performed. Detailed difference in growth condition is summarized in Table 1 of 2 C-C bond at 284.0 eV−1. The appearance of D peak has been observed in other reports of graphene/h-BN heterostructure2g optical phonon peak is evident at 1364 cm−1. Analyzing the 2D peak of graphene to detect the mismatch angle of graphene and h-BN has been reported recently−1, which indicates the mismatch angle is below 1°.We have demonstrated direct epitaxial growth of h-BN on graphene utilizing MBE. Signature triangular h-BN flakes with sizes as large as 20 μm were observed. Further improvement on the growth condition results in high-quality wafer-scale graphene/h-BN heterostructure single domain film. The epitaxial single/bi-layer graphene/few-layer h-BN structure is further confirmed by XPS, Raman spectroscopy, electron diffraction, and TEM imaging. The misorientation between the graphene and h-BN layers is less than 1°. While MBE synthesis of high-quality graphene/h-BN heterostructures is a critical step toward the realization of 2D electronics, reasonable electronic functionality of heterostructures is of greater concern. In the future work, two- and three-terminal devices will be fabricated and the electrical measurements will be carried out to evaluate their electrical performance.How to cite this article: Zuo, Z. et al.In-situ epitaxial growth of graphene/h-BN van der Waals heterostructures by molecular beam epitaxy. Sci. Rep.5, 14760; doi: 10.1038/srep14760 (2015).


Many microbial ecology studies have demonstrated profound changes in community composition caused by environmental pollution, as well as adaptation processes allowing survival of microbes in polluted ecosystems. Soil microbial communities in polluted areas with a long-term history of contamination have been shown to maintain their function by developing metal-tolerance mechanisms. In the present work, we review recent experiments, with specific emphasis on studies that have been conducted in polluted areas with a long-term history of contamination that also applied DNA-based approaches. We evaluate how the “costs” of adaptation to metals affect the responses of metal-tolerant communities to other stress factors (“stress-on-stress”). We discuss recent studies on the stability of microbial communities, in terms of resistance and resilience to additional stressors, focusing on metal pollution as the initial stress, and discuss possible factors influencing the functional and structural stability of microbial communities towards secondary stressors. There is increasing evidence that the history of environmental conditions and disturbance regimes play central roles in responses of microbial communities towards secondary stressors. Soil is known as a major sink for metals released into the environment. In many industrialized countries, soil pollution has become a serious problem . Metal p2 production pyrene and flooding stress applied as secondary stressors . This sun levels . The autn levels .et al. pyrene, salt, arsenic and flooding). The authors stressed that, without this knowledge, it would be difficult to pinpoint the factors determining the response of the community after the secondary stressors. We argue that knowledge about factors affecting the structure and the function of microbial communities is pivotal when attempting to predict the impact of secondary stressors on the stability of soil microbial communities.Previous “stress-on-stress” research in areas with long-term metal pollution history focused mostly on functional aspects of microbial communities, such as changes in respiration rates ,35,117 aet al.  showed hA number of studies published over the past decade highlighted the role of microbial diversity for ecosystem functioning ,119,120,It has been shown that microbial diversity is especially important for stability of narrow-scale functions, such as those associated with nitrification and denitrification ,125. GenSoil microorganisms perform many important functions, and the dynamics of different functional groups following the exposure to stressors can be different . Soil miet al. [−1) and unpolluted soils, Deng et al. [−1). They showed that the number of copies of 16S rRNA genes, microbial biomass carbon (Cmic), and substrate-induced respiration (SIR)  were more stable to additional metal spiking (CuSO4) than the number of copies of amoA gene and potential ammonia oxidation rate (classified as specific parameters).Contrasting results were observed when different microbial parameters were compared in the face of additional stressors. In general, previous studies indicated that the effect of stressors on the functional stability of the soil microbial community depends on the level of specificity of the function measured. It has been well documented that soil respiration is less sensitive to stressors than more specific parameters, such as enzyme activity. This is due to the fact that a wide range of organisms contributes to respiration, while only a small fraction of the total microbial community is responsible for nitrification ,112,135.et al.  reportedg et al.  studied Reports focusing on the responses of microbial communities towards a secondary stressor can be divided into two types. The first type consists of field observations after long-term (at least 10 years long) pollution with such metals as copper ,35,137, Previous studies have shown that the functional stability of microbial communities changes with soil properties ,116. SoiTo improve our understanding of the combined impact of metal pollution and secondary stressors on soil microbial communities, it is crucial to learn which microbial groups are selected in communities under strong pressure by metal stress and how associated costs of tolerance affect their responses in the face of additional stressors. There are several issues that still need to be addressed in order to evaluate the effect of metal pollution and secondary stressors on microbial communities in metal impacted soils. In this review, we showed that historical environmental conditions need to be considered when attempting to predict stability of microbial communities towards secondary stressors. Furthermore, microbial communities selected for metal resistance, in some cases, did not show a loss of their capabilities to cope with additional stress (due to acquired co-tolerance). Indeed, for some stress factors, the tolerance and resistance of soil microbial communities appeared elevated rather than decreased. Therefore, we may expect that if a microbial population has adapted genetically to long-term metal contamination, it may have higher tolerance and/or a higher ability to deal with additional environmental stressors. This is an important aspect for predicting responses of metal-tolerant communities to future environmental impacts, such as climate change, in areas with a long-term history of contamination.(1)The application of new DNA-based methodologies, allowing culture-independent assessment of microbial communities, has greatly enhanced our insights into the composition of these communities and their functional capacities.(2)There is increasing support that the historical environmental conditions and disturbance regimes are primary factors to influence microbial community stability towards secondary stressors. We argue that knowledge of factors controlling the composition and function of microbial communities is pivotal when attempting to predict the impact of secondary stressors on the stability of soil microbial communities. The potential influence of past events  on the development of community co-tolerance highlights the importance of historical factors in predicting the response of microbial communities to additional stressors.(3)Long-term “stress-on-stress” studies with single metals or defined mixtures may provide an integrated insight into the complex responses of soil microbial communities to secondary stressors at polluted sites. Following severe or long-term stress exposure, microbial community may not be able to recover within a short-term period.(4)The effect of soil physicochemical properties is important. The effects of metal pollution on microbial community structure is often confounded by natural soil properties such as pH and organic matter content.(5)Detailed knowledge on the functional and compositional properties of the microbial community prior to applying additional stressors may help to evaluate and interpret the microbial response to these stressors.(6)In contrast to DGGE and PLFA, far less attention has been given to sequence data to examine the direct or indirect interactions between microbial taxa in the face of stressors. We recommend that future “stress-on-stress” studies should investigate microbial communities under environmentally stressful conditions, using metagenomics and meta-transcriptomics together with carefully designed disturbance experiments and using different types of stressors. We expect that such studies will provide a deeper insight and clearer picture on whether predictable species assemblages occur following the combination of metal stress with other environmental stressors.In historically contaminated areas, functional redundancy buffers selective pollution effects, allowing soil microbial communities to perform their functional roles at relatively unchanged levels, despite the observed shifts in community structure. It may be, thus, difficult to evaluate ecological risks to such ecosystems based solely on measuring functional performance of their communities. In order to identify areas that are at high risk of losing their functionality due to pollution, microbial communities from such areas should be exposed experimentally to secondary stressors in order to see whether they show any reduction in functional capabilities. Such “stress-on-stress” studies should be designed with the following issues in mind: