The study of South American camelids and their domestication is a highly debated topic in zooarchaeology. Identifying the domestic species  in archaeological sites based solely on morphological data is challenging due to their similarity with respect to their wild ancestors. Using genetic methods also presents challenges due to the hybridization history of the domestic species, which are thought to have extensively hybridized following the Spanish conquest of South America that resulted in camelids slaughtered en masse. In this study, we generated mitochondrial genomes for 61 ancient South American camelids dated between 3,500 and 2,400 years before the present (Early Formative period) from two archaeological sites in Northern Chile (Tulán-54 and Tulán-85), as well as 66 modern camelid mitogenomes and 815 modern mitochondrial control region sequences from across South America. In addition, we performed osteometric analyses to differentiate big and small body size camelids. A comparative analysis of these data suggests that a substantial proportion of the ancient vicuña genetic variation has been lost since the Early Formative period, as it is not present in modern specimens. Moreover, we propose a domestication hypothesis that includes an ancient guanaco population that no longer exists. Finally, we find evidence that interbreeding practices were widespread during the domestication process by the early camelid herders in the Atacama during the Early Formative period and predating the Spanish conquest. The study of South American camelids (SACs) and their domestication has been a central subject in South American zooarchaeology since the 1970s. Many studies have focused on elucidating the domestication process as well as use and exploitation of camelid species in the past. These animals were extremely plastic, able to adapt to a great variety of environments found across the Andes and were even present in remote islands, emphasizing their importance in local ecosystems and for historic/prehistoric human populations. Camelids were key in the transition from hunter-gatherer to a mixed economy and played a central role in the cosmic vision of past Andean communities .Lama guanicoe) and vicuña (Vicugna vicugna), and two domestic species, llama (Lama glama) and alpaca (Vicugna pacos). Guanaco and vicuña are the most important endemic large herbivores in South America due to their ecological dominance in the upper Andean ecosystem and their importance for human populations in terms of their subsistence strategies  and the small sized vicuña and alpaca (genus Vicugna)  and astragalus  have beeVicugna) . HoweverVicugna) , the taxTo evaluate the presence of domestic camelids in the Early Formative period  in Northern Chile and to elucidate the origin and evolutionary history of SACs, we have used a combination of osteometry and mitogenomics. We collected ancient samples in the archaeological sites of Tulán in Northern Chile and modern samples collected throughout the current distributional range of the vicuña and guanaco  followinFor the ancient DNA study, we have selected camelid bone samples from four archaeological sites  from San Pedro de Atacama Desert, Northern Chile. The two main sites, Tulán-54 and Tulán-85, belong to the Early Formative period where the camelid domestication is already incorporated into the economic activities by human groups of this area. Tulán-54 is located in Tulán ravine at the Atacama Desert and it shows the development of ceremonial and public architecture. Archaeological remains from offerings and domestic activities evidence that Tulán-54 was an important place of cultural and economic transformation from hunter-gatherers to early pastoralist communities in the highlands of the Southern Andes during the Early Formative society (ca. 3500–2400 BP) . Tulán-8The comparison between the patterns of genetic variation of this dataset of ancient (61) and modern (66) mitogenomes along with the largest dataset of SAC mitochondrial D-Loop sequences (815) generated thus far provides a unique and deeper insight into one of the biggest unsolved mysteries of South American zooarchaeology, SAC domestication.Lama guanicoe and Vicugna vicugna (Lama glama/Lama guanicoe and between Vicugna pacos/Vicugna vicugna) and the use of models based on modern species dimensions, ignoring the large period of selection and change that cannot be recognized through current standards  for either of the bones measured  showed significant differences between the two groups of camelids . These comparisons were carried out between the two groups for each of the four morphological measurements , as well as on the product measurement of the two measurements for each bone (i.e. Bd (mm) * GLm (mm) for the astragali, and BFp (mm) * Dp (mm) for small specimens).Using a capture strategy to enrich our aDNA libraries for mitochondrial sequences, we generated 61 near complete mitogenome sequences from camelid bone samples collected in the Tulán ravine and Atacama Salar: 47 from Tulán-54, 12 from Tulán-85, 1 from Tulán-94, and 1 from Tulán-52. The average depth of coverage in the assembled mitogenomes ranged between 2.53x and 153.03x with a final length of 15,438 base pair (bp).The analysis with mapDamage showed the characteristic pattern of ancient DNA consistent with postmortem deamination damage in all aDNA samples . An incrV. vicugna and two alpacas (V. pacos) . In this clade, 12 ancient specimens (11 from Tulán-54 and 1 from Tulán-85) were morphologically identified as small sized camelids and two samples as big sized (C38 and C39 from Tulán-85). The haplotype diversity for the ancient samples was 1.00 and for the modern samples, the values oscillated within 0.971–0.993 (Table S8).The Bayesian phylogenetic analysis of the ancient and modern mitogenomes using the Bactrian camel as outgroup resulted in strong statistical support for four clades in our data . Clades The dated BEAST phylogeny recovered the same major clades as the MrBayes analysis, lending support to our overall tree topology . In addiThe Bayesian Skyline Plot (BSP) obtained from modern and ancient maternal sequences shows an increase in the Ne of the domesticated clades (Clades 1 and 3) around 8,000–6,000 yr BP in accordance with the putative initiation of domestication. Clade 2 (guanacos) and Clade 4 (vicuñas) also present evidence of a demographic expansion, however at a much earlier time ~ 50,000 yr BP . NeverthLama guanicoe and domestic camelid haplogroups: cacsilensis (66), cacsilensis-guanicoe (30), guanicoe (76), llamas (25), and alpacas (5) (Table S7).To investigate the relationship between the ancient and modern camelids, we analyzed 300 bp of the CR hypervariable I domain. Among the 849 samples analyzed, we detected 76 different polymorphic sites and 158 haplotypes divided into two main haplogroups . ThirteeL. guanicoe guanicoe and Lama glama haplogroups. The temporal network suggests a loss of ancient mitochondrial lineages, mostly in the vicuña (Table S9).A temporal network analysis of the 300 bp of the CR hypervariable I domain was performed using the same dataset as in the PopArt analysis. In total, 849 sequences were included, yielding 158 different haplotypes (42 from ancient sequences and 150 from modern sequences) of which only 11 haplotypes were shared by ancient and modern SAC despite the inclusion of a much higher number of modern mitochondrial sequences than ancient ones . Most ofLama, while for the small animals, the ancient samples fall in the 98th percentile of the distribution of random samples of Vicugna for both statistics.Control region haplotype diversity values were relatively similar between the modern samples (0.9765) and the ancient samples (0.9348). However, given the uneven sample size between modern and ancient samples, we randomly sampled with replacement 10,000 times the modern datasets conditional on a sample size like the ancient samples. For each of the random samples, we estimated the haplotype diversity and the number of substitutions observed. We used that information to draw a distribution of those two summary statistics of genetic diversity conditional on the ancient DNA sample size. The analysis of all the data  indicated that the ancient samples do indeed present a high genetic variation given their sample size as their diversity measures fall in the 93rd and 94th percentile of the distribution of the random samples from modern animals . For theHere, we present the most comprehensive dataset of mitogenomes from SACs to date. This includes a collection of sequences reflecting zooarchaeological samples (ancient DNA) from the San Pedro de Atacama Desert and dates between ~ 3,150 and 2,380 yr BP, and a wide collection of modern samples representing the four extant species. We used these data to compare patterns of genetic variation between taxa and across time to generate insights into hypotheses about the domestication of alpaca and llama. This dataset not only presents unexpected information about the domestication history of SACs, it also provides new insights into the loss of genetic diversity in the extant species.Lama animals and the smaller Vicugna animals is possible (Lama) animals and the small (Vicugna) animals (−4).Zooarchaelogy’s foundation is the ability to accurately identify species from animal remains in order to develop an interpretation of human and environmental interactions within an archaeological context . While tpossible . The bon animals . The staLama (95%) with the exception of two samples that grouped with the Vicugna vicugna clade (Clade 4). The converse also occurred with the majority of the small sized animal bone samples that clustered in Clade 4 (63%) with the exception of seven  that grouped with Lama (Clades 2 and 3).The phylogeny generated using 130 ancient and modern camelid mitogenomes places the vast majority of the ancient samples on the expected clades based on bone morphology . Namely,Few domestic animals have had their origin as contested as llamas and alpacas. While their wild relatives, the guanaco and the vicuña, have been determined to have diverged from their common ancestor two to three million years ago , the domLama alongside guanaco, modern llamas, and zooarchaeological samples of both large- and small sized animals. While llamas are a paraphyletic group with two main branches (Clades 1 and 3) within the greater Lama clade. Within Clades 1 and 3, alpaca groups within llamas supporting the hypothesis that llamas and alpacas were both domesticated from guanacos (hypothesis 3) while vicuña was possibly never domesticated.Our analyses place modern alpaca, with the exception of two samples, within the genus Such a scenario would assume, however, that no alpaca should cluster with vicuñas and that the clade formed by llamas and alpacas has the guanaco as sister taxon  which isLama mitogenome.The domestication hypothesis described above would imply that the alpaca was simultaneously domesticated alongside with the llama from the two guanaco ancestors based on the clustering of alpacas and llamas in our phylogenetic tree. While that could have happened, it is also possible that alpacas are the outcome of hybridization between llamas and vicuñas . AlpacasModern alpacas and llamas are the outcome of an extensive hybridization that has been attributed to the species near demise following the Spanish conquest , hybridiIn particular, However, despite advances in the field of ancient DNA, it still remains very challenging to obtain nuclear data from ancient samples, due to the fragmentary nature of the DNA on these samples and the low endogenous content and, so far, it has not been possible to be obtained from ancient SAC samples . In factFurthermore, although the wild, a hybrid lineages can consistently carry the same mitochondrial haplotype throughout time, they may change body size (e.g. become larger) through backcrossing as the host population genome becomes prevalent in the lineage due to Mendelian inheritance. Lastly, it is possible that the ancient DNA samples in Clades 1–3 represent mostly guanacos and not domestic camelids, suggesting that llamas were domesticated from two different guanaco clades (i.e. Clades 1 and 3) in line with Lama and Vicugna clades, alpaca may have derived in some part from both species with an important component of its genetic variation comprises guanaco, which indicates that hybridization between these species played a role in its evolutionary history.Although our results support the guanaco as the ancestor of llama, we propose a more complex evolutionary history for the alpaca. As small sized animals  are found in both the Lama and Vicugna as their distribution ranges probably shifted north and toward lower altitudes as the far South and mountaintops started to freeze. Contrastingly, the ancient domestic samples from Clades 1 and 3 present a roughly stable ancient demography (Clade 3) or a comparatively moderate population expansion at the onset of the last glacial period (Clade 1). Interestingly, the four clades all show a demographic decline starting near ~ 10,000 yr BP reaching its lowest point around ~ 6,000 years ago. These dates coincide with the establishment of human populations throughout the upper Andes ~ 9,000 yr BP  showed that substantial demographic expansions started in the middle of the previous interglacial and continued expanding throughout the last glacial period. Such an observation is consistent with an increase in habitable land for 00 yr BP  and the 00 yr BP . The bri00 yr BP . The lar00 yr BP .Taken together, the comparative analysis of both the demographic histories and levels of genetic diversity in both datasets indicates that the ancient camelids presented a much larger genetic pool than the modern ones. In the BSP, it can be seen that the plots obtained from the ancient samples  result in substantially larger effective population sizes and more ancient coalescent times , reflectV. v. mensalis) where guanacos (L. g. cacsiliensis) tend to form small family groups of 5–10 individuals , while in the southern ranges vicuña groups tend to be smaller (V. v. vicugna) and the guanacos (L. g. guanicoe) are larger than in northern locations. Such differences between the two species likely made the vicuña a preferential hunting target for the upper Andean human population, as they were easier to find in large quantities and because their groups were found in relative proximity to each other due to the environmental constraint of their habitat distribution. Lastly, by inhabiting a much more homogeneous habitat in the upper Andes (the High Plateau) reflecting the narrower gradient of the Andean slopes where they are found, vicuñas are more susceptible to environmental changes that affect their habitat as these likely impact their entire range. Nevertheless, while human-driven demographic changes are likely to have driven some of the changes observed in this data, the effect of environmental changes cannot be ignored, in particular during the middle Holocene climatic optimum (~8.2 kyr BP–4.2 kyr BP). This time span is marked by a period of strong hydroclimate shifts that led to the aridization of the Andes as rainfalls became more stochastic and temperatures increased , current climate change exacerbating the aridification of the upper Andes, land conversion for agriculture, and a general lack of appetite from local governments to invest in these native species of agricultural value are likely to drive these camelids into further trouble.The study area is located in the II Region of Chile (Antofagasta), specifically in the western slope of the Puna de Atacama (22°−24°S)  at the sThe site Tulán-54 is located on an esplanade on the southern edge of the Tulán ravine and Tulán-85 is located on the border of the Salar de Atacama salt flat at an altitude ranging from 3,200 masl to 2,300 masl in less than 30 km , enablinSamples from Tulán-54 were selected from inside and outside of the main and smaller ceremonial structure and outside  in order to cover different sections of the site (Table S1). Tulán-85 corresponds to an extended mounded stratified deposit , interpreted as an area of domestic activity associated to human newborn burials and few domestic structures, however no ceremonial architecture was found . SamplesWe recorded osteometric measurements from 61 out of the 77 bone samples from the archaeological sites using Vernier calipers to the nearest 0.1 mm, as described by DNA was extracted from 61 samples of phalanx and astragalus bones in a dedicated ancient DNA laboratory at the GLOBE institute, University of Copenhagen. From each specimen, 150 mg of bone powder was drilled using a Dremel drill to obtain bone powder for DNA extraction. DNA extractions were performed as in Double stranded libraries were built using 21.25 µl of the extracts following the protocol from https://arborbiosci.com/) as part of several MYbaits targeted enrichment kits. Hybridization by capture was performed following the MYbaits v3 manual, using 350–500 ng of library starting material. We performed a single round of enrichment at 55°C for 24 hr. Enriched libraries were eluted in a total volume of 30 µl. Post-capture amplification was carried out according to the suggested protocol using KAPA HiFi HotStart Uracil+ReadyMix and 12 cycles for the cycling PCR conditions. Enriched libraries were quantified using Agilent 2200 TapeStation System, pooled in equimolar amounts, and sequenced on an Illumina HiSeq 2500 using 80 bp single-end chemistry at the Danish National High-Throughput Sequencing Center.Our sequencing libraries underwent target-capture prior to sequencing in order to enrich our libraries of endogenous mitochondrial DNA sequences. We designed a camelid set bait which was composed of 120 nucleotides baits tiled every four bases across a representative sample of four living camelid mitochondrial genome sequences. The 6234 baits were then synthesized at MYcroarray (Vicugna pacos (GenBank: Y19184.1), and (iii) PCR duplicate removal. Seeding was disabled for mapping and low-quality reads of less than 30 bp were discarded. The BAM files with unique reads were loaded into Geneious v7 (PALEOMIX was used to perform basic read processing : (i) adaeious v7  to genereious v7 .L. guanicoe cacsilensis) and Southern guanaco (10 L. guanicoe guanicoe); 17 vicuñas: Northern vicuña  and Southern vicuña (11 V. vicugna vicugna) from throughout the current distribution across Peru, Argentina and Chile; 16 llamas from a slaughterhouse in Putre, Chile; and 17 alpacas from a farmer and breeder in Chile. Genomic DNA was extracted using the Puregene Tissue Core Kit A (Qiagen). For each individual, 1–3 μg of DNA was sheared into fragments of 150–700 bp prior to the construction of the sequencing library. Adapter ligation (or library build) was performed using the NNNPaired-end sequencing library kit with an insert size of approximately 300 bp. Libraries were sequenced to mid/low-coverage (11-14x) on an Illumina Hiseq X Ten platform.For comparison with the aDNA samples, blood and tissue samples were obtained from 66 georeferenced modern samples to generate complete mitochondrial genomes excluding the control region , the Southern guanaco (366 L. guanicoe guanicoe), the Northern vicuña , the Southern vicuña (35 V. vicugna vicugna), domestic alpaca (53 V. pacos), domestic llama (79 L. glama) and 10 hybrids . Additionally, three ancient guanaco mitogenomes from Isla Mocha (Chile) , one guanaco (NC_011822.1), one vicuña mitogenome (FJ456892.1), one llama mitogenome (AP003426.1) and two alpaca mitogenomes  were downloaded from NCBI and included in the analyses (Table S3).We included 300 bp mitochondrial control region sequences from 815 modern camelids representing the Northern guanaco , ribosomal RNA (rRNA) genes, and a partial control region sequence (CR). Partition Finder v1.1.1  was used6 generations, sampling every 1 × 103 generations and discarding the first 25% of steps of the MCMC as burn-in following visual examination of the stabilization of the likelihood values of the inferred tree. Trees were summarized with the majority-rule consensus approach, using posterior probability as a measure of clade support. The final consensus tree was visualized in FigTree v.1.4.2 (Phylogenetic relationships were estimated using the software MrBayes v3.2.6  and the  v.1.4.2 . The num v.1.4.2  and ape  v.1.4.2 . We estiC. dromedarius (NC_009849.1) and C. bactrianus (NC_009628.2) as outgroups. A Shimodaira-Hasegawa test for the evolution of the trio vicuña, guanaco, and dromedary was calculated in MEGA X    and showtree.org  divergenTo explore the relationship between the different subspecies and their wild relatives, we aligned the partial CR (control region) from the ancient and modern mitogenomes with a total of 849 specimens for the four species covering the distribution range across Peru, Chile, Argentina, and Ecuador using MAFFT  Table S and to i In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:This manuscript combines osteometric and ancient genetic data to address the persistent problem of camelid domestication in South America. It offers a large dataset of modern and ancient mitochondrial sequences from across South America and from the sites of Tulán-54 and Tulán-85 in northern Chile, respectively. By drawing samples from a time before recent major losses of camelid diversity, ancient DNA allows identification of lost lineages, and reveals layers of complexity in the process of domestication for both large and small camelid taxa. This has implications ranging from understanding the history of culturally iconic species to aiding in the conservation of threatened lineages.Decision letter after peer review:eLife. Your article has been reviewed by four peer reviewers, including Jessica C Thompson as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by George Perry as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Mariana Mondini (Reviewer #3); Pamela A. Burger (Reviewer #4).Thank you for submitting your article "Ancient DNA reveals the lost domestication history of South American camelids in Northern Chile and across the Andes" 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.eLife papers without additional data, even if they feel that they would make the manuscript stronger. Thus the revisions requested below only address clarity and presentation.We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, we are asking editors to accept without delay manuscripts, like yours, that they judge can stand as Summary:eLife, and the approach is both well-justified and a welcome connection between morphology and aDNA. The reviewers focus on eight major points that should be addressed in a revision. Special attention should be given to the points about the figures, which could be made more clear.Reviewers included area specialists in zooarchaeology and in genetics. All were in agreement that this is a well-written paper, with important implications for understanding camelid domestication in South America. All reviewers also agreed that the data are robust and well-presented. The content is appropriate for publication in The reviewers also wanted to make it clear that future work should involve samples drawn from a larger time span, or with more specific assignment within the ~800 year time span of the sites that dominate this sample. Specifically, it would be very useful to include earlier samples, before culturally induced interbreeding took place. Also, it would be crucial to include camelid fibers, especially those of the early type resembling modern llamas that is no longer found in extant wild populations, as they might correspond to the now extinct wild component of Clade 3. As the archaeological sample is from an area far from the current known range of alpacas – in wetter altiplano areas –, specimens confidently attributed to ancient alpacas that are not suspected to be hybrids and with less interbreeding than modern ones should also be compared in future. It is important that further studies also discuss ways to test the hypothesis of the cross of female llamas with male vicuñas to end up in "phenotypical alpacas" in San Pedro de Atacama ca. 3000 years ago.Essential revisions:1) Osteological measurements: There are issues here of both nomenclature and method:– Astragali have no lines of fusion and therefore an intermediate or small size can simply be a juvenile of a larger species. No criteria are mentioned for defining the condition of "adult" for the astragali. An astragalus of a young individual of the large group could overlap in size with that of an adult of the small group. If this were to occur, some specimens could be wrongly attributed to a different size group and this, as the authors have pointed out, has implications for the taxonomic discussion. Phalanges can be more useful in this way, but they do not seem to discriminate as well. Given the history of hybridization revealed by the mtDNA analysis, there needs to be more nuance in interpreting metric data from phalanges.– The bivariate plot of Figure 2A based on measurements of the phalanxes show two size clusters  but there are several cases  that are intermediate in size. This pattern needs a further explanation that it is not included in that section of the paper. Also Figure 2B based on the astragali shows several outliers that cannot be interpreted in a straightforward manner.– While osteometry is mentioned, there are also geometric morphometric studies that address shape instead of size to determine camelid taxa that could be mentioned. The section called "Morphometric analysis" should in fact be renamed as "Osteometric analysis," as it does not address shape.– Figures need to be adjusted so that the samples are clearer , and where the samples came from (and modern distributions) are better-justified.2) Archaeological context of the samples: This needs to be much clearer and more specific, and there seem to be superfluous images of some contexts  without explanation about their relevance to the sample. Specifically:– There is little discussion about what happened with the single specimen that was older, from TU-52, or the significance/insight of the contemporaneous specimen from TU-94, which was ostensibly sampled to develop the more regional view. It is really challenging to find these on the various figures without a lot of flipping back and forth between sections of the manuscript. Information provided on Tulán-52 and Tulán-94 sites is too short and uneven. The samples from these sites (2 from each?) should better be listed in Table S1 in Supplementary file 1. It is not clear why only two samples were taken from these two sites, especially Tulán-52, which represents an earlier period that was critical in SAC domestication. In presenting the region, note that mention of San Pedro de Atacama desert only appears in the Discussion.– What is the specific significance of the locality of Tulán with respect to this problem? How might the story change if a different locality had been sampled? The supplementary information on this is not very detailed and, as mentioned above, the significance of specimens from multiple sites at Tulán is not really explored.– What was the context of the sampled remains? They were inside or outside of ceremonial or inhabited areas, but were they from ceremonial contexts, or trash middens? Supplementary Figure 2B – what is the purpose of illustrating the human burial with the offering? Is it because this is the context from which the camelid remains derived?– Supplementary Figures 1 and 2: A "partial" (Tulán 54) and "panoramic" (Tulán 84) site views are provided. However, it would be useful to include a plan of both sites to know the spatial provenience of the bones discussed at a more detailed scale.– In the subsection “Biogeographical area”, different dates are provided for the Formative period; it would be clearer to state that it starts at c. 5000 BP and the period represented in these particular sites is c. 3360-2370 BP. The inference that human populations started selecting wild camelids to raise their own herds c. 3000 years ago should be supported with some references.3) Explicit discussion of limitations: The authors should be certain to clearly state the caveats of this study. They should be clear about the potential influence of factors, and their timing, that are independent of domestication on both size and molecular diversity. Specifically, these are limitations imposed by:– The osteometric and aDNA methods.– The archaeological sample relative to the research question .– Hybridization and later killing-off events.– Potential impact of climatic/environmental changes.– The extent to which any of the above is knowable.4) Interpretations of hybridization:In the subsection “Early Hybridization of domesticated camelids”, if modern genomes are extensively hybridized in the recent past, how valid are the modern samples used in this analysis? In other words how can a molecular taxonomic identification actually work – and be compared against the morphological results – if the modern reference collection of, say, a "guanaco" might actually have been hybridized in the recent past? And if only mitogenomes are used in this analysis, how can such hybridization truly be pulled apart? Then, the specific "hybrid specimens" described here are not really directly addressed in the Results sections; on what basis are these individuals determined to be hybrids? If it is simply the difference between morphology and mitogenome haplotype (which seems to be the case), then we should investigate which specimens were first phalanges and which ones were astragali, because the astragali seem to be much more effective at separating them out (see point #1). On the trees that code the specimens by size, I am reading this to mean that anything in the "intermediate" category was a phalanx, and therefore I'm not sure that this is a "hybrid" so much as a problem with the discrimination of the method. Alternatively, if they are truly hybrids, then there might not be expected to be a clear morphological result from the bone measurements.5) Discussion of implications of bottlenecks:– It is interesting that the vicuña, which according to the model reconstructed here was not a major source of domesticated stock in the past, is the one that has undergone the most genetic bottlenecking; could this potentially be as a result of human manipulation of other taxa that caused their populations to increase at the expense of wild vicuña habitat? What is the actual mechanism that the authors propose for the bottleneck in vicuña?– The Holocene SAC demographic decline is related to human behavior in the manuscript. It would be interesting to assess the impact of climatic and concomitant environmental changes since the late Pleistocene – including the Antarctic Cold Reversal and the Hypsithermal – on their demography as well. The reasons for vicuñas undergoing bottlenecks unlike guanacos is also worth further exploring. It will also be interesting to explore why vicuña demography has recovered so nicely in recent years in spite of that, while Andean and Chaco guanacos – excluding Fuego-Patagonian steppe populations – are now so patchily distributed that are considered to be endangered. It would be interesting to know how genetic variation corresponds to camelid subspecies in modern samples, and whether this variation can be traced back in the fossil samples.6) Adjustments to figures:L.g. cacsilensis is present in southern latitudes , distant from its northern type locality, and the Andes appear "to offer little impediment to movement"  it would be important to establish if in the past there has been an interconnection between both guanaco populations.– Figure 1: The map legend should stress on what kind of data it is based, whether on the documented provenience localities where the subspecies have been previously described from a taxonomic point of view, or from any other source of information. It would be also helpful to have some precisions on the time period represented: pre or postcolonial times. The sources of the distributional information should be cited. More importantly, the fact that there are these subspecies and their main differences should be introduced when presenting SACs and before the figure. The color-coding used for the map is not enough clear to distinguish the overlapping distribution of these four subspecies, as is the case of the southern vicugna and the guanaco. Also, there are extant guanaco populations in Córdoba, Santiago del Estero and Buenos Aires provinces in Argentina that do not appear in Figure 1. The map should show the georeferenced points from where the specimens used in the analysis (data from Table S3 in Supplementary file 1). The apparent attribution to the L. g. guanicoe subspecies in the map is a supposition since the guanaco of Northwest Argentina has not been studied genetically yet. As – Figure 2: In the section "Morphometric analysis" it says that measurements were taken from 61 specimens out of a sample of 75 bones, and that of those measured specimens 18 were not considered for morphometric analysis. In a previous section it says that the mitogenomes dataset comprises 60 ancient records. These numbers do not add up. This seems to suggest that DNA was extracted from bones that were not included in the morphometric analysis. If this is true it should be mentioned. It would give a better understanding of the complementary nature of the osteometric and genetic database and the research design. The dots are also difficult to differentiate, because the labels overlap so much and the dots are the same size and color; which ones are archaeological and included versus not included in the analysis? There also seem to be no modern camelid measurements for the astragalus?– Figure 3: Even with a zoom 200% it is impossible to read the posterior probability values of the support of the nodes. It is interesting that there are a number of "small-sized" archaeological specimens in Clade 2. Are these samples on astragali (better indicators) or phalanges (more variation)? There is a lot more variation in the sizes of adult phalanges and astragali in the archaeological sample than in the modern sample, and potentially this may have something to do with active moments of admixture and hybridization between the different clades. This relationship between the morphological data from the two different elements and how this would be represented under various hybridization scenarios could be better-addressed in the text (see point #4).– Figure 5 is really interesting and easy to read, but surprising that the small-sized archaeological samples group exclusively away from the vicuña.– Figure 6: Is there some way to indicate which taxa are associated with which haplogroups here, as well as linking the past with the present? It is a really useful figure, but difficult to understand the relationships between taxa (even if only the modern taxa are indicated). Perhaps the (lost) vicuña haplotypes be indicated by A vicuña figure, similar to Figure 5 – which is very clear.– Figure 7: the labelled panels  do not have any specific information about the samples to which they pertain.– Supplementary Figure 3: A minor thing maybe, but the underlying grey map seems to have different square shades of grey. This is a bit irritating. The legend next to the map is difficult to read, the quality of the letters is not good and the green and blue dots have grey squares around them.7) References:Check for missing references, incomplete ones and errors in them. There are some inconsistencies in how the authors are listed in the text and in the Bibliography, particularly with authors who have two last names/surnames. For example: "López, P." and "López Mendoza, P." are the same person; while "Mengoni-Goñalons" correctly in the text, is only "Goñalons" in the Bibliography. The authors might also consider how their names are listed here with respect to how they have published their names in other papers.8) Formatting:eLife style does typically place it at the end.– Although it is possible to place Materials and methods to be near the front of the manuscript, – The supplementary information should also appear together at the end of the manuscript, not divided up into sections within the main body of the manuscript.– Check for typos and formatting errors; date ranges should always be provided from the older to the younger age. Also check for grammar and sentence structure across the text, as many phrases need rewriting . The text needs extensive revision in this regard.– Figures and tables are not numbered after the order in which they appear in the text, and formatting is not even . While the text mentions that first phalanges were measured, the figures and tables should also read so . Letters in the pictures making up Supplementary Figure 2 do not match the legend; they mix with Supplementary Figure 1. Figure 3—figure supplement 3 is only mentioned in the legend of another figure and not in the main text. Essential revisions:1) Osteological measurements: There are issues here of both nomenclature and method:– Astragali have no lines of fusion and therefore an intermediate or small size can simply be a juvenile of a larger species. No criteria are mentioned for defining the condition of "adult" for the astragali. An astragalus of a young individual of the large group could overlap in size with that of an adult of the small group. If this were to occur, some specimens could be wrongly attributed to a different size group and this, as the authors have pointed out, has implications for the taxonomic discussion. Phalanges can be more useful in this way, but they do not seem to discriminate as well. Given the history of hybridization revealed by the mtDNA analysis, there needs to be more nuance in interpreting metric data from phalanges.In the case of the astragalus, growth occurs from a single primary center of ossification, after birth the bone continues growing until the bone is completely ossified. The reduction of bone porosity and well delimited medial intertarsal and plantar trochlear articular surfaces were used as criteria to select the samples. Modern measurement of subadult individuals of the big size camelid does not overlap in size with that of an adult of the small group . However, we cannot rule out the possible overlapping due to age in the smaller size camelid group .–The bivariate plot of Figure 2A based on measurements of the phalanxes show two size clusters  but there are several cases  that are intermediate in size. This pattern needs a further explanation that it is not included in that section of the paper. Also Figure 2B based on the astragali shows several outliers that cannot be interpreted in a straightforward manner.– While osteometry is mentioned, there are also geometric morphometric studies that address shape instead of size to determine camelid taxa that could be mentioned. The section called "Morphometric analysis" should in fact be renamed as "Osteometric analysis," as it does not address shape.– Figures need to be adjusted so that the samples are clearer , and where the samples came from (and modern distributions) are better-justified.The provenance of the ancient samples used in this study are included in Table S1 in Supplementary file 1. You can find more information about the measurements of each sample in Table S4 in Supplementary file 1.About the modern samples, Figure 1 has been adjusted to have a better visualization of the distribution and sampling of the modern samples used in this manuscript.2) Archaeological context of the samples: This needs to be much clearer and more specific, and there seem to be superfluous images of some contexts  without explanation about their relevance to the sample. Specifically:– There is little discussion about what happened with the single specimen that was older, from TU-52, or the significance/insight of the contemporaneous specimen from TU-94, which was ostensibly sampled to develop the more regional view. It is really challenging to find these on the various figures without a lot of flipping back and forth between sections of the manuscript. Information provided on Tulán-52 and Tulán-94 sites is too short and uneven. The samples from these sites (2 from each?) should better be listed in Table S1 in Supplementary file 1. It is not clear why only two samples were taken from these two sites, especially Tulán-52, which represents an earlier period that was critical in SAC domestication. In presenting the region, note that mention of San Pedro de Atacama desert only appears in the Discussion.We mention briefly in the main text, subsection “Archaeological samples” the presence of domestic camelids during the Late Archaic period suggested as well by osteometry and fibers analysis . Early Formative groups show strong continuity with Late Archaic ones expressed in architectural patterns and lithic material among others . Introduction.The principal focus of this project was the material from the sites TU-54 and TU-85. During the experimental work we included two samples from TU-52 and TU-94 to evaluate the DNA preservation of material from other archaeological sites from the same area for further genetic studies at Tulán Ravine. We agree with the reviewer that it would have been a good idea to further sample other sites. However, this was not possible at the time this project was conducted.The study area located in the western slope of the Puna de Atacama (22°-24°S) at the southeastern border of the Salar de Atacama salt is mentioned at the beginning of Materials and methods.– What is the specific significance of the locality of Tulán with respect to this problem? How might the story change if a different locality had been sampled? The supplementary information on this is not very detailed and, as mentioned above, the significance of specimens from multiple sites at Tulán is not really explored.We develop the description of the main formative sites (Tu-54 and Tu-85) in the text and only leave the figures in the supplementary material. As we pointed out in the main text, the Tulán ravine contains a complete occupational sequence from the Early Holocene to the recent times . It is characterized by a redundancy of occupations in a long span of time and Tu-54 is the only site with monumental architecture in the region during the Early Formative. This site could be considered as a local political and religious center. Furthermore, it shows the integration of macro-regional interaction networks between the cost and Northwest Argentina . These are the reasons why we chose this area to explore the history of domestication of South American camelids through ancient DNA.Discussion.– What was the context of the sampled remains? They were inside or outside of ceremonial or inhabited areas, but were they from ceremonial contexts, or trash middens? Supplementary Figure 2B – What is the purpose of illustrating the human burial with the offering? Is it because this is the context from which the camelid remains derived?Tulán-54 has been defined as a ceremonial center, where the sunken ceremonial structures had been filled with remains deposited over time to cover the entire structure, forming a stratified mound made mostly of camelid bones, although lithic, ceramic, and plant remains, among others, are also present. Stratigraphic analyses showed recurrent discarding events during the occupation of the site, suggesting the practice was repeated over time until it was completely covered to a maximum depth of around 2m Cartajena et al., 2019. Samples were collected at different levels and sectors in the sites, incorporating samples from inside the ceremonial structures and outside, related to domestic activities.The image with the human burial has been removed, it was used to illustrate the ritual context of the site, but was not in direct relation with the samples. The provenance of the samples was included in a column in Table S1 in Supplementary file 1.– Supplementary Figures 1 and 2: A "partial" (Tulán 54) and "panoramic" (Tulán 84) site views are provided. However, it would be useful to include a plan of both sites to know the spatial provenience of the bones discussed at a more detailed scale.The information of each sample was included in Table S1 in Supplementary file 1. We consider that it is not relevant to include a spatial plan of each site because our main goal was more focused on DNA analyses of camelid bones.– In the subsection “Biogeographical area”, different dates are provided for the Formative period; it would be clearer to state that it starts at c. 5000 BP and the period represented in these particular sites is c. 3360-2370 BP. The inference that human populations started selecting wild camelids to raise their own herds c. 3000 years ago should be supported with some references.14C ages have now been included in the main text and references included to support previous works. Additional references were included in the text:All Cartajena, 2013; Cartajena et al., 2019; Izeta, 2004; Loyola, Núñez and Cartajena, 2019a; Núñez, Grosjean and Cartajena, 2002; Núñez et al., 2005; Núñez et al., 2017a.3) Explicit discussion of limitations: The authors should be certain to clearly state the caveats of this study. They should be clear about the potential influence of factors, and their timing, that are independent of domestication on both size and molecular diversity. Specifically, these are limitations imposed by:– The osteometric and aDNA methods.The limitations of ancient DNA analyses were added in the Discussion, subsection “Early Hybridization of domesticated camelids”.The discussion about osteometric methods and their validity for taxonomic identification is explained in the Results.– The archaeological sample relative to the research question .We included an extended description of the sites Tulán-54, Tulán-85, Tulán-52 and Tulán-94, subsection “Archaeological samples”. We explained why we chose samples from that period of time, Early Formative and why the Tulán ravine in San Pedro de Atacama is a suitable place for looking at the domestication process of SAC from early times. Materials and methods.Moreover, we have included pictures of the sites and a map with the location of the sites. Figure 1—figure supplement 1.– Hybridization and later killing-off events.The problem with the hybridization and later killing-off events is explained in the Discussion, section “Early Hybridization of domesticated camelids.”– Potential impact of climatic/environmental changes.We refer to the impact of the climatic changes in the Discussion.– The extent to which any of the above is knowable.The exact extent as to which any of the above mentioned limitations is knowable is somewhat limited by our dataset as it is just a single marker from a relatively small geographical region. While we have still added some significant new hypotheses to the question of SAC domestication and evolution, the most comprehensive way to address these would be through the inclusion of a large palaeogenomic dataset from multiple sites across the Andes. We mention that the inclusion of ancient nuclear genomes may shed some light on these with more depth, however, such data is out of the scope of the current study. Discussion.4) Interpretations of hybridization:In the subsection “Early Hybridization of domesticated camelids”, if modern genomes are extensively hybridized in the recent past, how valid are the modern samples used in this analysis? In other words how can a molecular taxonomic identification actually work – and be compared against the morphological results – if the modern reference collection of, say, a "guanaco" might actually have been hybridized in the recent past? And if only mitogenomes are used in this analysis, how can such hybridization truly be pulled apart? Then, the specific "hybrid specimens" described here are not really directly addressed in the Results sections; on what basis are these individuals determined to be hybrids? If it is simply the difference between morphology and mitogenome haplotype (which seems to be the case), then we should investigate which specimens were first phalanges and which ones were astragali, because the astragali seem to be much more effective at separating them out (see point #1). On the trees that code the specimens by size, I am reading this to mean that anything in the "intermediate" category was a phalanx, and therefore I'm not sure that this is a "hybrid" so much as a problem with the discrimination of the method. Alternatively, if they are truly hybrids, then there might not be expected to be a clear morphological result from the bone measurements.The hybridisation history of South American Camelids is focused around the hybridisation events that occurred between alpacas and llamas, i.e. between the domestic forms and not between the wild camelids (Guanacos and Vicuñas). Hence any statement reflecting hybridisation events in the past are limited to those that occurred between llamas and alpacas.Three main sections for this argument:1) We do find significant differences between large and small animals with morphology. Discussion.-4).The bones from which no DNA was extracted were removed from the analyses as their results bear little consequence to our work presented here . Additionally, we carried out statistical tests between the different measurements carried out for each of the bones and showed that the large specimens are significantly different from the small specimens  Hybridization is largely a new thing and not an old event.“In particular, Fan et al., 2020, timed the hybridization history between South American camelids and found evidence for a post-colonial hybridization, and not an ancient one, thereby suggesting that the bulk of the taxonomic uncertainty driven by this process affects modern samples. Moreover, although rare, hybridization between llamas and guanacos , and separately between alpacas and vicuñas  have been previously reported . While the results presented here do not disagree with the hybridization patterns described elsewhere , the observation of hybrid animals among the zooarcheological samples (i.e. those samples that present a discordance between their taxon identification based on bone morphology and their molecular taxonomic identification), suggests that the hybridization history of the domestic camelids is yet far from understood, and could have also occurred prior to the European colonization of South America.”3) It is possible that Clades 1-3 are mostly guanacos and not domestic animals:“Furthermore, although the wild, a hybrid lineages can consistently may carry the same mitochondrial haplotype throughout time, they may change body size (e.g. become larger) through backcrossing as the host population genome becomes prevalent in the lineage due to mendelian inheritance. […] However, the presence of both domestic camelids in Tulán has been previously established through the analyses of bone remains , microscopic camelid fiber analyses , and the analyses of bone collagen stable isotopes that differentiate llamas ritually fed with maize from wild guanacos .”5) Discussion of implications of bottlenecks:– It is interesting that the vicuña, which according to the model reconstructed here was not a major source of domesticated stock in the past, is the one that has undergone the most genetic bottlenecking; could this potentially be as a result of human manipulation of other taxa that caused their populations to increase at the expense of wild vicuña habitat? What is the actual mechanism that the authors propose for the bottleneck in vicuña?We hypothesize that the stronger loss of variation in the vicuña is the outcome of the preferential hunting of vicuñas rather than guanacos by the early upper Andean communities, reflecting aspects of the ecology and behavior of vicunas:“This is not surprising when considering the relative homogeneity of the environment where vicuñas are found and how gregarious they are in comparison to guanacos. […] Such aridification is likely to have changed the habitable range for the vicuña further exacerbating the demographic pressure that it was already experiencing.”– The Holocene SAC demographic decline is related to human behavior in the manuscript. It would be interesting to assess the impact of climatic and concomitant environmental changes since the late Pleistocene – including the Antarctic Cold Reversal and the Hypsithermal – on their demography as well. The reasons for vicuñas undergoing bottlenecks unlike guanacos is also worth further exploring. It will also be interesting to explore why vicuña demography has recovered so nicely in recent years in spite of that, while Andean and Chaco guanacos – excluding Fuego-Patagonian steppe populations – are now so patchily distributed that are considered to be endangered. It would be interesting to know how genetic variation corresponds to camelid subspecies in modern samples, and whether this variation can be traced back in the fossil samples.We thank the reviewers for highlighting the Antarctic Cold Reversal and Hypsithermal, which we have now addressed in our paper (see point above).The issue of the recovery of the vicuña and the endangerment of the guanaco was also addressed in the last paragraph of the Discussion.The relationship between haplotypes shared by the ancient and modern samples is shown in Table S9 in Supplementary file 1.6) Adjustments to figures:– Figure 1: The map legend should stress on what kind of data it is based, whether on the documented provenience localities where the subspecies have been previously described from a taxonomic point of view, or from any other source of information. It would be also helpful to have some precisions on the time period represented: pre or postcolonial times. The sources of the distributional information should be cited. More importantly, the fact that there are these subspecies and their main differences should be introduced when presenting SACs and before the figure. The color-coding used for the map is not enough clear to distinguish the overlapping distribution of these four subspecies, as is the case of the southern vicugna and the guanaco. Also, there are extant guanaco populations in Córdoba, Santiago del Estero and Buenos Aires provinces in Argentina that do not appear in Figure 1. The map should show the georeferenced points from where the specimens used in the analysis (data from Table S3 in Supplementary file 1). The apparent attribution to the L. g. guanicoe subspecies in the map is a supposition since the guanaco of Northwest Argentina has not been studied genetically yet. As L.g. cacsilensis is present in southern latitudes , distant from its northern type locality, and the Andes appear "to offer little impediment to movement"  it would be important to establish if in the past there has been an interconnection between both guanaco populations.The Figure 1 was completely replaced by a new one with a more precise distribution of the current distribution of the guanaco (A), separated from the current distribution of the vicuña (B). The colors used in this figure are in correspondence with the rest of the figures along the manuscript.g. guanicoe  so we assume that the guanacos of Northeastern Argentina are as well.This figure shows the collection of modern samples from the places where the mitogenomes and the Control Region were obtained. Regarding the limit of the guanaco subspecies, there is a very wide contact zone that includes the northeast of the distribution, Monte Argentino and North-central Chile, but whose individuals belong predominantly to L. – Figure 2: In the section "Morphometric analysis" it says that measurements were taken from 61 specimens out of a sample of 75 bones, and that of those measured specimens 18 were not considered for morphometric analysis. In a previous section it says that the mitogenomes dataset comprises 60 ancient records. These numbers do not add up. This seems to suggest that DNA was extracted from bones that were not included in the morphometric analysis. If this is true it should be mentioned. It would give a better understanding of the complementary nature of the osteometric and genetic database and the research design. The dots are also difficult to differentiate, because the labels overlap so much and the dots are the same size and color; which ones are archaeological and included versus not included in the analysis? There also seem to be no modern camelid measurements for the astragalus?We fixed the mistake of the dataset, there are a total of 61 ancient mitogenomes, review Table S5 in Supplementary file 1.Figure 2A and B were remade and removed the dots that were not included in the ancient DNA analysis. Moreover we included modern camelid measurements for the astragalus for the four species in Figure 2B.– Figure 3: Even with a zoom 200% it is impossible to read the posterior probability values of the support of the nodes. It is interesting that there are a number of "small-sized" archaeological specimens in Clade 2. Are these samples on astragali (better indicators) or phalanges (more variation)? There is a lot more variation in the sizes of adult phalanges and astragali in the archaeological sample than in the modern sample, and potentially this may have something to do with active moments of admixture and hybridization between the different clades. This relationship between the morphological data from the two different elements and how this would be represented under various hybridization scenarios could be better-addressed in the text (see point #4).We are sorry for having submitted the low font version of the figure. Because the majority of the posterior probability values for the nodes’ supports are larger than 0.9, we opted for removing all support values of the three and only indicate those with a posterior probability support lower than 0.9. We have accordingly added a line to the figure legend indicating this and the percentage of the nodes that have a posterior probability larger than 0.9.We have added information on the sample sizes used to estimate the standard deviations obtained from bone measurements in the modern samples, which for three of the species correspond to 7 or fewer individuals, and only the llama had 14 samples measured. The issue about hybridisation scenarios was addressed in point 4.– Figure 5 is really interesting and easy to read, but surprising that the small-sized archaeological samples group exclusively away from the vicuña.For this network we only used the most variable region of the mitochondrial DNA. The results could have changed compared with the complete mitochondrial genome phylogeny.– Figure 6: Is there some way to indicate which taxa are associated with which haplogroups here, as well as linking the past with the present? It is a really useful figure, but difficult to understand the relationships between taxa (even if only the modern taxa are indicated). Perhaps the (lost) vicuña haplotypes be indicated by A vicuña figure, similar to Figure 5 – which is very clear.We added Table S9 in Supplementary file 1, with the information about which taxa are associated with which haplogroups and the ancient samples included in each haplotype.– Figure 7: the labelled panels  do not have any specific information about the samples to which they pertain.For this figure we used random modern samples conditional on the sampling size of the ancient DNA samples. The purpose of this analysis was to measure the genetic diversity between modern and ancient samples.– Supplementary Figure 3: A minor thing maybe, but the underlying grey map seems to have different square shades of grey. This is a bit irritating. The legend next to the map is difficult to read, the quality of the letters is not good and the green and blue dots have grey squares around them.Supplementary Figure 3 was removed. Nevertheless, location of the archaeological site and map of modern camelid samples included in the Bayesian phylogenetic analysis was included in the new Figure 1.7) References:Check for missing references, incomplete ones and errors in them. There are some inconsistencies in how the authors are listed in the text and in the Bibliography, particularly with authors who have two last names/surnames. For example: "López, P." and "López Mendoza, P." are the same person; while "Mengoni-Goñalons" correctly in the text, is only "Goñalons" in the Bibliography. The authors might also consider how their names are listed here with respect to how they have published their names in other papers.We have checked and fixed the errata in the bibliography.8) Formatting:– Although it is possible to place Materials and methods to be near the front of the manuscript, eLife style does typically place it at the end.Materials and methods has been moved to the end of the manuscript before the bibliography.– The supplementary information should also appear together at the end of the manuscript, not divided up into sections within the main body of the manuscript.Supplementary material has been moved to the end of the manuscript.– Check for typos and formatting errors; date ranges should always be provided from the older to the younger age. Also check for grammar and sentence structure across the text, as many phrases need rewriting . The text needs extensive revision in this regard.Thank you for the observation. We have checked and reviewed those errata.– Figures and tables are not numbered after the order in which they appear in the text, and formatting is not even . While the text mentions that first phalanges were measured, the figures and tables should also read so . Letters in the pictures making up Supplementary Figure 2 do not match the legend; they mix with Supplementary Figure 1. Figure 3—figure supplement 3 is only mentioned in the legend of another figure and not in the main text.We have checked and reviewed those errata.


The improvement of the carrier transport properties promotes by the turbostratic stacking structure. In the turbostratic multilayer GNR with 6 layers or more, although the Ion/Ioff ratio degraded, the conductance extremely improved with increasing the number of layers. This indicates that the turbostratic multilayer GNR with thicker number of layers becomes the significantly lower resistivity wire as a metallic characteristic. We revealed that the crossover point of the physical properties between the semiconducting and metallic characteristics is determined by the strength to screen the surrounding environment effects such as charged impurity on the substrate. Our comprehensive investigation provides a design guidance for the various electrical device applications of GNR materials.The electrical transport properties of a turbostratic multilayer graphene nanoribbon (GNR) with various number of layers (1–8 layers) were investigated using a field effect transistor with a single GNR channel. In the turbostratic multilayer GNR with 5 layers or less, the carrier mobility and I The edge structures and the width of GNR material play an important role in its electrical band structures9, and optical properties13. For example, the band gap width shrinks as the GNR width increases14. Therefore, the various synthesis methods of the GNR using electron beam lithography16, chemical cutting or etching of graphene18, nickel nanobars19 and precursor monomers as a bottom-up approach20 have been studied for the precise control of the structures.Since theoretical calculation using a tight binding model predicted that a quasi-one dimensional monolayer graphene nanoribbon (GNR) exhibits a finite energy band gap at the Fermi level22, and the high Ion/Ioff ratio (105–106) and carrier mobility exceeding 100 cm2/Vs are demonstrated using the field effect transistors (FETs) with the single GNR channel24. This result indicates that these GNRs have a semiconducting characteristic. On the other hand, the multilayer GNR can be synthesized by unzipping multi-walled CNTs26 and the plasma-enhanced chemical vapor deposition (CVD) growth on nickel nanobars27. Recently, the demonstration of the extremely low resistivity in the metallic multilayer GNR28 had a great impact on the interconnect applications of the GNR materials32. The completely different physical properties such as semiconducting and metallic characteristics observed in the GNR materials are properly utilized in these device applications, respectively. However, there is no clear design guidelines for controlling the semiconducting and/or metallic characteristics in the real GNR devices except for the edge structure and GNR width in the ideal system treated in theory. What is the dominate factor that determines the conductive properties of the GNR material embedded in the device structure? The semiconducting and metallic characteristics tend to be mainly observed at a monolayer (or bilayer) GNR24 and a multilayer GNR28, respectively. Therefore, we focus on the effect of the number of layers on the carrier transport properties of the GNR-FETs.The monolayer or bilayer GNRs obtained by unzipping of single- or double-walled carbon nanotubes (CNTs) provide the narrow width and smooth edges2/Si device substrate as a comparison with an intrinsic property of the monolayer graphene observed in a suspended structure33. Since the carrier concentration of the monolayer graphene with a linear dispersion is very low near the Dirac point, the carrier transport properties in the monolayer graphene-FETs are extremely sensitive to the surrounding environments such as the charged impurity and surface roughness34. On the other hand, in a multilayer graphene-FET, the carrier scattering induced by the surrounding environments is dramatically improved by the screening effect of the multi-stacking35. In the GNR system with a narrow width, the surrounding environment effect is considered to be extremely large compared with the graphene system.It is well known that the conductivity and carrier mobility of a monolayer graphene-FET extremely degrades on the SiO2/Si substrate. The turbostratic multilayer were synthesized by an overlayer growth of graphene on a GNR template prepared by unzipping from double-walled CNTs as previously reported36. We found that the carrier transport properties such as the semiconducting and metallic characteristics observed in the multilayer GNR with a narrow width (18–25 nm) are determined by strength to screen the charges of the impurity on the device substrate as a surrounding environment. This result indicates that the type of the device can be controlled by the number of layers in the multilayer GNR.In this study, to understand the surrounding environment effect on the carrier transport properties of the GNR, we investigated the transfer characteristics of the GNR-FETs with various number of layers (1–8 layers) on the SiO22 used as the starting material, and is slightly higher than the interlayer distance (0.34 nm) in the bulk graphite. Considering that the height of the monolayer graphene on the SiO2/Si substrate is 0.6–0.8 nm due to a very weak interaction between the graphene and substrate37, this result means that the monolayer or bilayer GNRs are efficiently synthesized by unzipping from the DWCNTs. After the CVD growth, the height of the GNRs are increased by ~ 1 nm, meaning the formation of additional two or three layers of graphene on the pristine GNR template. Figure Figure 38. Figure −1. The spectrum observed from the pristine GNR can be fitted by a single Lorentzian peak, indicating that the pristine GNR is constructed by a monolayer structure. The stacking structures in the grown multilayer graphene are mainly divided into two types: the ordered stacking (e.g. AB stacking) and turbostratic stacking. In a multilayer graphene with the ordered stacking, the peak shape in the 2D-band spectrum is decomposed by two Lorentzian peaks at ~ 2680 and ~ 2720 cm−1, respectively39. On the other hand, the peak shape observed from a turbostratic multilayer graphene, in which the stacking of the graphene layers is rotationally random in plane, shows a single Lorentzian at ~ 2700 cm−1, just as in monolayer graphene40. Therefore, the turbostratic ratio (R′) in the multilayer graphene can be evaluated by the curve fitting analysis of the Raman spectrum in the 2D-band region using three Lorenz curves as the peak positions at ~ 2680, ~ 2700 and ~ 2720 cm−1, as noted by IG’3DA, IG’2D and IG’3DB using the following equation41:The Raman spectrum in the 2D-band region provides an evaluation method to distinguish the different number of layers and stacking structures in multilayer graphene systems) as the following equation:sd and Vsd are the source-drain current and source-drain voltage, respectively. Lch and Wch are also the length and width of the single GNR channel in the FET, respectively. Although the width of the GNR tends to increase with the growth  among them as indicated by the inset in Fig. min) as an OFF state region reaches the detection limit value of current (~ 10 pA) in our source-measure unit, and the semiconducting characteristics are observed. The strong suppression of conductance in the OFF state region is caused by the formation of the transport gaps associated with the disordered structures42. On the other hand, at room temperature, the transfer characteristics measured at the grown multilayer GNR with 6 layers or more slightly depends on the temperature, resulting the degradation of Ion/Ioff ratio. This means that the suppression effect in the grown multilayer GNR significantly reduces with increasing measurement temperature  value at the ON state region. Figure n and R1 are the sheet resistance of monolayer GNR in the n-th layer and pristine monolayer GNR as a growth template. Assuming that the sheet resistance of the grown monolayer GNR in each layer is equal to that of the pristine monolayer GNR as a growth template, the change in the sheet resistance with respect to the number of layers evaluated from the calculation becomes to be a curve as indicated by the solid line in Fig. 44. The improvement is explained by reducing the carrier scattering due to the screening effect of the multi-stacking for the charges of the impurity on the SiO2/Si substrate  stacking structure degrades with increasing the number of layers because the electrical band structure near the Dirac point approaches a parabolic dispersion45. It has been reported that the carrier mobility observed in a few layer graphene with the turbostratic stacking structure is higher than that of the monolayer graphene46 because the electrical band structure keeps the linear dispersion due to a weak inter-layer coupling between the layers40. Therefore, it is considered that the anomalous carrier transport properties in the turbostratic multilayer GNR as a function of the number of layers is caused by the improvement of transfer conductance associated with keeping the linear dispersion and the screening effect.Figure min) value as an OFF state. As shown in Fig. min of the multilayer GNR with 6 layers or more at the OFF state region depends on the measurement temperature. The analysis of temperature dependence in conductance is a convenient way to identify the dominate factors of such carrier scattering as phonons and charged impurities47. Figure min) as a function of T−1. The temperature dependence of ln (σmin) measured from the grown multilayer GNRs with 6 and 8 layers is well fitted by the sum of the two-dimensional variable range hopping (2D-VRH) and thermal activation (TA) conductions48, expressed byVRH (T) and σTA (T) are the temperature dependence of conductance based on the 2D-VRH and TA conduction models, respectively. In the 2D-VRH model, the σVRH (T) has the following formula4951. In the TA conduction model, the σTA (T) is expressed by a and kB are the thermal activation energy and the Boltzmann constant, respectively. The TA conduction means that the carriers flow via a continuous electrical band structure. When the measurement temperature rises at a boundary of around 40–100 K as indicated by the vertical dashed lines in Fig. min in the pristine GNR and the grown multilayer GNR with 5 layers or less is below the detection limit value at room temperature 54. Moreover, we observe that the strength of the suppression effect differs greatly between the pristine monolayer GNR and the grown multilayer GNR, despite the fact that the width is almost the same between them  on the surface of the SiO2/Si device substrate. Since the amount of charged impurities on the device substrate is larger than a typical carrier concentration (~ 1011 cm−2) of monolayer graphene near the Dirac point, the conductance and the carrier mobility significantly degrade on the device substrate due to the carrier scattering by the charged impurities56. The similar situation applies to the GNR system. Since the charged impurities induce the localized potential barriers in the GNR with a narrow width, it is considered that the transport gap is significantly induced by the charged impurities.We focus on the origin of reducing the suppression effect in the grown multilayer GNR with 6 and 8 layers. The suppression effect of the carrier transport is induced by the transport gap that arises from quantum confinement and edge effects57. Therefore, the temperature range of the TA conduction in the multilayer GNR with a thickness of more than 1 nm seems to be strongly dependent on the crystallinity of the synthesized GNR rather than the number of layers. In fact, a similar carrier transport mechanism with the combination of TA and VRH conductions has been observed in the reduced graphene oxide material48, and the appearance temperature of the TA conduction strongly depends on the crystallinity. In the synthesis of multilayer graphene grown on graphene template via our CVD process, since the crystallinity tends to decrease as the number of layer increases43, it seems that the appearance of the TA conduction is observed at higher temperature in the 6-layer GNR-FET than the 8-layer GNR-FET.The appearance temperature of the TA conduction is expected to increase with increasing the number of layer. However, the opposite trend is observed as shown in Fig. 2 substrate when the density of charged impurity is 2 × 1011 cm−2. The potential profile is calculated within the Thomas–Fermi approximation. In the bottom layer attached on the substrate, the surface potential is strongly modulated by the charged impurities, indicating that the electron–hole puddles as the localized states near the charge neutrality point are formed by the potential barrier of the charged impurities58. In this case, the carrier transport property is dominated by the 2D-VRH conduction via the localized states. On the other hand, the modulation of the surface potential due to the charged impurities dramatically reduces in the top layer because the charges on the substrate is screened in the interlayer of the multilayer GNR. Since the electrical band structure in the top layer of the multilayer GNR is not modulated by the charged impurities, the carrier transport is dominated by the TA conduction via a continuous band structure without any localized states as indicated by Fig. 57. As a result, the significant improvement of the conductance in the turbostratic multilayer GNR is observed by the efficient screening effect as shown in Fig. We discuss the effect of the charged impurities on the carrier transport in the turbostratic multilayer GNR. Figure 59. In the simulation, the mobility is asymptotic to the value calculated by a model considering only phonon scattering, as indicated by the dashed line. Figure max (T)) in the pristine monolayer and the grown multilayer GNR-FET as the ON state region. The σmax (T) is normalized by the σmax (300 K) measured at room temperature. For the pristine monolayer GNR-FET, the σmax (T) is independent to the measurement temperature. It has been reported that the conductance observed from the monolayer graphene is independent to the measurement temperature because the density of the charged impurities on the substrate is larger than the thermally excited carriers around the Dirac point due to the linear dispersion35. The similar situation applies to the GNR system. As a result, the conductance of the pristine GNR shows a lower value due to the strong suppression of the carrier transport by the localized potential barriers of the charged impurities as indicated by Fig. max (T) in the grown multilayer GNR-FET significantly improves as the measurement temperature decreases as shown in Fig. max (T) is caused by reducing the phonon scattering. Since the carrier scattering of the charged impurities is greatly reduced by the screening effect of the multi-stacking, the dominate factor of the carrier scattering is phonon scattering. This leads to the anomalous enhancements of the conductivity and carrier mobility as shown in Figs. Figure the σmax 00 K meason/Ioff ratio and carrier mobility in the GNR-FET are improved with increasing the additional graphene layers with the turbostratic stacking. As a result, the transfer characteristic in the turbostratic multilayer GNR exhibits the excellent semiconducting property. When the grown multilayer GNR is 6 layers or more, the suppression of the conductance leading to the carrier scattering dramatically reduces by the efficient screening effect of the multi-stacking. As a result, the transfer characteristic in the multilayer graphene shows the metallic property with the significantly higher conductance without the transport gap.Figure on/Ioff ratio and carrier mobility were observed due to the suppression of the carrier transport and the turbostratic effect. When the number of layers exceeds 6 layers, the conductance at the ON state region extremely enhances by reducing the carrier scattering due to the screening effect for the charges of the impurity on the substrate. We conclude that the semiconducting and metallic characteristics in the narrow width GNR system can be controlled by modulating the strength of the screening effect to the surrounding environment such as charged impurity.We investigated the carrier transport properties of the turbostratic multilayer GNR with various number of layers. At the grown multilayer GNR with a thin film thickness of 5 layers or less, the excellent I22. The synthesized GNRs were dispersed on the SiO2 (300 nm)/Si substrate, and the samples were preheated at 350 °C for 20 min in air under atmospheric pressure to remove the poly  used as a surfactant for the stabilization of unzipped GNRs in the solution. Subsequently, the graphene layers were grown on the GNR template using a sloped-temperature CVD apparatus with ethanol61. The CVD apparatus can regulate the temperatures separately in three zones, resulting in individual control over the decomposition reaction of the carbon feedstock and the growth of graphene layers by activated carbon species. The temperatures used in this study were 900 °C and 720–744 °C for the decomposition and graphene layer growth, respectively.The pristine GNRs used as a growth template were synthesized by unzipping of double-walled CNTs (Tokyo Ohka Kogyo Co. Ltd.)sd) as a function of the gate voltage (Vg) in the FET using a standard formula64, expressed bych, Lch and Cg are the width of the GNR, the channel length and the gate capacitance, respectively. The gate capacitance is given by Cg = εiε0/d. The εi and ε0 are the relative permittivity of SiO2 and the permittivity of a vacuum, and d is the film thickness of the SiO2 (300 nm).The structural analysis of the synthesized GNR was performed by atomic force microscope (AFM: HITACHI AFM5100N) observations with the dynamic force mode and Raman spectroscopy (Horiba HR800UV) with a laser excitation of 532 nm . The GNR-FET devices were fabricated by a conventional photolithographic process. The electrical transport measurements were carried out for the FET devices with just only single GNR channel. The carrier mobility were evaluated from the source-drain current (I2 substrate with a rectangle shape of Lch × Wch was calculated using a semiclassical Monte Carlo approach considering the surface phonon scattering and the charged impurity scattering induced by the substrate66. The gapless energy dispersion at K1 and K2 points was used as the band structure because the inter-layer coupling interaction between GNR sheets to mimic the turbostratic stacking in the multilayer GNR.The electron mobility in a turbostratic multilayer GNR on the SiOSupplementary Information.


Based on the transformation acoustics methodology, the design principle for achieving an arbitrary shape magnifying lens (ASML) is proposed. Contrary to the previous works, the presented ASML is competent of realizing far-field high resolution images and breaking the diffraction limit, regardless of the position of the utilized sources. Therefore, objects locating within the designed ASML can be properly resolved in the far-field region. It is shown that the obtained material through the theoretical investigations becomes an acoustic null medium (ANM), which has recently gained a significant attention. Besides the homogeneity of ANM, which makes it an implementable material, it is also independent of the perturbation in the geometry of the lens, in such a way that the same ANM can be used for different structural topologies. The obtained ANM has been implemented via acoustics unit cells formed by membranes and side branches with open ends and then was utilized to realize an ASML with the aid of effective medium theory. It is shown that the far-field results of an ideal ASML abide well with the results of the implemented sample, validating the proposed design principle. The presented acoustic magnifying lens has a wide spectrum of possible applications ranging from medical imaging, and biomedical sensors to focused ultrasound surgery. To obviate this limit, acoustic super-lenses (ASL) have been proposed that are capable of achieving sub-diffraction-limit by partially magnifying evanescent waves3. However, ASL usually requires meta-atoms with highly dispersive characteristics, which are difficult to be realized in practice and are not suitable for broadband applications. To overcome the narrowband nature of ASL, acoustic hyper-lens (AHL) was propounded as an alternative for converting evanescent components associated with the deep sub-wavelength into propagating waves and; thus, making it possible to break the diffraction limit over a broad frequency bandwidth5. Nevertheless, AHLs are significantly sensitive to the position of the sources, implying that only objects located close to the circumference of the lens can be properly resolved. This has also been verified experimentally by Li et al.6 as only sources locating in the close vicinity of the input surface of the designed AHL can be appropriately resolved on the output interface. This inherent nature of the AHLs will consequently limit the applicability of such lenses for being used in practical scenarios. In addition to the location of the sources, to date, all the propounded works are merely limited to two specific geometries of cylindrical and planar, which consequently restrict the applicability of these lenses for being used in more general situations. Moreover, owing to the fixed functionality of these lenses, their corresponding magnification factors are set to stone after their implementation and cannot be altered afterward10. Therefore, achieving an acoustic magnifying lens (AML) that can overcome the aforementioned problems is still a challenge which needs to be addressed. Besides material engineering, other approaches such as the utilization of core-shell-shaped lenses have been propounded to overcome the ultrasonic diffraction limit. However, despite the material simplicity and feasible implementation, the obtained resolution is limited compared to the aforementioned techniques13.Imaging with the acoustic waves is one of the most widely used methods in applications such as industrial test and clinical diagnosis. However, the resolution of conventional imaging systems is inherently restricted by the diffraction limit, as the information carried by evanescent waves cannot be effectively delivered to the far field23, provides a set of powerful design tools that can be used for the creation of devices with an unprecedented ability to manipulate acoustic waves26. Following this approach, a new route to manipulate acoustic waves for high-resolution imaging and achieving AML has been opened recently27. However, the main challenge of TA-based AMLs is their inhomogeneous constructive materials that cause difficulties for their fabrication in real-life scenarios. Moreover, the same as other conventional approaches, TA-based lenses are shape dependent which leads the applicability of these platforms to be limited to particular scenarios29. Here, on the basis of coordinate transformation method, we will introduce the design principle of an arbitrary shape magnifying lens (ASML) that can efficiently transfer the acoustic sub-wavelength details from objects to far field region and thus breaking the diffraction limit. In particular, we will establish a rigorous theoretical formulation based on our recent work26 which leads to a transformation function that maps a region with a short radius to another domain with a longer radii. As a result of such a transformation, all the points within the smaller region are magnified by the  ratio of two radii, a parameter that has been dubbed as magnification factor throughout the paper. Then, we will obtain a specific kind of material, known as acoustic null medium (ANM)32, that when it surrounds the utilized sources (locating at a subwavelength distance), the total scattered field mimic as if the sources were located in a distance which is not subwavelength. The main advantage of the proposed method is its resultant materials which are homogeneous and independent of the the lens structural topology, indicating that regardless of the lens shape, one can use the same materials for different geometries. Several numerical simulations were performed in order to verify the effectiveness of the proposed ASML. Although previously the ANM has been designed with the aid of effective medium theory (EMT)33 together with exploiting natural materials, assuming tungsten hexafluoride as a background medium restricts its application in real-life scenarios. In particular, in our recent work26, we have assumed the background medium to be filled with a particular material, that in general is not always possible and as such it will restrict the applicability of the realized ANM. Therefore, to fill the long-standing gap between the theory of ASML and its design in the context of acoustic metamaterials, here, we have realized the obtained ANM by using nonresonant acoustic meta-atoms made of periodic cubic blocks with clamped elastic membranes and side branches, which compared to our recent publication, not only is more practical, as one can simply change its geometrical parameters to obtain the desired characteristics, but also is more simple and cost-effective. It is shown that by changing the thickness of the membrane and geometry of the side branches, one can achieve the desired density and bulk modulus of the ANM. Then, it is demonstrated that the realized ANM with the proposed metamaterial is competent of mimicking the behavior of an ideal ANM and thus applicable to be used for implementing an ASML. Finally, as a proof of concept, a practical and realistic ASML is designed and implemented with multi-layered structure made of the realized ANM. It is observed that the image of the utilized sources which were located in close vicinity of each other is properly resolved. The proposed ASML represents a significant step toward realizing super-resolution imaging devices that may greatly benefit the development of new-concept acoustic imaging and detection systems in the future.Transformation acoustics (TA), which its underlying physics comes from the well-established method of coordinate transformationd are placed on a plate and excited by an acoustic plane wave. Since these holes are located in close vicinity of each other, their images cannot be resolved in the far-field region due to the diffraction limit35. The goal is to design a coating layer on the basis of coordinate transformation methodology that is capable of obviating the diffraction problem and giving rise to two distinguished images in the far-field region. To this aim, the inner red region in the physical space, The schematic of potential application of the ASML in a imaging system is illustrated in Fig. mentclass2pt{minimd) between two holes , even though the sources are physically locating at sub-wavelength distance of d. This can also be viewed in the perspective of acoustic illusion, that is, we want to make an illusion for the outside viewer, in such a way that two acoustic sources separated by the distance of d in the physical space  By adjusting the geometrical features of HR which are schematically shown in Fig. Besides the stretched region, to implement the materials of the compressed domain with near zero bulk modulus, an arrays of repeated unit cells with shunted Helmholtz resonators (HR) are exploited which are demonstrated in Fig. In conclusion, we have proposed a method to design an ASML, which is capable of obviating the diffraction limit. The obtained materials through the propounded approach became ANM which are homogeneous and independent of the device geometry. This will consequently enable the proposed ASML to be capable of magnifying arbitrary shape regions without the demand of recalculating or redesigning the necessitating material. Moreover, owing to this feature of ANM, the proposed ASML can have reconfigurable magnification factor, which is of utmost importance in practical scenarios. The ANM is implemented with two acoustic meta-atoms named HR and cubic blocks with clamped elastic membranes and side branches. Then, as a proof of concept, an ASML was realized based on the retrieved parameters from the designed unit cells and with the aid of EMT. It was observed that the realization results were well abide with the theoretical predictions and numerical simulations. The propounded ASML can pave the way towards many functionalities and applications such as medical imaging, as well as focused ultrasound surgery.As it was mentioned, setting entclass1pt{minima40To further gain insight about the ANM, the dispersion relation in such a medium will be investigated. It is known that the dispersion expression for an anisotropic mass density can be expressed as