Despite the recent flurry of interest in various aspects of ancient urbanism, we still know little about how much traffic flowed in and out of ancient cities, in part because of problems with using commodities as proxies for trade. This article investigates another approach, which is to estimate these flows from the built environment, concentrating on transport infrastructure such as city gates. To do this, I begin by discussing a new model for how we would expect this kind of infrastructure to expand with population, before investigating the relationship between the populations of sites and the total numbers and widths of city gates, focusing on the Greek and Roman world. The results suggest that there is indeed a systematic relationship between the estimated populations of cities and transport infrastructure, which is entirely consistent with broader theoretical and empirical expectations. This gives us a new way of exploring the connectivity and integration of ancient cities, contributing to a growing body of general theory about how settlements operate across space and time. Although there have been considerable advances in our ability to investigate various aspects of human life through the collection of large amounts of data, it can still be a significant challenge to estimate socio-economic flows because of the wide range of channels through which these exchanges can occur . This isDespite the recent flurry of interest in various aspects of ancient urbanism, we still know little about how much traffic flowed in and out of ancient cities, in part because of issues with assessing the scale of daily commuting and seasonal migration, as well as problems with using commodities as proxies for trade. As Killgrove and Montgomery have pointed out, although it is clear from both literary and epigraphical evidence that the movement of individuals was a regular occurrence in ancient life, it is more difficult to quantify its exact scale . An obviAnother approach has been to use small finds, such as ceramics, as a reflection of the overall scale of trade , 5,6. ItThis means, in general, that the extent to which individual communities were dependent on daily commuting or seasonal migration or on commodities obtained from beyond their immediate hinterlands is far from clear. This gap in our understanding is obviously important to address, since a clearer understanding of these issues should have important implications not only for our view of the economic roles of ancient cities and the extent to which they were able to specialize or diversify at both local and regional levels, but also for wider debate about the nature of daily commuting and seasonal migration in pre-industrial settings and the extent to which the expansion or contraction of settlements in non-modern contexts mainly occurred through preferential attachment or random processes .Here, I explore another approach, which is to use the built environment itself to estimate traffic flows. This is only possible because of recent research, which is based on a conception of settlements as social networks embedded in the built environment, allowing us to design a series of models for the relationship between the estimated populations of sites and their infrastructure and socio-economic conditions (and how these attributes change as the number of people involved increases) , 15,16. An important exception is Altaweel and Palmisano’s discussion of sites in the Khabur triangle in northern Mesopotamia in the Late Chalcolithic and Bronze Age (ca. 4200–1200 BCE), which concentrated on the mound areas and mean hollow way widths (i.e. the remains of roadways) of a selection of sites . While tThis therefore raises the question of whether we can extend Altaweel and Palmisano’s work by using other forms of transportation infrastructure as a proxy for traffic flows and, in particular, whether we can identify any relationship between the sizes of ancient cities and the amount of traffic that passed through them. Greek and Roman cities are an ideal candidate for this, since they are not only uncommonly well documented, but were also often surrounded by physical walls, meaning that all traffic would have had to pass through city gates, which can be measured directly from maps and plans.This article investigates whether the built environment can be used to estimate traffic flows. It begins by reviewing current understanding about the sizes of cities and traffic flows and discussing a new model for how we would expect transport infrastructure, such as city gates, to expand with population. It then tests the hypothesis that the former will increase more slowly than the latter by exploring the relationship between the estimated populations of sites and the total numbers and widths of city gates, before examining their relationship with other network measures, such as degree centrality, betweenness centrality, and Eigenvector centrality, using ORBIS: The Stanford Geospatial Network Model of the Roman World (henceforth the ORBIS model) . It showA major area of interest in urban studies is how flows in networks scale with size . AlthougThere is also evidence for various kinds of scaling in modern transportation systems, based on the number of locations that can be accessed within a certain travel time budget . CommutiThe most well-known way of representing these ideas is the gravity model . AlthougNn and Nm are the sizes of the interacting entities, α and β are measures of the propensity of individuals to move or exchange commodities, d is a measure of the distance between them , and γ is a measure of friction , 4118, 4I then used standard techniques to identify the most important nodes in the network. The measures we are concerned with are degree centrality (the number of edges to a node), betweenness centrality , and Eigenvector centrality (the likelihood of a node) being connected to other well connected nodes in the network)) . The lasIn total, there is evidence for the numbers and widths of city gates for some 32 sites . These sites are drawn from throughout the Roman Empire, although they are slightly more concentrated in Italy and the north-west than the rest of the ancient world . This isI then assessed the relationship between the estimated populations of sites and the total numbers and average and total widths of city gates using ordinary least squares regression . These rSeveral aspects of this relationship stand out. In the first place, it is worth noting that it is extremely sublinear, meaning that, although the total numbers and widths of city gates does increase with population, it does so only very slowly. This suggests that city gates were used more intensively in larger sites, in line with other studies of infrastructure, so existing city gates would have accommodated some of the augmented traffic . As noteIt is also interesting to observe that both the total numbers and average widths of city gates increase with population at about the same rate, approximately 1/6, suggesting that half of the gains went into the provision of more entrances and exits into cities and half into the construction of wider city gates that could accommodate more traffic. This is in direct contrast to the common assumption that the number of city gates depended on the number of approach roads and not on the numbers of inhabitants or the size of the city . AlthougThe results also indicate that both the average widths of city gates and the average widths of streets within cities increased at around the same rate, with an exponent of 1/6 in both cases, making them consistent with earlier findings . This is2 of the overall relationship is high for historical and archaeological data, it nonetheless reflects a relatively high degree of variation. As we have argued elsewhere, the residuals result from a wide range of factors, including both error in how the numbers of residents have been estimated (which itself includes errors in how the sizes and densities of sites have been measured or estimated) and in how the numbers and widths of city gates have been measured [Although the Rmeasured . HoweverThe pre-factor of the overall relationship is 1.32 m. Allowing for some error, this conforms well with ruts from Pompeii, which indicate that the largest carts to use the streets with any regularity were between 1.32 and 1.47 metres, potentially reflecting a situation in which only one vehicle was able to pass through at a time , 32. ThiAlthough we do not have evidence for all cities, there are two cases, Rome and Pompeii, that allow us to investigate the distribution of traffic across city gates within the settlement. These both seem to be close to a power-law, potentially reflecting the fractal nature of traffic flows in modern cities .It is possible to estimate the network values of 341 sites, which are distributed throughout the ancient world . The results are somewhat disappointing, in that there is no evidence for a strong relationship between the estimated populations of sites or any of the centrality measures referred to above . This isAt the most fundamental level, city gates can be taken as a measure of interaction rates, since we would expect them to reflect the volume of people, goods, and information flowing into and out of cities. As a result, when taken together, the results of the present study could be interpreted as a reflection of the extent to which settlements and their hinterlands interacted, the scale of surpluses that could be exchanged between them , and the extent to which they can be regarded as being energetically interdependent. Although this view of ancient cities is very far from the classic accounts of ancient cities, which saw them as cellular, self-sufficient communities, it is more in line with recent ideas , 45,46. This has important implications for our view of the economic profiles of sites, including the extent of their specialization and diversification, since the data suggest that sites could have experienced a sufficiently large increase in traffic with increases in size to allow them to establish different local and regional roles. This raises the question of whether we can use the residuals to give us an index of how much traffic came into or out of each city, above and beyond what we would expect for a city of its size, and therefore as a reflection of its overall specialization and diversification, especially in terms of manufacturing, services, and trade. This is something that needs to be investigated in future work.The results of the present study might also have a bearing on our overall view of commuting, migration, and social networks. The traditional assumption has been that, since we would expect most people to have lived where they worked, there would have been no commuter traffic. Meanwhile, although it is normally assumed that urban growth relied on large amounts of migration (because of the likely mismatch between birth and death rates in cities), reasonably little is known about this before the modern era. The results discussed above, however, suggest that interaction rates not only increased with size, but also did so at a consistent rate, supporting the idea that flows of people were more common than usually thought. If this is true, it has implications for our understanding of the social networks of ancient settlements, since it implies that they had relatively heterogeneous populations.Finally, the results might shed some light on the mechanisms through which ancient cities expanded or contracted, since they suggest a systematic relationship between the estimated populations of sites and their potential traffic flows, which is more in keeping with preferential attachment than random growth. This is consistent with the rank-size distribution of cities in the Roman world, which is approximately log-normal , suggesting that these cities functioned as single entity . One of This article has asked whether we can use the built environment to estimate traffic flows, concentrating on transport infrastructure such as city gates. I have presented results that suggest that there is a systematic relationship between the estimated populations of sites and both the total numbers and average widths of city gates, which might be taken as a proxy for traffic flows. This relationship is extremely sublinear, indicating that city gates were used more intensively as cities increased in size, in line with wider theoretical and empirical work. However, since we would expect these city gates to be a reflection of the average rate of traffic flow, we would also expect the total volume of traffic to have been slightly superlinear, reflecting the social and economic gains that come with agglomeration. Another striking aspect of the results is that the average widths of city gates and the average widths of streets increased at the same rate, suggesting that the interaction rates between individuals in cities, and between cities and their surroundings, scale similarly, allowing us to integrate existing theories about the internal and external dynamics of cities for the first time. In particular, the article extends recent theoretical and empirical work on ancient and modern cities by showing that outward-facing transport infrastructure functions in a similar way to the internal street networks documented in earlier papers. This raises the questions of how unique ancient cities were in this respect and whether we can take these relationships as a universal feature of both ancient and modern settlements. This article has also highlighted the importance of investigating the nature and scale of the connections between ancient cites, as well as the need for a much more comprehensive model for the transport network of the ancient world and more detailed evidence for the movement of individuals and commodities through human remains and small finds . In the meantime, this article provides a simple approach that can be used to guide future work. It can also be applied to any other contexts where cities were surrounded by walls or other defences, e.g. medieval Europe, imperial China, etc. . This ofS1 Appendix(XLSX)Click here for additional data file.


Malus sylvestris, a crop wild relative of Malus domestica, is a major contributor to the cultivated apple genome and represents a potential source of interesting alleles or genes, particularly pest resistance traits. An original approach was used to explore the trophic interaction between M. sylvestris populations and its pest, the rosy apple aphid (Dysaphis plantaginea). Using 13 microsatellite markers, population genetic structure and level of crop-to-wild introgressions were inferred between M. sylvestris seedlings from three sites in Europe , and M. domestica seedlings. Genetically characterized plants were also used to analyze aphid feeding behavior and fitness parameters. First, aphids submitted to two genetically close M. sylvestris populations (the Danish and French) exhibited similar behavioral parameters, suggesting similar patterns of resistance in these host plants. Second, the Romanian M. sylvestris population was most closely genetically related to M. domestica. Although the two plant genetic backgrounds were significantly differentiated, they showed comparable levels of sensitivity to D. plantaginea infestation. Third, aphid fitness parameters were not significantly impacted by the host plant’s genetic background. Finally, crop-to-wild introgression seemed to significantly drive resistance to D. plantaginea independent of host plant population genetic structure, with hybrids being less suitable hosts.The European crabapple  In this case, new breeding strategies making use of wild untapped genetic diversity could become a promising opportunity to provide farmers with crops less dependent on chemical inputs without heavy drawbacks to productivity2. Crop breeding programs often rely on wild species that are either phylogenetically close to a crop and/or that have played a primary role in the crop domestication history. Such plants have been named “crop wild relatives” (or CWR). Although the use of CWR in breeding programs has the potential to fulfill numerous agronomic needs (e.g. yield increase or abiotic stress adaptation), the major focus of CWR research concerns their ability to enhance crop resistance against pathogens and pests3.Intense farming practices can lead to harmful impacts on the environment and human health. It is therefore urgent to promote eco-friendly agricultural management while feeding a growing world populationMalus domestica Borkh appears to be an ideal model system for reducing the environmental impacts of food production. Indeed, M. domestica is one of the most important fruit tree crops in the world (http://faostat.fao.org/). In Europe, the apple tree is severely attacked by several pests and pathogens, and therefore apple production relies heavily on the use of pesticides. To reduce reliance on pesticide applications, apple breeding programs should test potential sources of resistance alleles such as the three potential local wild apple species in Eurasia. Population genetics analyses revealed that the cultivated M. domestica originated from the wild apple Malus sieversii Ledeb. in the Tian Shan mountains located in Central Asia5. From there, the cultivated apple continued its journey along the Silk Routes where it hybridized with local wild apple tree species: first to a little extent with Malus orientalis Uglitz. in the Caucasus, and later on (~ 1500 YA), massively in Europe with Malus sylvestris (L.) Mill.4. Malus sieversii is therefore considered the progenitor of the cultivated apple while M. sylvestris is considered the second main contributor of apple genetic diversity through recent wild-to-crop introgressions5. Through introgression and phylogenetic closeness, M. sieversii, M. orientalis, and M. sylvestris are all considered CWR of cultivated apple. The need to assess their value as future sources for future breeding programs is urgent because these species are currently threatened by local crop-to-wild gene flow9.In the context of breeding programs relying on CWR genetic traits, the cultivated apple tree M. domestica wild relatives has been carried out mostly against pathogens, whereas studies describing resistance to pests are less frequent10. Among these, the M. floribunda Siebold clone 821 proved to be a major source of resistance genes to apple scab Venturia inaequalis (Cooke) Winter11, fire blight Erwinia amylovora Burrill12 and the rosy apple aphid (RAA), Dysaphis plantaginea Passerini13. However, only a handful of studies have investigated resistance to pests and pathogens of CWR involved in the domestication of the apple. One study14 compared the levels of resistance against fire blight among 51 different genotypes of M. sieversii collected in Kazakhstan and Kyrgyzstan. Similarly the resistance of 194 M. sieversii accessions belonging to four distinct genetic groups15 was evaluated concurrently with nine different M. domestica cultivars16. Various fire blight resistance levels were observed among M. sieversii genetic clusters with two of them exhibiting the highest resistance levels among tested genotypes. Furthermore, a distinct resistance mechanism was revealed when several wild accessions were compared with cultivated apple cultivars using shoot inoculation in orchards. Specifically, fire blight infection rates were lower for several M. sieversii accessions but when successful, infection led to greater damage in these trees. Concerning resistance against insect pests, another study17 quantified the resistance of 19 M. domestica cultivars and two of its CWR, M. sylvestris and M. kirghisorum, to the florivorous apple blossom weevil, Anthonomus pomorum L.. The authors compared weevil resistance levels between the cultivated apple to the wild species. The species M. sylvestris and M. kirghisorum Al. Fed. & Fed. appeared to be more sensitive to A. pomorum but also supported a more abundant community of the weevil’s natural enemies.The search for resistance on M. sylvestris appears to be largely underexploited. For European apple production, M. sylvestris breeding presents several advantages including a shared local environment with M. domestica. Previous population genetic analyses of M sylvestris populations using microsatellite markers revealed five genetic clusters spread throughout Scandinavia, Western Europe (mostly in France), Eastern Europe, Central Europe, and Italy7. These five populations may possess adaptive alleles associated with specific environmental conditions or local parasites. However, responses to pathogens and pests among these wild apple genetic groups have yet to be explored.Among the pool of CRW apple species with putative benefits for cultivated apple breeding programs, D. plantaginea (RAA) is the major aphid pest of the cultivated apple in Europe, Maghreb, and North America. This aphid species feeds on sap drawn from the phloem and develops at the apex of branches and/or on the most recently developed leaves. In addition to sap extraction, RAA saliva secretion provokes leaf-rolling and impairs shoot growth, greatly reducing yield18. Plant defenses against aphids include various strategies disrupting aphid preference, particularly through their host-plant colonization process19 that can be characterized by the potential success throughout six behavioral phases: (1) long- and short-range of host-plant perception, (2) plant contact and assessment of surface cues, (3) epidermal probing, (4) stylet pathway activity, (5) phloem penetration and salivation, and (6) phloem acceptance and ingestion. This colonization is finalized by entering a reproductive phase which can be characterized by fecundity and adult survival traits as aphid performance. In this study, a host plant was considered resistant to RAA from the moment it negatively impacted the preference and/or performance of the pest.The rosy apple aphid M. sylvestris European populations7 and 14 cultivated apple plants derived from breeding crosses of M. domestica cultivars. (2) The 56 plants sampled were genetically characterized using 13 microsatellite markers4. Using population genetics inference, their genetic statuses were determined . The degree of crop-to-wild introgression was also assessed among these plants. These genetically characterized plants were then used for behavioral and physiological assays that tested for putative RAA resistance in a CWR of M. domestica. (3) For each plant population identified, aphid preference was tested based on feeding behavior measured with the electropenetrography (EPG) technique. (4) As a proxy for measuring fitness, adult fecundity, survival, and adult weight measures were used to determine aphid performance on each of the identified plant populations20.The current work explores the RAA behavior/physiology on a crop wild relative of the cultivated apple, taking into account intra-specific CWR population structure and level of crop-to-wild gene flow. This manuscript describes four research objectives: (1) An original sample collection was built from 42 wild apple plants grown from field-collected seeds derived from three of the five known M. domestica, referred to as “Dom”, N = 14, Table M. sylvestris plants were grown from field-collected seeds. These wild apple seeds originated from three out of the five known European wild apple populations . Each population was represented by a single sampling site, and within each site, each seed was sampled on a single mother tree, so that each seedling has a different parental origin. Though M. domestica is usually grafted, new plants were grown from seed to eliminate the rootstock effect.A total of 56 apple plants were grown from seeds and sampled for this study. Cultivated apple plants resulting from crosses between various cultivated apple varieties were used (M. sylvestris European populations or crop-to-wild/wild-to-wild hybrid).After field sampling, seeds were stored at -20 °C before vernalization for the experiment. Seeds were then vernalized for three months at 4 °C in the dark, then grown in controlled conditions for two months before being individually transferred to 3 L pots containing commercial sterilized potting soil. Potted plants were grown in a growth chamber for four weeks under the following conditions: 20 ± 1 °C, 75 ± 5% Relative Humidity (RH), and a 16:8 light:dark (L:D) photoperiod. The 56 plants were then genotyped using 13 previously published microsatellite markers (see below) to confirm their genetic status  was used and provided by INRAE which were sampled as a population in spring 2018 from an apple tree at the Agrocampus Ouest orchard  . This aphid population was mass reared without differentiating individual aphid clones on M. domestica cv. “Jonagold” plants obtained by in vitro multiplication21. Pots containing three plants (90 × 90 × 70 mm) were placed in a Plexiglas cube (50 cm). Mass rearing and experiments were performed in growth chambers under 20 ± 1 °C, 60 ± 5% RH, and a 16:8 L:D cycle.A single colony of M. domestica cv. “Jonagold” plants inside Plexiglas aerated boxes (36 × 24 × 14 cm) for ten days then used as the young adult RAA for the behavioral/performance experiments.Synchronized first instar nymphs were obtained by placing parthenogenetic adult females on plantlets for 24 h before removing them. They were then reared on 4. PCR were performed in a final reaction volume of 15 ml  (See4 for more details). The final volume was achieved with distilled water. A touch-down PCR program  was used. Genotyping was performed on the GENTYANE platform (INRAE Clermont-Ferrand) using an ABI PRISM X3730XL, with 2 ml of GS500LIZ size standard (Applied Biosystems). Alleles were scored with GENEMAPPER 4.0 software (Applied Biosystems). Only multilocus genotypes with < 10% missing data were retained.Genomic DNA was extracted with the NucleoSpin plant DNA extraction kit II  according to the manufacturer’s instructions. Microsatellites were amplified by multiplex PCR, with the Multiplex PCR Kit . We used 13 microsatellite markers, Ch01f02, Ch01f03, Ch01h01, Ch01h10, Ch02c06, Ch02c09, Ch02c11, Ch02d08, Ch03d07, Ch04c07, Ch05f06, GD12, and Hi02c07 in four multiplexes 22. STRUCTURE makes use of Markov Chain Monte Carlo (MCMC) simulations to infer the proportion of ancestry of genotypes from K distinct clusters. The underlying algorithm attempts to minimize deviations from Hardy–Weinberg and linkage disequilibria. STRUCTURE was run from K = 1 to K = 8, ten independent runs were carried out for each K and 500,000 MCMC iterations were used after a burn-in of 50,000 steps. CLUMPAK 23 was used to look for distinct modes among the 10 replicated runs of each K. STRUCTURE analyses were run for the full dataset , and included as well 40 M. domestica genotypes as a reference for the cultivated apple gene pool6. We determined the strongest level of genetic structure using ΔK24, as implemented in the online post processing software Structure Harvester25. However, the K identified by this criterion often does not correspond to the finest biologically relevant population structure27. A lack of consideration of intraspecies genetic structure in STRUCTURE analyses can bias the interpretation of introgression rates. We therefore visualized the bar plots and chose the K value for which all clusters had well assigned individuals while no further well-delimited and biogeographically relevant clusters could be identified for higher K values.The genetic status of each seedling was assessed using the individual-based Bayesian clustering method implemented in STRUCTURE 2.3.3K chosen, wild plants assigned to the cultivated gene pool with a membership coefficient > 0.1 were classified as crop-to-wild hybrids . Once crop-wild hybrids removed, plants assigned to a given wild gene pool with a cumulated membership coefficient > 0.9 were defined as “pure wild” individuals. Plants assigned to the wild gene pool with a cumulated membership coefficient < 0.9 to a given wild apple gene pool were defined as wild-wild-hybrids The pure, crop-to-wild and wild-wild hybrids were included as factors in the statistical analyses. Pure seedlings were then assigned to a population . Pure populations from the same geographic origin  which showed (1) weak genetic differentiation with other wild populations (2) low number of individuals were merged. The “population” was then used as a factor for statistical analyses on physiological and behavioral assays. Population genetics statistics were estimated with Genodive28 for each “pure” wild apple population including expected and observed heterozygosities, Weir and Cockerham F-statistics, Jost’s D, and deviations from Hardy–Weinberg equilibrium.Once the best 29. Individual aphids were connected to the Giga-8 DC-EPG amplifier, each being placed on the abaxial side of a new growing leaf of an individual plant. The recordings were performed continuously for 8 h during the photophase inside a Faraday cage. Acquisition and analysis of the EPG waveforms were carried out using the PROBE 3.5 software . Parameters from the recorded waveforms were calculated with the EPG-Calc 6.1.7 software30. They were based on different EPG waveforms31 corresponding to: (Pr) stylet activity within plant tissues; (C) stylet pathways in plant tissues except phloem and xylem; (E1) salivation in phloem elements; (E2) passive phloem sap ingestion; (G) active xylem sap ingestion; and (F) derailed stylet mechanics. A total of eight plants per M. sylvestris population  or M. domestica (Dom) genetic group were used for the EPG measurements. EPG records were obtained from 25 aphids for M. domestica, and from 27 aphids for each M. sylvestris population.The feeding behavior of the RAA was investigated using the electrical penetration graph (EPG) methodM. domestica, and 28 to 29 adults for each of the three M. sylvestris populations.Two to three clip-cages were installed on 12 plants per genetic group identified in this study. Each cage contained an individual, synchronized aphid and was enclosed on a newly grown leaf. For each synchronized adult, observations were assessed every 24 h for 10 days. Survival  and daily fecundity  were collected for 25 adults for To measure aphid weight, newly larviposited nymphs were enclosed for nine days in clip-cages on newly grown leaves similar to the above. For each plant genetic background, up to 20 aphids (i.e. young adults that had not larviposited yet) were then collected and stored in a freezer at – 80 °C. Each individual aphid was weighed using an electronic precision balance .https://www.r-project.org/). Generalized linear models (GLM) with a likelihood ratio and Chi-square test were used to assess whether there was an effect of the host plant on aphid feeding behavior and performance. The apple tree genotype was included as the main factor. Data on daily aphid fecundity and some EPG parameters describing the number of occurrences of a particular phase (e.g. “n_E2”) were not normally distributed (count data), accordingly a GLM was carried out using respectively a quasi-Poisson and a Poisson distribution; a quasi-likelihood function was used to correct for over-dispersion, and Log was specified as the link function in the model. EPG data on feeding phase durations (e.g. duration of phloem sap ingestion “s_E2”) and aphid weight were not normally distributed, so a GLM using a Gamma (link = “inverse”) distribution was carried out. Analysis of the time before the first probe (“t.1Pr”) and before the first phloem sap ingestion (“t.1E2”) and adult survival has been carried out using the Cox proportional hazards (CPH) regression model, which is adapted to treat time-dependent parameters. Absence of an EPG reading were treated as missing values. The assumption of validity of proportional hazards was validated using the function “coxph” . To assess whether the crop-to-wild hybrid status had a plant-mediated effect on RAA feeding behavior and performance, the same statistical tests were carried out with the hybrid statuts (i.e. “wild pure” or “wild-crop hybrid”) as the fixed factor while restricting the data set to the M. sylvestris populations only.All statistical analyses were performed using the R software version 3.6.2 . A Monte-Carlo permutation test (999 replicates) was conducted to test for the significance of the differences of median of EPG phases duration, daily fecundity and weight between aphids submitted to these two host groups. Analysis of the time before the first probe (“t.1Pr”) and before the first phloem sap ingestion (“t.1E2”) and adult survival has been carried out using the CPH regression model. The function “randtest” (package R: “ade4”: https://cran.r-project.org/web/packages/ade4/ade4.pdf) was run to access the significance of the observed differences.Finally, because of their close genetic relatedness, the Danish and French wild apples plants  were grouped together, as well as the https://cran.r-project.org/web/packages/emmeans/emmeans.pdf).The fit of all GLM was controlled by a visual evaluation of residuals and QQ plots. Concerning QQ plots, the distribution of the series were considered to follow the chosen theoretical law if the points of the graph were roughly aligned on a straight line. Any other structuring of the points (curvature(s), many distant points, etc.) indicated the opposite. GLM post-hoc comparisons were carried out by pairwise comparisons using estimated marginal means  the Romanian seedlings, divided into three genetic groups , (3) the French seedlings divided into two genetic groups , and (4) the Danish seedlings formed a single distinct genetic group (green). Note that the French and Danish samples only split from K = 11. This weak genetic structure was further validated by the relatively low FST and Jost’s D values among those two groups  M. a priori as M. sylvestris, four genotypes  showed signs of introgression from M. domestica  = 0.09 and FST(REF_NA-SYL_RO) = 0.11, respectively, P < 0.001), and the Danish and French wild apple populations were still the most genetically closely related, followed by the Romanian and the French wild apple populations , the total duration of stylet activity in the plants  lasted on average about 6 h (out of 8 recorded hours) and was not significantly different among aphids, whatever the genetic background of their host plant . This global activity was composed of an average of 13 probing events , again without any significant difference between aphids on the host plants with different genetic background . This is despite the fact that the mean number of probes tended to be smaller on M. domestica host plants. Similarly, there was no significant difference between the time to first probe  . The pathway phase  comprised on average of 60% of the total activity. There was no significant difference for its total duration   or for the number of occurrence  .Feeding phases and associated analytical results are described in Table M. sylvestris plants compared with aphids submitted to M. domestica and Romanian M. sylvestris plants  . Aphids fed on Danish and French M. sylvestris plants salivated three times less compared to aphids fed on M. domestica and Romanian M. sylvestris plants. Aphids submitted to French and Danish M. sylvestris plants displayed a smaller number of salivations within phloem compared to aphids on M. domestica whereas aphids submitted to French M. sylvestris plants had a significantly shorter number of salivations within phloem compared to those on Romanian M. sylvestris plants  . The mean duration of phloem ingestion  was significantly shorter for aphids on the Danish M. sylvestris compared to aphids on M. domestica and the Romanian M. sylvestris plants . The mean proportion of the time dedicated to phloem ingestion (E2) was variable depending on the host plant genetic background: from 9% (Syl_Dk) to 24% (Syl_Ro/Dom) within the general probing activity. The number of phloem ingestion  was significantly smaller for aphids submitted to the French M. sylvestris compared to those submitted to M. domestica and the Romanian M. sylvestris plants .Concerning the phloemian phase (parameters 6–10), most parameters revealed differences among aphids fed on trees belonging to the three wild apple populations and the cultivated gene pool. The mean duration of salivation within phloem was significantly shorter for aphids submitted to French and Danish M. domestica or Romanian M. sylvestris plants (around 3 h) than on Danish and French M. sylvestris plants (around 4 h), though no significant difference was observed . Considering xylem ingestion  aphids submitted to Danish M. sylvestris plants did not ingest raw sap, whereas a few aphids ingested xylem on the French and Romanian M. sylvestris plants. Altogether, not enough aphids displayed this behavior to conduct statistical analysis. Almost half of the aphids presented stylet derailment  for an average total duration of roughly 1.5 h, that was not statistically different between the different host plant genetic backgrounds .Finally, the duration of the time needed by an aphid to reach the phloem  tended to be shorter on M. sylvestris populations  revealed that the total duration of phloem salivation  and the total duration of phloem sap ingestion  were significantly longer for the Dom/Syl_Ro pair. Regardless M. sylvestris populations, the duration of phloem sap ingestion phase was significantly affected by the host plant hybrid status and was shorter for hybrids compared to pure M. sylvestris .Considering the genetic proximity of the Danish and French ns Table , as well2 = 1.49, Df = 3, P > 0.05) between aphids raised on the four plant genetic backgrounds. Similarly, there was no significant difference for survival . Aphid weight was significantly impacted by the plant genetic background: the weight of aphids raised on M. domestica was significantly greater  than that of aphids raised on the Danish M. sylvestris plants. Again taking into consideration the genetic proximity between the Danish and French M. sylvestris as well as the Romanian M. sylvestris and M. domestica, the two-by-two pairing of datasets (Syl_Dk/Syl_Fr vs Dom/Syl_Ro) revealed no significant difference for all fitness parameters. Regardless M. sylvestris population, aphid weight was significantly affected by the host-plant hybrid status: aphids raised on hybrids displayed smaller weights compared to those on pure M. sylvestris , whereas neither fecundity nor survival were impacted.The impact of host plant genetic background on RAA fitness and associated statistical analyses are presented in Table M. sylvestris wild apple populations. Accordingly, behavioral assays of aphids submitted to plants from these two populations showed similar patterns suggesting antixenosis resistance32. Likewise, the Romanian M. sylvestris host population was strongly differentiated from M. domestica but was also the most closely genetically related to the wild apple population. Consistent with this observation, the Romanian wild apple population and M. domestica showed comparable levels of sensitivity to RAA. Crop-to-wild introgression appeared to drive resistance to RAA independent of population genetic structure.This is the first study reporting differences in phytophagous pest preference that are congruent with the genetic relationship between a wild relative and a cultivated plant with which it has introgressed. Population genetic analyses also revealed weak genetic differentiation between the Danish and French 33, with five main populations in Europe of which, an Eastern, a French and a Scandinavian. We showed that the French and Danish populations were the genetically closest and sharing the highest number of wild-wild hybrids. Accordingly, we can note that aphids showed similar patterns of feeding behavior and performance when submitted to the two most closely related wild apple populations (the Danish and French populations). When aphids were subjected to the Romanian wild apple population and the cultivated apple, similar patterns of aphid behavior and performance were recorded and were associated with higher preference for the Romanian host-plant than aphids submitted to the Danish and French populations. Aphid behavior was actually congruent with the level of genetic differentiation between the Romanian wild apple and the cultivated apple. Once the recent crop-to-wild hybrids were removed, the Romanian population appeared to be the closest wild apple relative to cultivated M. domestica. Genetic proximity of populations is known to drive patterns of resistance against pathogens in the wild apple M. sieversii16. Previous studies revealed variable resistance against pests and pathogens among M. domestica CWR16. In this study, the genetic differentiation between the two paired groups (Syl_Dk/Syl_Fr vs Dom/Syl_Ro) might be associated with a phenotypic differentiation associated with the differences observed in terms of RAA feeding behavior. The genetic proximity of the French/Danish and Romanian/cultivated apple may reflect common evolutionary history, however further investigations are required concerning the evolutionary history of the cultivated apple in Europe. In particular, the relative contributions of each wild apple population, especially the Romanian, to the cultivated apple gene pool remains unknown. Addressing this issue would require much larger sampling among European apple seeds.This study revealed a putative link between aphid preference and the genetic structure among wild and cultivated apple populations. The population structure inferred here stingingly matched the one previously observed for the European wild apple19. A delayed aphid stylet activity is considered to be due to epidermic factors, as the second phase of host selection involves the assessment of plant surface cues by the aphid. Features such as a thick cuticle or the presence of trichomes are physical parameters that may play a role in aphid resistance34. Stylet derailment was displayed on every plant genetic background in the same range of mean duration and the pathway phase was not influenced by the plant genetic background. This means that putative mild physicochemical resistance is present of mesophyll tissues in both wild and cultivated apple. In contrast with the above, significant differences were observed between the four plant genetic backgrounds in phloem-related behavior. The phloemian activity was significantly reduced in terms of the duration of both salivation and ingestion for aphids submitted to the Danish and French M. sylvestris populations. Since the average time to reach the phloem was not significantly different between the four plant genetic backgrounds, these differences did not appear to be linked to physical characteristics but due to the phloem chemical composition. Comparison of ascorbic acid glycoside (AAG) content in M. domestica, M. sylvestris and M. sieversii apple fruits revealed that accessions of M. sylvestris were distinguished by higher concentrations of AAG35. A difference in terms of phenolic compounds among M. sylvestris populations could be a possible factor explaining the contrasted phloemian activities observed. In fact, phenolic profile of various M. domestica cultivars apple fruits can be linked to field RAA resistance36. Further studies involving choice assays towards the four genetic backgrounds should provide a better understanding of RAA preference, and especially of long and short range host-plant perception. Despite the differences recorded in the feeding behavior, no differences were observed in two of the three RAA fitness parameters  regardless of host-plant genetic background. As high proportions of aphids could initiate reproduction before accessing the phloem37, the results concerning fecundity may be consistent with the absence of significant differences in pathway phase parameters. Aphid biomass assays revealed that only aphids submitted to the cultivated apple tree had greater weights than those submitted to the Danish M. sylvestris. This is consistent with the fact that the shortest sap ingestion was observed for aphids submitted to the Danish M. sylvestris, whereas aphids reared on M. domestica exhibited the longest sap ingestion. Sap ingestion is known to be positively correlated with growth38, thus the contrasted preference of RAA was not reflected in RAA performance, except for adult weight.Not considering genetic proximity among populations, but only population structure, behavioral analyses with EPG demonstrated a generalized activity for the rosy apple aphid, which did not depend on the genetic background of the host plant. Our results showed that whatever the plant genetic background, the time to first probe was not delayed, meaning that the possible influence of epidermal barriers and/or putative plant volatile organic compound (VOC) repulsive effects could be excluded; VOC on leaf surface could indeed impact aphids behaviorM. sylvestris on RAA preference and performance. Previous studies already detected substantial crop-to-wild gene flow in apple trees in Europe8. Here, we confirmed the occurrence of ongoing crop-to-wild gene flow for the European wild apple. We detected 11% of crop-to-wild hybrids in our dataset, which is half less than previous estimates (23%)7. Several reasons can explain this discrepancy, including the narrow spatial geographic area investigated here (four locations versus 62 locations previously) and the lower number of samples used (here 42 vs 1889 M. sylvestris trees previously). Note however that the aim of our study was not to investigate the large-scale crop-wild gene flow in M. sylvestris but assessing the genetic status of the seedlings used for aphid physiological and behavioral assays to control this effect in the statistical models. Yet, in comparison to previous studies, the detection of crop-wild hybrids in seeds collected in 2016–2017 are proof that recent ongoing crop-to-wild gene flows are still at work in apples, especially in the French populations. Indeed, previous studies rather investigated historical crop-to-wild gene flow as they did not sample seeds but much older mother trees. It is also interesting to note that we detected a clear effect of the hybrid status on RAA preference, in terms of phloem sap ingestion, and on RAA performance, in terms of aphid weight. Our bioassays on aphids revealed that pure M. sylvestris were more suitable hosts to RAA than crop-to-wild hybrids. A previous study revealed that crop-to-wild hybrids showed higher plant growth and pollination rates compared to pure wild apples8. Here our results would suggest that higher fitness of crop-to-wild hybrids is not only expressed for early developmental traits but is also associated with higher resistance abilities to RAA attacks. However, this question would require further investigation.For the first time, our study also shed light on the impact of domestic introgression in Malus sylvestris could represent a putative genetic source of resistance for Malus domestica breeding programs. The M. domestica genetic group studied here could be considered as susceptible when compared with the resistance/susceptibility of M. domestica to RAA demonstrated by a previous study also using EPG39. Regarding general probing activity and phloemian phases, the mean values of electropenetrography parameters obtained here for aphids submitted to the M. domestica genetic group appeared to be close to values obtained on a susceptible cultivar (M. domestica cv. Golden Delicious) in comparison to a resistant one (M. domestica cv. Florina)39. The latter has been identified as strongly resistant to the RAA in numerous studies42. In our study, we observed a gradient of resistance to the RAA for the European wild apple, but less marked than for M. domestica. Among M. domestica cultivars, resistant cultivars impacted RAA preference through both epidermic and phloemian factors39. Indeed, compared to that of aphids on susceptible controls, the feeding behavior of aphids on the resistant Florina cultivar revealed shorter durations for general probing (Pr), phloem salivation phase (E1), xylem sap ingestion (G). Also, none of the aphids ingested phloem sap (i.e. the E2 phase was null) and a significantly longer total duration of stylets derailment was observed when submitted to this resistant cultivar. In our case, M. sylvestris did only impact RAA preference through the phloemian phase, with most individuals able to ingest sap, although lasting for short duration. Therefore, RAA preference in the European wild apple studied here only involved one factor, the phloemian phase, in contrast to what was previously observed among M. domestica cultivars. Besides, the two-factor aphid response on the M. domestica host is also associated with lower RAA performance42. Strikingly, our study showed that only the Danish M. sylvestris population negatively impacted both RAA preference and performance compared to M. domestica. Thus, the Danish M. sylvestris population may be more resistant against RAA than M. domestica and would appear to represent a potential source of resistance for M. domestica breeding programs, although this CRW candidate did not impact RAA fecundity or survival. However, our results are consistent with previous investigations of pests and pathogens resistance in Malus domestica CWR which were involved in its domestication, in which some wild accessions were as sensitive as the cultivated ones17. Resistance against the pest of CWR would be mainly indirect, as they support greater communities of natural enemies17. Thus, as we did not proceed to field validation of our results, we may have overlooked some components of CWR resistance. Investigating RAA performance for several aphid generations would also be worth carrying out on the Danish M. sylvestris population, as this host negatively impacted aphid behavior and fitness in terms of weight and these negative effects may have a greater impact over generations. A greater diversity of new resistance genes or alleles against RAA may be present in CWR gene pool involved in the apple domestication. CWR are however largely neglected when it comes to studying their resistance to RAA. To better understand resistance differences at an interspecific level, more CWR species have to be included in experiments, such as M. orientalis and M. sieversii. However, this would involve exposing RAA to apple CWRs that are absent in its natural environment. Future studies may also investigate cross infestations of aphid populations from different parts of Europe onto CWR populations to truly test for RAA local adaptation. To test for the influence of maternal priming, it would also be interesting to compare relative preferences among the aphids reared on M. domestica to the relative preference among aphids reared on M. sylvestris.Finally, it is worth questioning to what extent M. domestica plant productivity. But above all, the present study reveals that the search for resistant CWR must not only be based on a genetic structure of wild populations but also on the crop-to-wild gene flow that appears to substantially drive resistance to RAA. In that sense French wild apples, which showed high level of crop-to-wild gene flow, may be good candidates for future breeding programs.To conclude, this work tested for the first time preferences and survival of a main pest of apple trees, among genetically distinct groups including wild and cultivated host plants. Identification of resistance adaptations among wild genotypes may help design strategies to improve Supplementary Information 1.


For many years, breeders of companion animals have applied inbreeding or line breeding to transfer desirable genetic traits from parents to their offspring. Simultaneously, this resulted in a considerable spread of hereditary diseases and phenomena associated with inbreeding depression.Our cluster analysis of kinship and inbreeding coefficients suggests that the Thai or traditional Siamese cat could be considered as a subpopulation of the Siamese cat, which shares common ancestors, although they are considered as separate breeds. In addition, model-based cluster analysis could detect regional differences between Thai subpopulations. We show that by applying optimal contribution selection and simultaneously limiting the contributions by other breeds, the genetic diversity within subpopulations can be improved.In principle, the European mainland Thai cat population can achieve a genetic diversity of about 26 founder genome equivalents, a value that could potentially sustain a genetically diverse population. However, reaching such a target will be difficult in the absence of a supervised breeding program. Suboptimal solutions can be obtained by minimisation of kinships within regional subpopulations. Exchanging animals between different regions on a small scale might be already quite useful to reduce the kinship, by achieving a potential diversity of 23 founder genome equivalents. However, contributions by other breeds should be minimised to preserve the original Siamese gene pool. Worldwide, there is growing concern about the loss of biodiversity, which is caused by the ever-increasing human population. The quantitative reduction of biotopes and over-exploitation of wild species result in the fragmentation of populations and reduction in their sizes which, in turn, increase the degree of inbreeding. Finally, inbreeding may result in inbreeding depression, which manifests itself by reduced reproductive fitness and reduced ability to adapt to changing environmental conditions. All these factors negatively influence the possibility to restore the size of populations to a reasonable level. Thus, the risk of extinction increases .Closer to home, this also applies to the breeding of domestic animals and companion animals. For many years, inbreeding has been an accepted method to transfer desirable genetic traits to the offspring in a predictable way, thereby increasing the economic or aesthetic value of the animals. However, this approach that deliberately reduces the heterozygosity level, simultaneously increases the risk of combining deleterious genes, and thus results in increasing the prevalence of several hereditary diseases or inbreeding depression which negatively influence the health of the animals and the population at large.The Siamese cat is a very ancient breed that originated from the Kingdom of Siam (nowadays Thailand), and was already known in the 14th century . The breA few breeders started to re-introduce the classical type and, in 1995, the traditional Siamese was once again accepted by some registries. The new population was built on founders that were imported from several countries around the world, and on selected Siamese cats of European origin, which had a less extreme conformation. The registered breed name Siamese remained connected to the modern type and from that time on, the classical type became known as the Thai cat, but this definition is not accepted worldwide. In the United States of America, the most important registry, the Cat Fanciers’ Association (CFA), does not recognise the Thai as a breed  whereas Meanwhile, heavy selection pressure and misfortunate outcrosses with other breeds resulted in an increased prevalence of several hereditary diseases within the contemporary Siamese cat population, e.g. progressive retina atrophy (PRA)  and traiIn this article, we report on the historic development and present status of the genetic diversity within the Siamese and Thai cat breeds that are present on the European mainland. We carried out a cluster analysis to reveal the relationships between both breeds and to explore regional differences in the distribution of their genetic diversity. We also calculated the mean kinship of breeding animals per cluster, which provides a more useful parameter to maintain or improve genetic diversity within the current Thai breed. Our aim is to preserve a healthy cat population without any exaggeration in appearance and that is related to the original Siamese cats that were present in Europe until about 1970.For this study, pedigree information was obtained from associated breeders, registries and on-line databases , 7 especThe data collection is known to be incomplete for the periods after the Second World War, when the registries had to be rebuilt, and towards the end of the 20th century when the traditional Siamese cats were not accepted anymore for registration or for entering shows, since they did not meet the new breed standards. The percentage of unknown parentage is about 9 %.The database of the CFA Siamese Breed Council  lists 49Since subsequent generations show a strong overlap and full demographic details are not available, throughout this work we define a population as the parents of the kittens, born in two consecutive years.J, under study:J are identical-by-descent. From this parameter, gene diversity (GD) is calculated ascondGD(J) of a birth cohort J as the probability that two alleles randomly chosen from the population are not identical-by-descent, under the condition that both alleles descend from native founders. When conservation of the original gene pool is an objective of a breeding program, this definition is a more adequate descriptor of the gene diversity within a population, which contains contributions from other breeds. In this situation, the aim is to maximise the conditional gene diversity while simultaneously limiting the genetic contribution by other breeds.In this work, we base our results on the concept of mean kinship as developed by Ballou  and Lacyr method . The avementclasspt{minimaFGE. This is the minimum number of unrelated founders required to create a population having the same genetic diversity as the currently investigated population. The FGE is approximated by:NGE(J) can be defined as the minimum number of unrelated native founders required to create a population having the same conditional gene diversity as the currently investigated population [J, has to be solved to minimise the population mean kinship in the next generation [Alternatively, genetic diversity can be described in terms of founder genome equivalents or mentclasspt{minimaneration :7\documeng (SQP)  was usedFGE, potential diversity, As for Statistical analysis was carried out using the R the statistics program version 3.5.3  includinPrior to further analysis, individuals that are not founders but for which parent information was missing were excluded by setting a threshold year, after which the breed name of these animals is set to Unknown. The parameter “lastNative=1940” was used for the Siamese and “lastNative=2005” for the Thai. Furthermore, individuals with less than 3 equivalent complete generations were excluded.Optimise both male and female contributionsLimit the increase in inbreeding by setting an upper boundary to the mean kinship according to the equation 1/(2NeL) . The effLimit the contribution by other breeds by setting a lower boundary to the contribution by the native breed. The contribution by native individuals should be larger as compared to the current population.To identify subpopulations, we performed a hierarchical cluster analysis of the current breeding animals, which were the parents of the kittens, born in two consecutive years. The input datasets consisted of 200 Thai (from the period 2016–2017) or 462 Siamese individuals (from the period 1998-1999). The function “hclust” was applied, in which the unweighted pair group (UPGMA) method [We investigated two OCS scenarios, i.e. by using the “min.pKin” objective function to minimise the kinship in the next generation and the “min.pKinatN” objective function while simultaneously restricting the contribution by other breeds, which minimises the kinship at native alleles. In principle, the latter approach can be used to reduce the influence of other breeds to next generations of the breed under investigation. Minimisation was performed, using the sequential least-squares quadratic programming (SQP) algorithm, which is available through the nloptr package , by sett package .Alternatively, we explored the use of a model-based cluster analysis applied to the same input data , 23. In Clustering solutions were visualised using the “clusplot” function. This function performs a principal component analysis (PCA) on the input data and uses the subdivision into groups as second input to identify the clusters by drawing ellipses around the data points (supervised learning).Preliminary results on smaller datasets were validated using the PMx software version 1.3.20150713 .The growth of the population of Siamese cats is evident from the number of breeding individuals until 1995 , reaching 4.2 and 7.0 respectively in 1998-1999. The situation is different for the Thai breed (Trace c and d). The FGE peaks at 23.8 in 2007 but rapidly decreases to 15.2 in 2016-2017 and the NGE shows a maximum of 5.4 around 2009, and drops to 3.1 in 2016-2017. Considering the Thai as a distinct breed, it has received a significant contribution from the Siamese cats with moderate conformation during the early years when the breed was founded. The difference between the FGE and NGE within the Thai breed (Trace e) represents the known contribution of Siamese cats to the Thai breed. It runs slightly above the FGE level within the Siamese breed between 1940 and 1960. If one considers Thai cats as a subpopulation of the Siamese breed, the Thai NGE could reflect the yet unknown lineage from the original Siamese population in Europe. The Thai NGE of the current population dropped by 9.7 units compared to the Siamese NGE in 1946, i.e. when it reached a maximum of 12.8 and before the modern Siamese was developed.Figure A preliminary pilot study, which we conducted in 2016, on a selection of 124 Dutch individuals, showed a lower inbreeding coefficient  of 85 % (i.e. a maximum loss of 15 %). These parameters are readily provided by commercial animal keeping software packages. Recently, it has become clear that this policy is difficult to maintain, mainly because there is only a limited number of sires available. The analyses based on kinship, presented in this report, provide alternative ways to maintain or improve genetic diversity within the European Thai cat population. In conservation breeding, achieving an iversity . As the iversity , breakin1/(2∗Ne) . A possiAlthough in 1998−1999, the genetic status of the Siamese population was less favorable, the genetic diversity of the first model-based subpopulation, consisting of 112 animals could be improved from 10.0 to a potential diversity of 18.0 founder genome equivalents by applying OCS . Breeds are recoded using the EMS abbreviations. Unfortunately, the country of origin is registered together with the pedigree name. This information gets lost upon recoding pedigree names to numeric data.Additional file 2. Completeness of Thai cat pedigrees. R script for analysing pedigree completeness per generation for males and females separately.Additional file 3. Historic development of mean kinship and inbreeding. R script to calculate and plot historic kinship and inbreeding of the Thai and Siamese breeds, using. Additional file Additional file 4. Historic development of mean kinship and inbreeding. R script to calculate and plot historic kinship and inbreeding of the Oriental Shorthair breed, between 1955 and 1999, using Additional file Additional file 5. Comparison of distribution of mean kinship and inbreeding. R script to plot the distribution of kinship and inbreeding coefficients of the Thai and Siamese breeds, using Additional file Additional file 6. Cluster analysis of the Thai breed. R script for hierarchical (UPGMA) and model-based cluster analysis of the Thai breed . This produces Table 1 and Figs. Additional file 7. Cluster analysis of the Siamese breed. R script for hierarchical (UPGMA) and model-based cluster analysis of the Siamese breed , containing the ancestors of the newly registered Thai breed. This produces Table 2. The figures showing the optimal number of clusters are left out from the main text.Additional file 8. Historic breed composition of the Thai breed. R script for analysing the contribution of other breeds to the Thai breed.


This study aimed to evaluate the genetic diversity of six groups of native Brazilian goats using a panel of single sequence repeats (SSRs). Results indicated a definite genetic differentiation among the Brazilian goat herd, which indicates the existence of at least four breeds according to the international concepts .The genetic diversity of six Brazilian native goats was reported using molecular markers. Hair samples of 332 animals were collected from different goat breeds  from five states of Northeast Brazil . A panel of 27 microsatellites or single sequence repeats (SSRs) were selected and amplified using a polymerase chain reaction (PCR) technique. All populations showed an average allele number of over six. The mean observed heterozygosity for Brazilian breeds was superior to 0.50. These results demonstrated the high genetic diversity in the studied populations with values ranging from 0.53 (Serrana Azul) to 0.62 (Repartida). The expected average heterozygosity followed the same trend ranging from 0.58 (Serrana Azul) to 0.65 (Repartida), and the values obtained are very similar for all six breeds. The fixation index (Fis) had values under 10% except for the Moxotó breed (13%). The mean expected heterozygosity of all Brazilian populations was over 0.50. Results indicated a within-breed genetic variability in the Brazilian breeds based on the average number of alleles and the average observed heterozygosity. The interbreed genetic diversity values showed proper genetic differentiation among local Brazilian goat breeds. Many domestic species originating in Europe were introduced to America in the colonial period. Brazilian goat breeds are derived mainly from Portuguese settlers with animals since the 16th century . The BraIn addition to early Iberian introductions, Asian breeds  were introduced. Recently, goats of European breeds  and African breeds (Anglo-Nubian and Boer) have been imported into Brazil to improve milk and meat productions.Brazil has more than ten million goat heads, according to FAOSTAT . ApproxiThe local Brazilian goat breeds Moxotó, Canindé, Serrana Azul, Marota, Repartida, and Graúna, are animals of multiple functions and adapted to the climatic conditions and smallholder farming systems. They are rustic animals with less nutritional requirements, raised extensively, and survive in areas where they predominately forage foe food with low nutritional value, unsuitable for foreign breeds. These breeds are the only source of animal protein for low-income populations in the semiarid northeast. Despite the historical and social importance of these breeds, they have been undervalued for decades due to their low contribution to the national economy and their concentration in the most impoverished Brazilian region. This fact has contributed to limited scientific knowledge about these valuable genetic resources.Another risk factor is that in the last years, the introduction of highly specialized foreign breeds in the Brazilian herds has caused a fast replacement and genetic erosion of the local ones, as observed by  and 7]..7]. ThisAccurate knowledge of genetic resource diversity is fundamental for properly targeting conservation strategies and using these resources. Genetic characterization of animals through molecular markers had it shown efficient to quantify genetic variation in different populations.This study aimed to use molecular markers (microsatellites) to determine the genetic variation between local Brazilian goats.Random samples were collected from 332 Brazilian goats: 60 Moxotó, 50 Canindé, 55 Serrana Azul, 68 Marota, 52 Repartida, and 47 Grauna . DNA wasThe PCR products were accomplished both by the internal size standard and by the same reference sample to correct the few variations in allele size assignation among runs. GENETIX v4.04 software  was usedis) of Brazilian goat breeds are presented in is) had values under 10% except for the Moxotó breed (13%).Genetic variation within breed and fixation index (Fe) of all Brazilian populations was over 0.50. The Serrana Azul was the breed with the lowest value (0.53). The matrix of DA genetic distances [ST among population pairs are showed in ST values.The mean expected heterozygosity . The differences can be observed in the dendrogram built through the UPGMA method . Two difThe numbers on the nodes are percentage bootstrap values from 1000 replications of resampled loci. The factorial analysis  showed mIbnelbachyr et al. , evaluatThe average number of alleles was higher than those found in different studies with goats in several regions globally, including some breeds in the Americas  and the Recent studies evaluating the genetic diversity of Brazilian goat breeds using microsatellite markers observed high levels of observed and expected heterozygosity and suggested substantial genetic diversity in the evaluated breeds.The average heterozygosity observed in our study was higher than that reported by Silvestre et al.  (0.385) According to Jianmin and Wenbin , in imprIS value was higher than 0.10. The FIS values in the studied populations shown genetic homogeneity. The high FIS value in the Moxotó breed (0.13) may be due to structure with subdivision into herds and low gene flow among the herds. Our samples were collected from four different herds that were geographically and reproductively isolated, and each of these populations is generally closed with a reduced number of sires, which favors inbreeding. In conservation programs, inbreeding levels should be taken into account, mainly when the same male is used for a prolonged period.Only in the Moxotó breed, the FIS values obtained in our study were contrasting with those found by other authors in goat populations [The low Fulations ,26,27,28ST = 0.075) among all populations was predictable, recent studies have already indicated this degree of differentiation of our Brazilian goats [ST values between 0.05 and 0.3 are typical of differentiated livestock breeds. In the European goat breeds, the FST lies around 5%.The degree of genetic differentiation .The genetic diversity and differentiation among the local Brazilian goat breeds must be safeguarded through in-situ and ex-situ conservation actions, integrated into local development programs.


Chilo partellus by crossing the hibernating (HD), aestivating (AD), post-hibernating (PHD), post-aestivating (PAD), and nondiapause (ND) strains. Reciprocal crosses were also made to gain full understanding of diapause regulation and the maternal effects, if any. Data were recorded on fecundity, egg hatching, larval survival, diapause induction and termination, adult emergence, and morphometrics of larvae, pupae and adults in the parents , F1 hybrids, and the reciprocal crosses. Genetic analysis showed that AD strain is general combiner, which also improved egg hatching, larval survival, diapause termination, adult emergence and proportion of females in the progenies. Incidence of diapause was highest in HD × AD, whereas termination was greatest in PHD × AD. However, ND strain and its reciprocal crosses with other strains did not exhibit any noticeable developmental response associated with diapause. Specific combining ability analysis suggested that where PHD and AD strains exist together there will be likely reduction in diapause incidence, increased survival with greater fitness and faster multiplication of their progenies resulting in outbreak of C. partellus. Degree of dominance estimates revealed that diapause, developmental and morphometric traits in C. partellus are governed by over dominance gene effects, and mainly depend on parental diapause history.Diapause is an endocrine controlled arrested metabolic state to delay development or reproduction under unfavorable conditions. To gain an understanding of importance of diapause for ecological adaptation, it is important to study regulation of diapause in insects. We examined genetics of diapause in  This happens to control the physiological processes and morphological development during particular stage of life cycle. The insects undergoing diapause pass through a series of physiological events such as suppression of development and reproductive functions, arrested metabolic activity to conserve the reserves, and resumption of normal developmental process on the onset of optimum climatic conditions7. Differences in genetic basis of various components of diapause also vary in different insect species8.Diapause is an endocrine controlled physiological state of arrested metabolic activity during a particular stage of insect development to survive under predictable adverse climatic conditions6. Abiotic factors like cooling and freezing, and rates of temperature change influence developmental and physiological alterations11, which also lead to several morphological changes such as body color, length, weight and width of various hard sclerotized structures like head capsule, mandible and body appendages15. Moreover, wider geographic distribution leads to behavioral and physiological differences in populations inhabiting different ecological niches16. Existence of ecotypes in different insect species is widely prevalent, and the inheritance of such traits may be due to long-term genetic differentiation or direct physiological response to environmental conditions18.Diapause being an adaptive but genetically regulated trait provide phenotypic plasticity to insects in response to environmental conditions1 progenies) in some insect species such as linden bug, Pyrrhocoris apterus L19., spider mite, Tetranychus pueraricola Ehara & Gotoh20, and flesh fly, Sarcophaga bullata (Parker)21. The incidence and duration of diapause in many insect species is under polygenic control22. Sex-linkage, maternal or paternal effects, and epistasis also play a significant role in diapause regulation in several other insect species26. Genetic and genetic-environmental interaction has been found to be involved in induction and termination of larval diapause in Ostrinia furnacalis (Guenée)27.Diapause has been reported to follow a simple Mendelian inheritance (3:1 segregation ratio in the FChilo partellus (Swinhoe) is widely distributed in tropical and temperate areas in Asia and Africa, and is a serious pest of maize and sorghum28. It undergoes facultative diapause as mature larvae inside the stems or stubbles of sorghum and maize30. Our earlier studies have generated comprehensive information on critical threshold conditions for induction and termination of diapause, duration of diapause, phenology of diapausing larvae, supernumerary moults, effects of diapause on post-diapause development and reproductive physiology, population buildup after diapause, and temperature-based development model of diapausing larvae in C. partellus32. Recent studies although revealed strong gene by environment interaction effects on diapause induction and incidence in some lepidopteran insect species, where inheritance patterns and dominance were found dependent on the photoperiod34. These studies further suggested that the use of varying photoperiod and temperature regimes, and different cross-mating combinations are integral to better understand the inheritance of diapause in insects. In this view our recent studies revealed that both temperature and photoperiod combine are critical for induction and termination of diapause in C. partellus15. Dhillon and Hasan32 elaborated that the larvae of C. partellus pass through hibernation under North Indian and aestivation under South Indian environmental conditions. The cross-mating among the adults of diapause and nondiapause strains and their F1 progenies within and across geographical regions is very likely, and there is a possibility of genetic polymorphism within and/or among the C. partellus populations. Thus, to understand such a complex population regulation mechanism, there is an urgent need for intensive genetic research on insects to explore alternative ways to manage insect pests of economic importance. None of the earlier studies attempted such a comprehensive genetic research to understand the genetic regulation of diapause and associated developmental traits in C. partellus under different geographical and population scenarios. Therefore, present studies were planned to investigate: i) the genetic components of diapause regulation and developmental traits, ii) effect of parental genetic background on diapause and related traits, and iii) effect of frequency of type(s) of pre-existing strain(s) on diapause incidence, developmental traits, and likely rate of C. partellus population buildup under given climatic conditions.The spotted stem borer, C. partellus. Similarly, incidence of diapause  and diapause termination  was also significantly different among the parents and the diallel cross progenies , percentage egg hatching , larval survival  and adult emergence   and post-diapause  for each diallel cross, and expressed as proportion of offsprings that were females. Data on developmental parameters, morphometrics during different developmental stages, and sex ratio in the progenies of parents and the diallel cross populations were subjected to one-way analysis of variance (ANOVA) followed by Tukey–Kramer honestly significant difference (HSD) test using SPSS v. 15.0 statistical package . Analysis of variance, component analysis for estimation of degree of dominance, proportion of dominant and recessive alleles, proportion of alleles with positive and negative effects, heritability, and combining ability analysis were performed as per the method suggested by Hayman67 using Windostat Version 9.2.Mean values of all the test parameters of progenies of parents and the diallel crosses were used for statistical analysis. Sex ratio was calculated according to Wilson and Hardy


Hermetia illucens) is renowned for its bioconversion ability of organic matter, and is the worldwide most widely used source of insect protein. Despite varying extensively in morphology, it is widely assumed that all black soldier flies belong to the same species, Hermetia illucens. We here screened about 600 field-collected and cultured flies from 39 countries and six biogeographic regions to test this assumption based on data for three genes  and in order to gain insights into the phylogeography of the species.The black soldier fly , Oriental (N = 4), and the Eastern Palearctic (N = 4) populations. We reviewed museum specimen records and conclude that the evidence for introductions is strong for the Western Palearctic and Afrotropical regions which lack distinct, private haplotypes.Our study reveals a surprisingly high level of intraspecific genetic diversity for the mitochondrial barcoding gene COI (divergences up to 4.9%). This level of variability is often associated with the presence of multiple species, but tested nuclear markers (ITS2 and 28S rDNA) were invariant and fly strain hybridization experiments under laboratory conditions revealed reproductive compatibility. COI haplotype diversity is not only very high in all biogeographic regions , but also in breeding facilities and research centers from six continents (10 haplotypes: divergences up to 4.3%). The high genetic diversity in fly-breeding facilities is mostly likely due to many independent acquisitions of cultures via sharing and/or establishing new colonies from field-collected flies. However, explaining some of the observed diversity in several biogeographic regions is difficult given that the origin of the species is considered to be New World (32 distinct haplotypes) and one would expect severely reduced genetic diversity in the putatively non-native populations in the remaining biogeographic regions. However, distinct, private haplotypes are known from the Australasian  of the fly strains used in artificial fly-breeding and share these data in research publications as well as when sharing cultures. In addition, fast-evolving nuclear markers should be used to reconstruct the recent invasion history of the species. Hermetia illucens   are large flies with brownish wings and a pair of characteristic translucent pale patches (windows) on the abdomen [https://phoenixworm.com/) [Black Soldier Flies (BSF),  abdomen –5. Not s abdomen ). Indeedrm.com/) , 10. Manrm.com/) . HoweverH. illucens specimens from Australia collected in 1915 [H. pallidipes Hill, 1919) [H. illucens is thus from 1948 . According to Marshall et al. [H. illucens by the1940s. This includes Hawaii, the Solomon Islands, New Caledonia, Mariana Islands, Palau, and Guam. Overall, it is striking that many of the earliest known localities outside of the New World are on islands or close to the coast. This may indicate that worldwide trade played a major role in the passive spread of BSF [The native range of BSF is considered to be New World, from northern parts of South America to the northern-most native populations occurring in the southwestern parts of the US (at least up to 40° N) . However in 1915 , 17 havel, 1919) . The oldl et al.  many Pacd of BSF . For exaHelobdella. By the time of discovery, the lack of timely genotyping had done severe harm to the scientific literature because results had been filed under incorrect scientific names [Hirudo medicinalis. Many leeches used for medical purposes were revealed to belong to species that were not approved for use [Species with exceptionally large distributions are frequently revealed to consist of species complexes or comprise several evolutionary significant units that deserve recognition once they are studied using systematic investigation using genetic methods in order to protect their genetic integrity –21. Fragic names . Shortly for use . Yet, BSWe here carry out a first systematic survey across the species’ current range with the aim toreconstruct the phylogeography of the target species. This is particularly challenging because the species has apparently been introduced repeatedly due to international trade in many regions over the last decades. Additional complication stem from the species’ being prone to escape from rearing cultures, and the international trade by commercial breeders (e.g. providing online ordering utilized by e.g. exotic-pet keepers). We therefore here compare the genetic diversity of field-collected flies  with samples obtained from rearing cultures maintained for academic or commercial purposes. In addition to mitochondrial data, we assess the genetic diversity for two fast-evolving nuclear markers  nucleotide sites were variable that coded for amino-acid changes in 5 of the 219 residues (2.3%). Our study screened > 500 fly rearing culture samples and barcoded 75 field-collected samples. These data were complemented with 49 publically available DNA barcodes from sequence databases which represented additional haplotypes and/or geographic localities  and added 49 barcodes from public databases  and recorded ten COI haplotypes from the rearing cultures , while all 28S loop sequences were invariant.Mating trials under laboratory conditions revealed a lack of reproductive isolation between the populations, as hybrid offspring were produced and successfully back-crossed with the parental population. The hybrid populations were also fertile and some have now been maintained for > 10 generations. Backcrosses were not attempted for these.Our data reveal many unexpected results for this widely used workhorse of insect protein production. They highlight that there is currently a surprising lack of background information for a species that is globally, and extensively, used and shared for scientific research, animal feed production and organic waste recycling.Helobdella and Hirudo. The high COI diversity is structured in both expected and unexpected ways. The species is considered native to the New World; i.e., the high genetic diversity we recorded for the New World samples corresponds well with expectations, while the remaining regions should mostly have subsets of the New World genetic diversity. This is indeed the case for the Afrotropical and the Western Palearctic populations, which contain a limited number of haplotypes. It appears likely that these populations were indeed introduced. However, it was surprising to find several distinct, highly diverged haplotypes in Australasian, Oriental and Eastern Palaearctic populations, and this diversity remains unexplained if the species indeed originated in the New World and only recently dispersed to these regions. Possible explanations include still insufficient sampling of the Neotropical diversity or a broader geographic origin of the species.Our study revealed very high COI barcode variability, but this variability is not mirrored by the two invariant nuclear markers that were included in our study. This high COI diversity also does not translate into reproductive incompatibility although the divergence levels (close to 5%) well exceeds what is typical for flies . This imBSF has not only unusually high levels of genetic diversity for COI, but it is also morphologically very variable with regard to body size, wing and abdominal coloration, and the shape of translucent abdominal windows. Wing coloration varies and ranges from almost black with a blueish-metallic shine over a clear brownish-opaque insignia in the inner part of the wing to almost pale-translucent. The amount of white versus black coloration on the head shows great variation, while the black and white pattern on the legs seem to be more consistent. There is a tendency for males to have a reddish to red abdomen, while females rarely display red coloration on the abdomen. Much of this variation can be found within the same laboratory cultures and is thus likely due to polyphenism, but more systematic study across cultures representing different haplotype groups is needed.We documented that the distinct haplotypes of the commercial US breeding strain Figs. . BarcodeArguably, the most convincing evidence for human-mediated introductions come from specimens collected at latitudes that are unlikely to support breeding populations , 29–31. Drosophila stock centers). The scientific utility for the genetic characterization and source documentation in commercial breeding of domestic livestock (e.g. pigs and cattle) is obvious, and genomics has not only proven useful for reconstructing the domestication history of the different breeds, but also for understanding the molecular mechanisms underlying traits of interest [The BSF is now being actively domesticated worldwide , but theinterest . A tremeinterest , and addinterest . Knowinginterest , and it This study screened a comprehensive number of both cultured and wild caught BSF samples from six continents and 39 countries, including numerous from the putatively native New World and continents where BSF is an alien species. Our findings clearly highlight the need for additional sampling of New World populations as well as the Australasian and Oriental regions. In particular, the discovery of the haplotypes which are currently only known from Australasian and Oriental populations would strengthen the hypothesis of a New World origin of the species.If a divergence rate of 2.3% per million years for insect mitochondrial DNA  were useA high genetic diversity has considerable implications for scientific research and commercial use of the species. Assessing the genetic makeup of cultures would seem a necessary fly culture management tool, considering the possible advantages of a high genetic diversity of the BSF strains coupled with different dominant characteristics that a breeding program could capitalize on. We therefore urge all commercial users and researchers to generate barcodes for their cultures given that this is low cost. The DNA and specimens should also be kept for future screening of additional nuclear markers. Based on our results, the mtDNA COI barcode is sufficiently informative to be a first step marker for any genetic characterization of BSF strains or populations. The available genomic studies on the BSF are still limited , 41, butncbi.nih.gov) and BOLD (boldsystems.org)  from rearing facilities in Europe, Africa, Asia, Australia and United States. Field-collected (“wild”) fly samples were collected in both urban and pristine habitats from Africa, Asia, Australasia, Europe, and the Americas, but it is often difficult to rule out that they are escaped flies from cultures. Our data were complemented with publicly available COI barcodes retrieved from GenBank  of larval tissue was used for DNA extraction with the Nucleospin Tissue DNA extraction kit . Ready-to-Go PCR beads  were used for amplification of the COI barcode using the universally conserved primer pairs LCO-1490 and HCO-2198 [Helsinki lab. HCO-2198  of all sField-collected samples: Fly specimens were obtained from field collections or from the authors’ conserved collections  and the same primers were employed for subsequent bidirectional Sanger sequencing reactions. The laboratory work was done at FMNH Luomus DNA Lab , and sequencing at Sequencing Service Laboratory . The raw sequences were edited for base-calling errors and assembled using Sequencher vs 5.0 .Singapore lab. We screened 259 specimens from 13 countries representing all six continents . Virgin hybrid flies of both sexes were separated and then back-crossed to virgin flies from their parental cultures. The following reciprocal pairings were carried out: 20 Spanish ♂♂ × 20 Hybrid ♀♀; 20 Hybrid ♀♀ × 20 Singapore ♂♂. A second batch of experiments involved 311 New World ♂♂ with 310 ♀♀ from Singapore and 408 New World ♀♀ with 407 Singapore ♂♂.Additional file 1 : Supplementary Figure S1. Median-Joining haplotype network of combined rearing culture and field-collected samples for the complete COI sequences. The biogeographical region of the haplotypes is color-coded, and the size of circles is proportional to the number of individuals per haplotype. The black circles represent putative un-sampled haplotypes. The branch lengths are largely proportional to the numbers of mutational steps separating the haplotypes, and the number(s) of mutational steps are indicated with hashmarks on the branches. All rearing culture samples are indicated with a black ►. The most abundant COI haplotypes are indicated with Roman numerals I-III with the geographical sources listed in Table Additional file 2 : Supplementary Table S1. GenBank accession numbers for each distinct haplotype and locality/culture. Specimen repository indicated for pinned fly specimens .Additional file 3 : Supplementary Table S2. List of haplotypes.


Skin thickening and joint contractures are often the main presenting features of the disease and may be mistaken for juvenile scleroderma.Myhre syndrome is a genetic disorder caused by gain of function mutations in the SMAD Family Member 4 SMAD4 gene, suggesting a diagnosis of Myhre syndrome. Securing a molecular diagnosis in this case allowed the cessation of immunosuppression, thus reducing the burden of unnecessary and potentially harmful treatment, and allowing genetic counselling.We report a case of a 13 year-old female presenting with widespread skin thickening and joint contractures from infancy. She was diagnosed with diffuse cutaneous systemic sclerosis, and treatment with corticosteroids and subcutaneous methotrexate recommended. There was however disease progression prompting genetic testing. This identified a rare heterozygous pathogenic variant c.1499 T > C (p.Ile500Thr) in the Myhre Syndrome is a rare genetic mimic of scleroderma that should be considered alongside several other monogenic diseases presenting with pathological fibrosis from early in life. We highlight this case to provide an overview of these genetic mimics of scleroderma, and highlight the molecular pathways that can lead to pathological fibrosis. This may provide clues to the pathogenesis of sporadic juvenile scleroderma, and could suggest novel therapeutic targets. SMAD4) gene causing progressive, proliferative fibrosis, occurring spontaneously or following trauma, in addition to a unique set of clinical phenotypic features described below [SMAD4. We discuss the therapeutic implications of establishing a genetic diagnosis in this case and provide an overview of genetic mimics of scleroderma.Myhre syndrome is a genetic disorder often presenting in infancy, caused by a gain of function mutation in the SMAD family member 4 . These histological features are typically encountered in scleroderma histopathology with the exception of the hyperkeratotic epidermis, which is less often seen [2/week) started. There was deterioration in joint contractures (further loss of range of movement) and spreading of skin changes observed despite treatment. When reviewed for a second opinion at GOSH, a genetic diagnosis was suspected and genetic testing via Sanger sequencing was undertaken for some conditions that cause skin thickening, dysmorphic features and congenital heart disease. Genetic testing revealed a previously well described rare heterozygous c.1499 T > C (p.lle500Thr) class 5 variant in SMAD4 [PTPN11, LMNA, and MMP14) revealed no other pathogenic variants [Clinical examination revealed diffusely thickened skin affecting the full length of her limbs and trunk, but sparing her face; weight 41 kg (25th centile for age), height 139 cm (2nd centile for age). She was normotensive at time of review. Delayed puberty was noted and the patient had no menarche at the age of 13 years old. There were multiple joint contractures, but no active arthritis. Cutaneous telangiectasia, fingertip ulceration and calcinosis were absent. She was also noted to have mild dysmorphic features: small eyes and ears, a broad nasal tip, a long and prominent chin, bilateral clinodactyly and mild two-three toe syndactyly. Nailfold capillaroscopy was abnormal, with evidence of dilated capillary loops, tortuosity, micro-bleeding, and widespread dropout in a pattern compatible with scleroderma-spectrum connective tissue disease ten seen , 13, 14.in SMAD4 , suggestvariants –26. PareWe present the case of a 13 year-old with a scleroderma-like condition, ultimately diagnosed with Myhre syndrome, a genetic disorder that may mimic juvenile scleroderma  signalling [Myhre syndrome is caused by mutations in gnalling –4. Skin gnalling . Some pagnalling –34. We sSeveral other conditions may also mimic juvenile scleroderma Table . Skin thRegarding the methodology of genetic screening, our case again illustrates the importance of next-generation sequencing (NGS) methodologies in this context. Mainly due to lack of routine NGS methods, initial routine genetic testing of candidate genes by Sanger was performed for this patient. This was a time consuming, costly, and mainly “clinician best guess” driven approach, which resulted in diagnostic delay of several months. Whole exome and genome sequencing and targeted gene panels now allow rapid, simultaneous detection of multiple genes, and are increasingly being used as diagnostic tools and to explore the pathogenesis of monogenic diseases –61. ThesUnderstanding the genetic basis of these genetic diseases with sclerodermatous features is not only crucial to secure diagnoses, improve prognostication and to facilitate genetic counselling but may also provide clues to the pathogenesis of sporadic cases. For instance, several of the genetic mimics of scleroderma involve the TGF-β pathway , 62–64. Myhre syndrome is a rare genetic disorder that causes skin thickening and joint contractures, and may be misdiagnosed as juvenile scleroderma (systemic sclerosis). Many other genetic conditions can similarly mimic the clinical manifestations of juvenile scleroderma and should be considered in the differential diagnosis of juvenile scleroderma. Onset in infancy and comorbidities such as structural heart disease, large vessel vasculopathy, dysmorphic features, developmental delay, and hearing loss are important clues to a genetic diagnosis. Clinical application of NGS is likely to transform the genetic diagnostic approach to young patients with scleroderma-like diseases and suggest targeted therapies for some cases. Therapeutic targets for sporadic cases of juvenile scleroderma are also likely to emerge, given the overlapping disease mechanisms for all these conditions leading to vasculopathy, skin and organ fibrosis.Additional file 1 Supplemental Table 1. Features of juvenile localised scleroderma, juvenile systemic sclerosis and Myhre syndrome.