The use of molecular markers can support the management of endangeredpopulations and should be combined with appropriate breeding strategies to improveproductive traits avoiding the decline of the breed. The genetic variability at 10microsatellite loci were investigated in a sample of 100 unrelated Markhoz goats. The investigated population was reared at the Sanandaj Markhozgoat Performance Testing Station in Kurdistan, Iran. Markhoz goat, a multipurpose breed,is one of the most valuable genetic resources in Iran. All the studied loci were found tobe polymorphic and a total number of 52 alleles were identified with an average number ofalleles of 5.2. Moreover, some population genetic indices, such as observed and expectedheterozygosity, observed and expected number of alleles, Shannon's index, Nei's expectedheterozygosity, and polymorphism information content were also calculated. Despite thedecreasing population size, Markhoz goat genetic diversity is still conserved. The breedseems to have a good level of genetic variability and, as a consequence, a potentialmargin of adaptability to environment and for future genetic improvement. Goats are able to provide high-quality productsunder diverse climatic conditions, being resilient to extreme and capricious environments. Moreover, local goat breeds playa significant role in the sociocultural life of the rural community and in preservingmarginal areas. .Undoubtedly, the effective management of genetic resources of farm animalsrequires comprehensive knowledge about breed characteristics, data on thesize and structure of the populations, and the geographicaldistribution of genetic diversity within and between breeds .Markhoz goat breed is one of the most valuable genetic resources in Iran. This breed iswell adapted to the environmental conditions of arid and semi-arid areas where it is usedas a multipurpose animal for the production of milk, meat, hair and hide . Markhoz goat has a coat called mohair being the only mohair goat breed inKurdistan province where it represents the main economic income for goat producers.Twenty years ago, about 25 000 Markhoz goats were raised in Kurdistan province of Iran.Latest census data reported a total population size of 1669 does and bucks . This rapid number decrease is due to many different reasons such asdiscouragement in youth and family members, shortage of shepherds, nutrition problems,existence of haired goat breeds with higher income, low income of goat raising and lowfinancial feasibility for herders. Although Markhoz goat is not at risk of extinction,when considering the downward trend, this breed may face the risk of genetic erosion dueto natural disasters and epidemic diseases .Therefore, given the importance of this breed and the need to identify geneticvariability in the Markhoz goat population, it is necessary to assess the geneticdiversity within this breed. In fact, genetic diversity is fundamental for long-termsurvival of populations providing the raw material for adaptation and evolution,especially when environmental conditions have changed . Thus, thegenetic characterization of native breeds is the first step for their conservation. Theuse of highly variable molecular genetic markers, such as microsatellites, is one of themost powerful tools to study genetic diversity because of its high polymorphic degree,random distribution across genome and neutrality with respect to selection . Therefore, the present study aimed to genetically characterize the Markhoz goatbreed using specific microsatellite markers to investigate the genetic diversity withinthis population and to improve future breeding strategies and conservation programmes.2Individual blood samples (10 mL each) were collected in K3-EDTA tubes and stored atTen microsatellite markers, previously described as polymorphic in goat, wereinvestigated: BM1312, LSCV06, ILSTS082, IDVGA64, LSCV25, BMS1248, BM2830,IL2RA, LSCV36 and MAP2. The studied microsatellites are located onchromosomes 1, 2, 5, 13 and 19 (Table 1).PCR amplifications were carried out in a 25 Population genetic indices, namely observed and expected heterozygosity, observed and expected number of alleles, Shannon's index andNei's expected heterozygosity  were performed by POPGENE32software version 1.32 . Moreover, polymorphism informationcontent (PIC) was calculated according to Botstein et al. (1980).3Microsatellite markers used in this study were previously assessed in different goatbreeds . Table 2 shows the measuresof genetic diversity at each microsatellite locus in Markhoz goat population. All 10microsatellite loci were successfully amplified and found to be polymorphic. A total of52 different alleles were identified with observed allele numbers ranging from 4 to 6 anda mean of 5.2 alleles per locus, being similar to findings obtained by Ganai and Yadav(2001), Gour et al. (2006) and Ramamoorthi et al. (2009) in some Indian goat breeds. Theinvestigated microsatellites showed a good level of polymorphism. The average effectivenumber of alleles was 4.12 varying from 3.46 (BMS1248) to 5.36 (LSCV25). Similar resultswere found by Dixit et al. (2011) who observed a mean effective number of alleles of 4.2in Kannai Adugoat; Kumar etal. (2009) who reported a value of 4.7 in Gohilwadi goat and Verma etal. (2010) who found a mean effective allele number of 4.0 in Sangamneri goat breed.Considering that at least four alleles were detected for each microsatellite locus in ourpopulation, this is in line with the selective standard of the microsatellite loci givenby the Secondary Guidelines for Development of National Farm Animal Genetic ResourcesManagement Plans using reference microsatellitegiven by FAO (2004) for proficient judgment of genetic distance between breeds. Thus, allstudied microsatellite markers exhibited sufficient polymorphism for evaluatingintra-population genetic variability in Markhoz goat breed.Heterozygosity is a good measure of the genetic variability within a population. In oursample, the most polymorphic locus was LSCV25 with six alleles and the highest value ofexpected heterozygosity was 0.817. On the other side, MAP2 was the least polymorphic withfour alleles and expected heterozygosity of 0.721. The average expected heterozygosityobtained was 0.756, being lower than the observed one (0.982). This slight excess ofheterozygosity was also reported by Ramamoorthi et al. (2009) in Barbari goats. Thediscrepancy between the observed and expected heterozygosity can be attributed to therandom mating among the individuals of the population. However, the available literaturegenerally reports lower values of observed heterozygosity . Considering the higher observedheterozigosity in the present population and the lack of allelic fixation at theinvestigated loci, it is possible to state that Markhoz breed has a wide margin ofgenetic improvement.PIC is a parameter indicative of the degree of informativeness of a marker. The PIC valuemay range from 0 to 1. In the studied population, the average PIC value was 0.719 rangingfrom 0.653 (ILSTS082) to 0.789 (BM1312). According to the classification of PIC , this populationpossessed high genetic diversity. PIC values calculated in the present investigation werecomparable with those reported by Ramamoorthi et al. (2009) (0.686) in Barbari goats,Serrano et al. (2009) (0.743) in Spanish Guadarrama breed, Verma et al. (2010) (0.711) inSangamneri goats, Dixit et al. (2012) (0.770) in Indian goat breeds and by Zhang etal. (2012) (0.781) in Chinese goat breeds. More recently, Radhika et al. (2015) reportedan average PIC value of 0.77 in Indian native and crossbred goats, and Wang et al. (2017)stated a mean PIC of 0.716 in Chinese dairy goats. Conversely, lower PIC values werereported in Indian and Pakistani goat breeds , in goats from Korea and China  and in SouthAfrican Angora goats .The variability in PIC values found in literature may be due to different mating methodsused in the studied populations. In the present study, the high PIC values prove that themicrosatellite markers used are highly polymorphic and can be well utilized for molecularcharacterization of goat germplasm.Shannon's index measures the biodiversity level in a population  and itresulted in sufficiently high values for the studied goats with a mean value of 1.50,ranging from 1.32 (MAP2) to 1.73 (LSCV25).Nei's expected heterozygosity indicates the level of gene diversity within the population. In the Markhoz breed it ranged from 0.62 (LSCV25) to 0.81 (LSCV06 andILSTS082) with a mean value of 0.73. These values were within the range of 0.3–0.8 asdetermined by Takezaki, and Nei (1996) for markers to be useful to assess geneticvariation in a population. Similar results were reported by Rout et al. (2008) in someIndian goat breeds, Dixit et al. (2011) in Kanniadu goats, Verma et al. (2010) inSangamneri breed and by Rout et al. (2012) in Jamunapari goat.It is important to underline that it is quite difficult to compare results from otherstudies as different microsatellite sets have been investigated by different scientists.Thus, comparisons may only give suggestive indication of diversity in a population.Nevertheless, to study the genetic variability in a population plays an important role indeveloping rational breeding strategies for domestic species.4Genetic diversity is the main component of the adaptive evolution mechanism because ofits preeminent role for the long-term survival probability of all species. Despite thedecreasing population size, Markhoz goat genetic diversity seems still conserved. Infact, considering that the sampled population included unrelated animals, the breed seemsto have a good level of genetic variability and, as a consequence, a potential margin ofadaptability to environment and for future genetic improvement. Nevertheless, furtherstudies should be conducted to deeply evaluate the genetic variability of the Markhozgoat breed.


Brasilianthus carajensis (Melastomataceae), an endemic herb from Amazonian Savannas. Using RAD sequencing we identified a total of 9365 SNPs in 150 individuals collected across the species’ entire distribution range. Relying on assumption-free genetic clustering methods and environmental association tests we then compared neutral with adaptive genetic structure. We found three neutral and six adaptive genetic clusters, which could be considered management units (MU) and adaptive units (AU), respectively. Pairwise genetic differentiation (FST) ranged between 0.024 and 0.048, and even though effective population sizes were below 100, no significant inbreeding was found in any inferred cluster. Nearly 10 % of all analysed sequences contained loci associated with temperature and precipitation, from which only 25 sequences contained annotated proteins, with some of them being very relevant for physiological processes in plants. Our findings provide a detailed insight into genetic diversity, neutral and adaptive genetic structure in a rare endemic herb, which can help guide conservation and management actions to avoid the loss of unique genetic variation.Conserving genetic diversity in rare and narrowly distributed endemic species is essential to maintain their evolutionary potential and minimize extinction risk under future environmental change. In this study we assess neutral and adaptive genetic structure and genetic diversity in  Rare and narrowly distributed plants are particularly sensitive to the loss and fragmentation of their natural habitats, which ultimately result in reduced genetic diversity and a weakened ability to adapt to changing environments. In this study we evaluated the genetic health of a narrowly distributed herb from Amazonian Savannas. Relying on Next-Generation Sequencing technologies we were able to take a detailed glance at a representative portion of the plant’s whole genome, assessing genetic diversity, genetic composition and adaptations to local environmental conditions. Our results provide clear guidance on how to avoid the loss of unique genetic variation in this unique plant. The assessment of population genetic structure has frequently been employed to delineate conservation units  and adaptive units , stands out for being a monotypic plant genus from the Amazonian Savannas of the Carajás mineral province , encompassing the entire species’ distribution range (−1. DNA samples were shipped to SNPSaurus (http://snpsaurus.com/) for sequencing and bioinformatic processing. The nextRAD libraries were prepared . Genotype calling was done using Samtools and bcftools. Alleles with a population frequency of less than 3 % were removed, and loci that were heterozygous in all samples or had more than two alleles in a sample  were discarded.We used the genomic data generated by on range . We sampon range  without ap tools . Second,r2vcftools R package (https://github.com/nspope/r2vcftools). Loci were filtered for quality (Phred score > 50), read depth (30 – 240), linkage disequilibrium  and deviations from the Hardy–Weinberg equilibrium (HWE). For HWE filtering, the data were initially subdivided into large geographic groups , and loci showing strong deviations (P < 0.0001) in any of these groups were excluded. Additionally, we removed loci potentially under selection conducting a genomic scan analysis implemented in the R package LEA  and setting false discovery rates to q = 0.05, using the Benjamini–Hochberg algorithm . The resulting colour patterns represent the similarity in adaptive genetic composition. Finally, to visualize admixture in neutral genetic clusters, we calculated ancestry coefficients using the snmf function from the LEA package and plotted them for each individual, as in classical STRUCTURE-like figures (The assumptions underlying commonly employed genetic clustering software like STRUCTURE (a Bayesian approach) or ADMIXTURE (based on maximum likelihood estimation) include absence of genetic drift, existence of HWE and lack of LD in ancestral populations . We theradegenet . In DAPC figures .HE), inbreeding coefficients (F), nucleotide diversity (π) and effective population size (Ne). While the former three were calculated using the ‘het’ option in VCFtools implemented in r2vcftools  was also estimated using r2vcftools. Additionally, we calculated Tajima’s D, representing the difference between the mean number of pairwise differences and the number of segregating sites. Negative values of Tajima’s D show that a cluster has an excess of rare alleles, indicating a recent selective sweep or a population expansion after a recent bottleneck. Positive values appear when rare alleles are lacking, therefore suggesting balancing selection or a sudden population contraction. In a population of constant size evolving under mutation-drift equilibrium Tajima’s D is expected to be zero . We selected a set of orthogonal environmental variables, running a PCA on all 19 WorldClim bioclimatic variables plus elevation . We used 1000 iterations, a burn-in of 10 000 and 10 runs per environmental variable  and false discovery rates were set using the Benjamini–Hochberg algorithm at a rate of q = 0.05  to obtain the corresponding protein sequences coded by genes contained in the flanking regions of our candidate SNPs. We used all six frames with standard code (codon table), regions (start–end), trimming (yes) and reverse (no). We then ran a functional analysis using InterPro  containing putative adaptive SNPs were submitted to the EMBOSS Transeq  between neutral clusters ranged between 0.024 and 0.048 and substantial admixture was detected [see].From a total of 9365 SNPs identified in the 150 sampled individuals, we kept 1911 neutral SNPs after filtering for quality, depth, LD, HWE and clusters , whereasclusters . While eclusters . Clusterttleneck . PairwisP-values for genotype–environment associations ranged between 0.05 and 1 × 10−8, and revealed stronger associations with precipitation of the wettest quarter and minimum temperature of the coldest month [see]. Only 25 sequences contained InterPro annotations and two annotated genes were shared between all environmental variables (P = 0.001), with individuals from Serra Norte and Bocaina showing genotype associations with higher temperatures and higher precipitation levels [see].Nearly 10 % of all analysed sequences contained putative adaptive SNPs , most ofariables . Adaptivariables , and a lariables . RedundaB. carajensis collected across the species’ currently known distribution range. While neutral genetic structure indicated the presence of at least three discrete genetic clusters, six adaptive genetic clusters were found. Despite the small estimated effective population sizes, genetic diversity was similar to that found in other plant species occurring in the region, and none of the neutral clusters showed significant inbreeding. In addition, around 10 % of the analysed sequences contained putative adaptive SNPs associated with environmental variables, revealing different selective pressures in a narrowly distributed species. Below we discuss our findings, and show how combining information derived from neutral and adaptive genomic markers can help guide conservation efforts.This study assessed genetic diversity, neutral and adaptive genetic structure in 150 individuals from B. carajensis were generally higher or similar to those observed for other patchily distributed herbs, shrubs and trees , the lack of inbreeding, the low levels of genetic differentiation and the existence of admixture among the studied populations suggest there is no short-term risk of inbreeding depression   using neutral markers.P-values of candidate SNPs for each environmental variable, generated by latent factor mixed models (LFMM).plaa003_suppl_Supplementary_FiguresClick here for additional data file.plaa003_suppl_Supplementary_TablesClick here for additional data file.https://doi.org/10.6084/m9.figshare.8224175.v3Genotypes, sequences and geographic coordinates for all samples are publicly available in FigShare:


Previous studies indicated existing, albeit limited, genetic-geographic population substructure in the Dutch population based on genome-wide data and a lack of this for mitochondrial SNP based data. Despite the aforementioned studies, Y-chromosomal SNP data from the Netherlands remain scarce and do not cover the territory of the Netherlands well enough to allow a reliable investigation of genetic-geographic population substructure. Here we provide the first substantial dataset of detailed spatial Y-chromosomal haplogroup information in 2085 males collected across the Netherlands and supplemented with previously published data from northern Belgium. We found Y-chromosomal evidence for genetic–geographic population substructure, and several Y-haplogroups demonstrating significant clinal frequency distributions in different directions. By means of prediction surface maps we could visualize (complex) distribution patterns of individual Y-haplogroups in detail. These results highlight the value of a micro-geographic approach and are of great use for forensic and epidemiological investigations and our understanding of the Dutch population history. Moreover, the previously noted absence of genetic-geographic population substructure in the Netherlands based on mitochondrial DNA in contrast to our Y-chromosome results, hints at different population histories for women and men in the Netherlands. An extensive database of genetic variation and detailed insight in genetic-geographic population substructure is essential for forensic investigation, epidemiology and historical, archaeological, evolutionary and genealogy studies within a nation (see for example ). For thConsidering the non-recombining part of the Y-chromosome, Roewer et al. described a significant division between the north and the south of the Netherlands, based on Y-chromosomal short tandem repeat (YSTR) haplotypes from 275 samples from five different locations, but this division was not detectable based on SNP data . MoreoveDespite the aforementioned studies, YHG data from the Netherlands remain scarce and do not cover the territory of the Netherlands well enough to allow a reliable investigation of genetic-geographic population substructure. Such a Dutch YHG database would be a valuable addition to the already available autosomal and mitochondrial DNA information for various reasons. First of all, in contrast to the autosomes the Y-chromosome is uniparental. This means its genetic variation is influenced by, but also indicative of, male-specific demographic processes and is therefore very useful in reconstructing population histories. Also, in the forensic world genome wide data analysis is still scarcely applied (and standardized) and therefore uniparental markers remain essential for inferring bio-geographic ancestry of suspects or unidentified victims. Therefore, regional knowledge on genetic-geographic substructure of the Y-chromosome is of great value.2 of land [2 of land [The phylogenetic resolution of the current YHG tree is now sufficiently high  to be ab of land , althoug of land . The goa of land , 14 and  of land .For our study, we used 2085 blood-donor samples from male donors that are reported to be unrelated and residing in a-priori selected locations. This is the same dataset as published in Westen et al. for autosomal STR data  and WestBecause of the sampling strategy, excluding several of the major cities that harbor large recent since 1950) immigrant populations , this da50 immigrTo increase the study area and enhance the possibility to detect geographic patterns, a dataset of 773 males from the northern Belgian provinces West-Vlaanderen, Oost-Vlaanderen, Antwerpen, Vlaams-Brabant, the Brussels region, and Limburg, using the actual living place at the time of sample collection (the “present” dataset), as previously described by Larmuseau et al. [The samples from the Flanders dataset were collected with a different sampling strategy than the Dutch dataset and mostly single samples are available per location. Therefore, several methods for detecting population substructure and gradients were not applicable to the Flanders dataset.FST on YHG proportions among sampling locations. This matrix was computed with ARLEQUIN software version 3.11 with standard settings [We used two different approaches in order to search for genetic-geographic patterns in the Dutch dataset. First, classical multidimensional scaling (MDS) analysis was performed with R software version 3.4.4  to represettings . The ressettings . Second,FST genetic distance matrices between populations while controlling by geographic distance, by conducting a partial Mantel test with PASSaGE version 2 software [The above mentioned previous study by Lao et al.  on genomsoftware . Since tp-value < 0.05) of the spatial autocorrelogram of each YHG was estimated with 999 permutations and Bonferroni correction was applied. This test was only applied to the Dutch dataset. Second, we estimated the linear relationship between the proportion of each YHG and geographic coordinates with a generalized linear model (GLM) using a binomial logit link in R with the “vegan” package. Bonferroni correction was applied on the resulting p-values for the correlation  +1 for not tested haplogroups)). Gradients that are significant before Bonferroni correction, but no longer after, will be referred to as marginally significant. This test was applied to both the Dutch and the combined dataset.To detect geographic patterns for individual YHGs, we applied two statistical methods on (sub)YHGs with a proportion of ≥1% (corresponding to a minimum of 20 samples in the Dutch dataset). First, we computed Moran’s I spatial autocorrelograms with binary weight tests for each YHG using PASSaGE version 2 software, with ten distance classes of equal width, assuming randomly distributed data and excluding the largest distance class. Statistical significance  and R1b-S116 Total .Data generated in this study have been uploaded to the public database YHRD under accession number YA002897 , 26.YHGs were manually assigned to all 2085 samples of the Dutch dataset. In total, 32 different YHGs were observed. YHGs I (28%) and R (62%) are by far the most common. Within YHG R, subgroups R1b-L48 (15%), R1b-M405 (14%), and R1b-S116 (9%) are the three most common ones   Fig. .Fig. 4a r value = 0.268, p-value = 0.002 after 999 permutations)  Fig. .FST between the two datasets after controlling by geographic distance .When comparing the new YHG data with the previous genome-wide autosomal SNP data from Lao et al. , we did The spatial analyses of individual YHGs identified departure from spatial randomness for several of them. The spatial autocorrelograms suggest statistically significant spatial trends compatible with a cline  after BoFrom all the YHGs for which prediction surface maps were created, only YHG R1b-M529 is more or less evenly distributed over the Netherlands. All other YHGs show more distinct patterns of distribution, either clinal or non-clinal and more patchy Figs. . The preThe map for YHG R1b-M405 shows a gradient that runs from the northeast to the southwest, while only a marginally significant westward gradient was observed with the GLM. For YHG R1b-L48 the map suggests a gradient in a northwestern direction, rather than the significant northward gradient as indicated by the GLM results. For YHGs R1b-S116 Total, R1b-S116, and R1b-U152, the maps show similar patterns and suggest more southeastwards gradients, rather than the southward gradients as suggested by the GLM results.Furthermore, the prediction surface maps suggest the presence of other complex patterns that could not be identified with the statistical analyses we performed, or were not significant, mostly due to low proportions. For YHG E1b-V13, we observe a depression-like pattern with highest proportions in the southwest and northeast and a band of lower proportions running from the northwest to the southeast. YHG J2-M172 shows a complex pattern with dispersed patches of higher and lower proportions. For R1a-SRY10831 we see two distinct areas of higher concentrations in the center and northeast of the country. YHG R1b-U198 seems to follow a depression-like pattern, somewhat similar to E1b-V13, with highest proportions in the southwest and northeast and a band of lowest proportioning the center. We see a more or less random distribution for R1b-L47, except for a distinct vertical band of low proportions in the northern half of the country and also rather distinct patches of high proportions in the northeast and center. Finally, the map for R1b-M167 indicates a clear concentration of low proportions in the southeast and increasing proportions towards the northwest.The Y-chromosomal genetic diversity present in the Dutch dataset showed clear spatial differentiation. What is remarkable, considering the small size of the Netherlands, is that additionally we see a non-random spatial pattern, either by formal testing or visually in the prediction surface maps, for nearly every YHG tested (≥1%). Roewer et al. already detected population substructure in the form of a division between two samples from the south of the Netherlands and three samples from the central-west and north. This was based, however, on YSTR data and could not be replicated with YSNP data from a smaller selection of that same sample set . The conThe population substructure and gradients for many of the individual YHGs we found in our study are in strong contrast with the apparent lack of genetic-geographic patterns for mtDNA data, as previously reported for a subset of the same sample set we used for our study . This coThe dissimilarity between the geographic-genetic patterns we observed for the Y-chromosome and the one found for genome-wide autosomal SNP data by Lao et al. , based oA solid explanation, however, for the differences in the geographic-genetic patterns between autosomal, mitochondrial, and Y-chromosomal SNP data requires additional data from past periods by means of ancient DNA and multidisciplinary research, including historians and archaeologists. Larmuseau et al. already hinted at the value of temporal analyses with several studies where they reconstructed historical patterns based on a combination of present-day samples and genealogical data and found strong indications for temporal fluctuations of YHG proportions , 8.When we additionally include the Flanders dataset in our analyses, we detect almost the same gradients with formal testing. This is not surprising since all but one of the significant gradients we observed in the Dutch dataset run in a (more or less) longitudinal direction and the inclusion of the Flanders dataset extends the territory mostly southwards. However, two exceptions concern YHGs R1b-M405 and R1b-M529. For R1b-M405, we detected a marginally significant westward gradient in the Dutch dataset, but no significant gradient in the combined dataset. This is a remarkable observation, since it is one of the three main subgroups in both the Dutch and Flanders datasets. For R1b-M529 we did not detect a significant gradient in the Dutch dataset, while we do detect a  significant south(west)ward gradient in the combined dataset.All together our results are in line with the findings of Larmuseau et al.  of signiThere are two other comparable examples of YSNP based population studies in the vicinity of the Netherlands; one for Germany and Poland by Kayser et al.  and one In the French study by Ramos-Louis et al. on samples from seven regions they reported no clear indications for population substructure, other than Bretagne being a genetic outlier . This inTo further put our results in a broader context, we compare the  significant YHG gradients in the Dutch dataset with published geographic distribution patterns in Europe. According to Rootsi et al.  YHG G-M2The  significant trends that are observed in our study in the Dutch dataset in most cases seem to more or less follow the European-wide trends for YHG distributions. The fact that our results resemble these European trends could be interpreted as the result of European-wide events. However, recent studies on both modern , 36 and In summary, our detailed analysis of YHG distribution in the Netherlands, based on 2085 geographically dispersed males resulted in an informative database. By combining several statistical methods, we have been able to detect Y-chromosome based genetic-geographic population substructure and significant gradients for several individual YHGs in the Netherlands, some of which extend to the south into the northern part of Belgium and eastward into Germany. These observations indicate the value of subtyping YHGs to a highly detailed level in micro geographic regions and add to the existing knowledge based on genome-wide data and mitochondrial data. It is therefore of great use as a reference in forensic casework in the Netherlands in relation to genetic ancestry and geographic origin assessments of suspects or unidentified victims. Moreover, the geographic patterns we observed, in addition to genome wide data, stress the importance of taking geographic origin into account in sampling strategies for control groups and comparing data from subpopulations in epidemiological studies.The discrepancies between patterns of population substructure of mitochondrial and Y-chromosomal DNA data point to different population histories for women and men in the Netherlands. However, solid explanations for the observed spatial patterns require further multidisciplinary research, including historians and archaeologists and detailed YHG data, similar to what we present here, from past Dutch populations via ancient DNA analysis.supplementary informationsupplemental table S4


On the Qinghai–Tibet Plateau, known as the roof ridge of the world, the yak is a precious cattle species that has been indispensable to the human beings living in this high-altitude area. However, the origin of domestication, dispersal route, and the divergence of domestic yaks from different areas are poorly understood.Here, we resequenced the genome of 91 domestic yak individuals from 31 populations and 1 wild yaks throughout China. Using a population genomics approach, we observed considerable genetic variation. Phylogenetic analysis suggested that the earliest domestications of yak occurred in the south-eastern QTP, followed by dispersal to the west QTP and northeast to SiChuang, Gansu, and Qinghai by two routes. Interestingly, we also found potential associations between the distribution of some breeds and historical trade routes such as the Silk Road and Tang-Tibet Ancient Road. Selective analysis identified 11 genes showing differentiation between domesticated and wild yaks and the potentially positively selected genes in each group were identified and compared among domesticated groups. We also detected an unbalanced pattern of introgression among domestic yak, wild yak, and Tibetan cattle.Our research revealed population genetic evidence for three groups of domestic yaks. In addition to providing genomic evidence for the domestication history of yaks, we identified potential selected genes and introgression, which provide a theoretical basis and resources for the selective breeding of superior characters and high-quality yak. The domestication of animals provided a stable source of food, labor, and hides, which played an important role in the lifestyle changes of humans from hunter gatherer to agricultural settlement . On the Previous studies have investigated yaks at archaeological –9, mitocCompared with other livestock, domestic yaks have a lower degree of domestication. Domestic yaks have a wide range of interactions with wild yaks and genetic exchange occurs and is hard to avoid. In addition, genetic exchange among domestic yaks from different areas occurs frequently because of human activities. The phenotypic and character differences of domestic yaks are not conspicuous. To examine intraspecific genetic diversification and geographical distributions of genetic lineages, we performed whole genome resequencing of 91 domestic yaks distributed throughout the QTP, from XiZang, QingHai, SiChuang, YunNan, GanSu, and XinJiang. Analyses of population genetics, selection, and demographic history built a database of genetic diversification resources for domestic yaks and revealed a series of interesting discoveries regarding their domestication and dispersal.We generated whole-genome sequences of 91 domestic yaks from 31 different locations and 1 wild yak from the QTP . The unusual olfactory might helped them to adapt to their environment and avoid predators. We also identified variations in gene regions specific to each group of domestic yaks. Although the differences among groups were not obvious, the group-specific variation at the genomic level was significative and will help to provide an insight into the relevant unique traits. In our samples, the most obvious phenotypic character divergence is the pure white hair specific to the Tianzhu group . We idenTo identify the genetic relationships between domestic yaks, we constructed a phylogenetic tree of yak SNPs using the neighbor-joining method with wild yaks as an outgroup Fig. . The resThe branch containing BQ, JC, JD, CT, ZD, and DQ separated first from wild yak. These areas are on the southeastern edge of the QTP, and, except for ZD, they are located at very similar latitudes. The Changdu area , at the center of these regions, was historically the populated area of the QTP. In conclusion, we suggest that the Changdu area was most likely the origin of the domesticated yaks and that they first dispersed to the east and west to BaQing and JingChuan, respectively.The second branch in the phylogenetic tree contained NR, GD, RD, SR, LZ, CN, ZB, SZ, SS,  and showed a small genetic distance from the first branch. These areas represent the core region of Tibet, including the central, west, and south of Tibet, suggesting that following successful domestication, yaks were gradually brought to the hinterland of the QTP. We defined this as the second dispersal of domestic yaks through the east of Tibet to the south and west.The third branch of phylogenetic tree contained XJ, JL, SB, LWQ, HH, GN, TZ, MW, DT, NY, GY, and BZ. Most of these are located at the edge of QTP in Qinghai, Gansu, Sichuang, Shanxi, Yunnan, and XinJiang. This suggested that the third dispersal of domestic yaks was from the center to the periphery of the plateau. Moreover, we found that the diversity of the third branch was higher than that of the other two branches. This is not only related to the hybridization of yaks in trade, but also to interspecies hybridization with wild yaks or cattle , 19, forΠ value for domestic groups was 0.93–1.2 × 10–3 was similar to that for the wild yaks (1.1 × 10–3), which is consistent with previous research [Π of group 3 and the lower θΠ of group 2 support the inferences related to trade and geographical enclosure. Linkage disequilibrium analysis suggested that the wild group exhibited a rapid decay rate and a low level of LD, whereas the group 3 yaks showed an overall slow decay rate and a high level of LD  and Bayesian model-based clustering analysis were employed to examine the phylogenetic groupings and provide any additional evidence. The PCA did not show significant separation among the samples of domestic yak, consistent with a single origin of domestication and the close genetic distance shown in the phylogenetic tree  and molybdenum ion binding (GO:0030151). Several disease pathways were enriched such as type I diabetes mellitus, graft-versus-host disease, rheumatoid arthritis, asthma, Leishmaniasis, and autoimmune thyroid disease   and 0.000856952 (group 2), but the widely geography range, disordered phylogeny of three sample from same region and the highest gene exchange among group 3 with Tibetan cattle, wild yaks and other domestic yaks didn’t support it as the origin of domestication. In contrast, the group 1 have an earlier divergence and higher genetic diversity than group 2. The inference of origin is consistent with the history of human transformation from a nomadic society to an agricultural society on the QTP, and is consistent with the previous suggestion through mitochondrial DNA by Guo et al. [In common, comparing to populations derived through subsequent migration colonization, the populations near to a centre of initial origin are expected to show higher haplotypic and nucleotide diversity as they maintain more ancestral variation. However, this genetic signature might be blurred by the recent gene flow after domestication. Group 3 had a highest θo et al. . In detaIn our result, we found the introgression from Tibetan cattle to yaks was far less than that from yaks to Tibetan cattle. It suggested that the successful adaptation of the cattle to the plateau environment depends on the gene introgression from yak. The introgression between Tibetan cattle and domestic yaks is more frequent than that between Tibetan cattle and wild yaks, we believe that this is related to domestication and breeding in that domestic yaks have more opportunities to exchange genes with Tibetan cattle. We also found the introgression from wild yaks into domestic yaks was far less than that from domestic yaks into wild yaks, this was related to the selective breeding of domestic yaks, however, the small population of wild yaks, the poorer living environment, and the bottleneck effect  could alIn the areas of introgression between domestic yak and wild yak, we found 2608 (domestic to wild) and 307 genes (wild to domestic). These two sets of genes showed similar pathway enrichment results  sampled in 32 different regions of the Qinghai-Tibet Plateau. Pair-end libraries were constructed with insert sizes of 500 bp and sequenced on an Illumina Hiseq 2000 Sequencer . In addition, previously published resequencing data from 17 wild yak samples were downloaded from NCBI and used in this analysis . To perf2 between any two loci using PopLDdecay [2 value was calculated for pairwise markers in a 500-kb window and averaged across the whole genome. The demographic history of yaks from geographically diverse populations was inferred using a hidden Markov model approach as implemented in pairwise sequentially Markovian coalescent (PSMC) analysis [An individual-based Neighbor-Joining phylogenetic tree was constructed from the population scale SNPs using TreeBest  softwarepLDdecay . The aveanalysis  based onFor each group, FST values in the top 0.1% region were taken to indicate significant selection and FST values in the top 1% were taken to indicate selected regions. To detect regions with significant signatures of selective sweep, we considered the distribution of the θ π ratios  and FST values. We used an empirical procedure and selected windows simultaneously with significant low and high θ π ratios (the 2% left and right tails) and significant high FST values (the 2% right tail) of the empirical distribution as regions with strong selective sweep signals along the genome, which should harbor genes that underwent a selective sweep. Protein-coding genes in these outlier windows were treated as candidate positively selected genes. Moreover, EigenGWAS  was usedFor a comprehensive analysis of introgression in yaks, we added data from 8 Tibetan cattle to the population SNP dataset through the same GATK pipeline. The combined VCF dataset was phased using read-aware phasing with SHAPEIT v2.r904 –52. SubsAdditional file 1. Additional Figures and Tables. Figures S1–S10, Tables S1–S8 and S10–S13: additional data to support the manuscript (see text for references).Additional file 2: Table S9. The introgression statistics of each individual.


Discrete breed ideals are not restricted to delimiting dog breeds from another, but also are key drivers of subpopulation differentiation. As genetic differentiation due to population fragmentation results in increased rates of inbreeding and loss of genetic diversity, detecting and alleviating the reasons of population fragmentation can provide effective tools for the maintenance of healthy dog breeds.Using a genome-wide SNP array, we detected genetic differentiation to subpopulations in six breeds, Belgian Shepherd, English Greyhound, Finnish Lapphund, Italian Greyhound, Labrador Retriever and Shetland Sheepdog, either due to geographical isolation or as a result of differential breeding strategies. The subpopulation differentiation was strongest in show dog lineages.Besides geographical differentiation caused by founder effect and lack of gene flow, selection on champion looks or restricted pedigrees is a strong driver of population fragmentation. Artificial barriers for gene flow between the different subpopulations should be recognized, their necessity evaluated critically and perhaps abolished in order to maintain genetic diversity within a breed. Subpopulation differentiation might also result in false positive signals in genome-wide association studies of different traits.Purebred dogs are, by definition, reproductively isolated from other breeds. However, similar isolation can also occur within a breed due to conflicting breeder ideals and geographic distances between the dog populations. We show here that both of these examples can contribute to breed division, with subsequent loss of genetic variation in the resulting breed lineages. Breeders should avoid creating unnecessary boundaries between breed lineages and facilitate the exchange of dogs between countries. Canis familiaris L.) are the oldest domesticated animals, comprising of more than 400 highly diverse contemporary breeds [breeds originates from the Victorian era and current breeding practices largely still reflect this thinking [Domestic dogs , we show here that all of the studied breeds show subpopulation differentiation, either by their geographical origin, selection for performance or morphological traits. We conclude that the subpopulation differentiation should be taken into account in the breeding programs of the studied breeds to conserve their genetic diversity.We analyzed the genetic differentiation in Belgian Shepherd, English Greyhound, Italian Greyhound, Finnish Lapphund, Labrador Retriever and Shetland Sheepdog using multi-dimensional scaling (MDS)  and STRUFST and lower heterozygosity (Hz) values . In essges Fig. b, d. SimFST can be seen as a measure of an increase in the “quality” genes. In these cases, mixture of differentiated subpopulations could create an inferior outcome, similar to the decay of locally adapted allelic combinations known to occur in the wild, also known as outbreeding depression [The loss of genetic diversity can pose a threat to breed health , 26–29. pression  or migrapression . HoweverKnowledge of the genetic differentiation of populations and gene flow between them not only offers insights into breed history and current breeding practices, but also highlights how subpopulations might most beneficially be used for the maintenance or restoration of genetic diversity. Optimally the breeding of desired traits should aim to mimic the natural selection operating in wild populations, selecting uniformity for some parts of the genome, while maintaining diversity elsewhere , 33. TheOur results show that geographical isolation and founder effect are the main drivers of subpopulation differentiation among Italian Greyhounds and Shetland Sheepdogs, whereas selection for show and sports or working lineages explained the genetic structure among English Greyhounds and Labrador Retrievers. The genotyping analysis could also detect the predicted breed type structure in Belgian Shepherds and revealed unexpected, relatively recent split in the Finnish Lapphund population due to breeder preferences. Our study exemplifies the use of genetic data in detecting population structure among dogs to identify and measure the degree of subpopulation differentiation. The findings could be then applied in breeding programs to facilitate gene flow between isolated subpopulations, as well as to raise awareness of practices that are potentially harmful for the maintenance of genetic diversity within a breed. Responsible dog breeding requires careful balancing between the selection for the desired traits and health, while avoiding excess loss of genetic diversity due to drift.The Belgian Shepherd, English Greyhound, Italian Greyhound, Labrador Retriever, Finnish Lapphund and Shetland Sheepdog breeds were selected for the study as they were known or suspected to have either distinct breed varieties (Belgian Shepherd) or differential breeding practices depending on the intended use of the dogs Table . Breeds Dogs included in the study were sampled as a part of submission for routine commercial screening provided by Genoscoper Laboratories Oy, Helsinki, Finland . Additional information on country of origin, breed variety, and owner-reported characterization of the dog’s use  was collected. The pedigree background of the Finnish Lapphund samples was validated manually based on publicly available breed registry information, assigning the dogs into subpopulations depending on whether their breeder adhered to the herding lineage requirements set forth by the breed community or not.Genetic analyses were carried out on DNA extracted from owner-collected, non-invasive buccal swab samples. All dog owners provided consent for the use of their dog’s DNA sample for research purposes.The genotypes were obtained using a custom-designed Illumina Infinium  genotyping microarray for 1319 neutral SNPs, evenly distributed across the 39 canine chromosome pairs  with a mFST values were calculated based on all individuals using ARLEQUIN v.3.5 [https://mydogdna.com/ by searching for the breed of interest and choosing the “genetic relationships” tab. The population structures were analyzed using STRUCTURE v.2.3.4. with 5000 MCMC repetitions and burn-in period of 5000 [Samples included in this study reached a call rate of at least 95% of the analyzed markers. The median heterozygosities, basic population genetic parameters and IN v.3.5 . The genIN v.3.5 , which i of 5000 , 37. The of 5000 . The obtAdditional file 1.


Seed dispersal is crucial to gene flow among plant populations. Although the effects of geographic distance and barriers to gene flow are well studied in many systems, it is unclear how seed dispersal mediates gene flow in conjunction with interacting effects of geographic distance and barriers. To test whether distinct seed dispersal modes  have a consistent effect on the level of genetic connectivity  among populations of riverine plant species, we used unlinked single-nucleotide polymorphisms (SNPs) for eight co-distributed plant species sampled across the Rio Branco, a putative biogeographic barrier in the Amazon basin. We found that animal-dispersed plant species exhibited higher levels of genetic diversity and lack of inbreeding as a result of the stronger genetic connectivity than plant species whose seeds are dispersed by water or wind. Interestingly, our results also indicated that the Rio Branco facilitates gene dispersal for all plant species analyzed, irrespective of their mode of dispersal. Even at a small spatial scale, our findings suggest that ecology rather than geography play a key role in shaping the evolutionary history of plants in the Amazon basin. These results may help improve conservation and management policies in Amazonian riparian forests, where degradation and deforestation rates are high. FST; The proportion of total genetic variability that resides among populations  have a consistent effect on the level of historical and contemporary  genetic connectivity among populations of riverine plant species . We alsoAn adequate understanding of gene flow patterns and genetic diversity in the Amazon can help guide effective conservation and management policies  of riverThe Rio Branco is a medium-sized river that has been used as a model system in various studies due to its size. This is due to the fact that narrow and wide rivers tend to affect gene flow in various taxa in the same way, regardless of their dispersal modes. The Rio Branco in particular has been shown to represent an important barrier to gene flow for birds e.g.,  and primAdenocalymma schomburgkii (DC.) L.G. Lohmann (Bignoniaceae), Amphirrhox longifolia (A. St.-Hil.) Spreng. (Violaceae), Anemopaegma paraense Bureau & K. Schum (Bignoniaceae), Bignonia aequinoctialis L. (Bignoniaceae), Pachyptera kerere (Aubl.) Sandwith (Bignoniaceae), Passiflora spinosa (Poepp. & Endl.) Mast. (Passifloraceae), Psychotria lupulina Benth. (Rubiaceae), and Tanaecium pyramidatum (Rich.) L.G. Lohmann (Bignoniaceae). These species were chosen due to their varied dispersal modes  and combined this information with data available from the same location for A. longifolia, P. spinosa, and P. lupulina  on population structure, while considering distinct dispersal modes. Toward this end, we collected new genomic data for five plant species  is the largest tributary of the Rio Negro; it is located on the Guiana Shield, within a sediment-rich “white-water” river ecoregion, and flows southward into the Negro-Amazon Rivers , A. longifolia (n = 78), A. paraense (n = 84), B. aequinoctialis (n = 78), P. kerere (n = 84), P. spinosa (n = 78), P. lupulina (n = 78), and T. pyramidatum (n = 78). Those individuals were distributed among 14 sampling locations situated on both banks (left and right) of the Rio Branco during the wet seasons of 2015 and 2016  while reducing the number of samples randomly, from six to two using a custom script in R (described by A. schomburgkii an FST (n = 6) = 0.014, 95% CI  compared with an FST (n = 2) = 0.013, 95% CI , for A. paraense an FST (n = 6) = 0.015, 95% CI  compared with an FST (n = 2) = 0.022, 95% CI , for B. aequinoctialis an FST (n = 6) = 0.052, 95% CI  compared with an FST (n = 2) = 0.051, 95% CI , for P. kerere an FST (n = 6) = 0.022, 95% CI  compared with an FST (n = 2) = 0.010, 95% CI , and for T. pyramidatum an FST (n = 6) = 0.032, 95% CI  compared with an FST (n = 2) = 0.033, 95% CI .For each sampling location, sample sizes ranged from two to six individuals . The comngifolia , P. spinlupulina . To ensuA. schomburgkii, A. paraense, B. aequinoctialis, P. kerere, and T. pyramidatum. The genomic libraries were created using a double digest RADseq (ddRAD) protocol . The genomic libraries were sequenced on Illumina HiSeq 2500 flow cell  to generate single-end 100 bp reads at The Centre for Applied Genomics, Toronto, Canada. More details of the protocol can be found in A. longifolia, P. spinosa, and P. lupulina  and reads missing the expected EcoRI cut site (options –barcode_dist 2 -q -e ecoRI). All sequences were processed in ustacks to produce consensus sequences of RAD tags. The program ustacks takes a set of short-read sequences from a single sample as input and aligns them into exactly matching stacks. For each species, a maximum-likelihood framework (m) to create a stack was set at three sequences, the maximum distance allowed between stacks was two nucleotides, and the maximum number of stacks allowed per de novo locus was three. The stacks assembly enabled the Deleveraging algorithm (-d), which resolves overmerged tags, and the Removal algorithm (-r), which drops highly repetitive stacks and nearby errors from the algorithm. The alpha value for the SNP model was set at 0.05. As reported by Files containing the raw sequence reads were analyzed separately for each plant species in Stacks 1.35  using deramework  was applk tests using the sequential Bonferroni procedure  and the inbreeding coefficient (Wright’s Fixation Index F) for each population. Population genetic statistics were averaged across loci using the R package diveRsity , we performed Pearson product-moment correlation test of uHE and HO with the geographic distance along the river course using the package ggpubr implemented in R 3.3.1 , we analyzed the five abiotically dispersed plant species for which new data was obtained using the methods described in In order to investigate how the river affects the population structure of plant species with different seed dispersal modes , we assessed the historical and contemporary genetic structure and connectivity of 1.We assessed the genetic structure and the connectivity patterns between sampling locations along and across the river using complementary genetic analyses. For all plant species, we investigated the historical relationships among sampling locations using RAxML 8.2  in post-processing. As the most likely number of k populations was unknown, it was treated as a simulated variable along with the MCMC simulations (1 ≤ k ≤ N), where N represents the number of sampling locations for each plant species, which ranged from 13  to 14 . The modal number of genetic groups of the best run  was considered as the number of genetic clusters (K).To more precisely characterize the geographic distribution of genetic variation, we applied a Bayesian model that uses a Markov Chain Monte Carlo (MCMC), as implemented in the R package GENELAND 4.0.6 . BayesiaFST; To investigate whether species with different dispersal modes displayed congruent patterns of genetic structure, we estimated pairwise genetic differentiation FST;  using thFST/(1 – FST); 4 permutation test of significance for the coefficient of correlation. A Mantel test was also applied to pairs of sampling locations situated (1) across river banks and (2) along each bank of the Rio Branco.In order to investigate the similarity between genetic and geographic distance, we conducted a test for isolation by distance (IBD)  between 4 random permutations.Finally, to examine the effect of the Rio Branco on the partitioning of genetic variation among sampling locations, we performed a Molecular Analysis of Variance AMOVA; . We defim) using a Bayesian inference framework implemented in BayesAss3-SNPs 1.1 (n) of six individuals per sampling location  of six, four, and two individuals in two populations. The results indicated that there are no differences in the migration rates (m) even when a small number of individuals  are used in the analysis .We obtained estimates of contemporary bidirectional migration rates  did not vary much among populations of A. schomburgkii, A. longifolia, A. paraense, B. aequinoctialis, P. kerere, P. spinosa, P. lupulina, and T. pyramidatum  to 0.376 (P. lupulina), while the mean observed heterozygosity ranged from 0.113  to 0.410  to 0.025 ± 0.007 95% CI (A. schomburgkii). Due to the excess of observed heterozygotes, the mean fixation index was negative for all animal-dispersed plant species  for A. schomburgkii, 0.015  for A. paraense, 0.052  for B. aequinoctialis, 0.025  for P. kerere, and 0.033  T. pyramidatum. For the three animal-dispersed plant species the average pairwise estimate of FST was 0.021  for A. longifolia, 0.031  for P. spinosa, and 0.031  for P. lupulina . Results were also not significant when applied between pairs of sampling locations across river banks . Likewise, results of simple matrix correlation between genetic and geographic distance were insignificant when applied separately along each bank of the Rio Branco , but significant congruence between water- and wind-dispersed plant species . These results indicated that the biotically dispersed plant species are more connected in the Rio Branco. All these findings were consistent when different datasets  were considered  and 3L , while the expected proportion of migrants from 4L into 2L , and 3L  were much smaller. Asymmetrical migration rates were also detected among some pairs of sampling locations for the animal-dispersed plant species A. longifolia, P. spinosa, and P. lupulina . For P. kerere, exclusively, we found a significant and positive association between contemporary migration rates and geographic distance, although this association was restricted to the right river bank of the Rio Branco  were observed for all but one plant species . Howevero Branco . This reLife-history traits, such as dispersal mode/ability can have an effect on the species’ response to geographical and evolutionary processes e.g., , with coA. schomburgkii, A. paraense, B. aequinoctialis, P. kerere, and T. pyramidatum) and those reported for three animal-dispersed plant species  shape the genetic structure of shrub plant species with different seed dispersal modes.The population structure observed for the five abiotically dispersed plant species studied here  in this large-sized river  and P. lupulina  showed higher levels of genetic variation  than A. longifolia. The effectiveness of seed dispersion in A. longifolia – a plant species with a mixed seed dispersal mode  – can be influenced by flood dynamics throughout the year, which may be more restricted in periods of non-flooding, especially if the plant is located far from the river banks. Thus, the contribution of ballistic dispersal may have a negative effect on the levels of intrapopulation genetic diversity in A. longifolia. Indeed, animal-dispersed plant species tend to have higher levels of genetic diversity than ballistic-dispersed plant species , life-history traits such as pollination mode and growth form  these results can help guide conservation and restoration policies in Amazonian riparian forests areas, where degradation and deforestation rates are rising . Yet, siAmphirrhox longifolia, Passiflora spinosa, and Psychotria lupulina populations are available for download from the Dryad Digital Repository (https://doi.org/10.5061/dryad.f53j0dk). For Adenocalymma schomburgkii, Anemopaegma paraense, Bignonia aequinoctialis, Pachyptera kerere, and Tanaecium pyramidatum populations, the SNP datasets are available for download at https://doi.org/10.5061/dryad.573n5tb6c.SNP datasets for the AGN and LGL designed the study and coordinated sample collection. AGN conducted molecular work, performed analyses, and led the writing of the manuscript with input from all co-authors. LLK provided analytical support and troubleshooting. CWD provided laboratory assistance. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Bos indicus) and identified new alleles and haplotypes. Compared to other indicine studies, the high Y-haplotype diversity found in Indian cattle supports the hypothesis of greater genetic variability across the centre of origin decreasing along migratory routes with increasing distance. Hence, a considerable paternal genetic diversity of Indian cattle appears to have been lost in transboundary commercial indicine breeds. The Khillar and Gir are the most diversified populations where the first tends to be the well-differentiated traditional breed carrying strikingly distinct Y-lineages with typical BM861-158 bp allele, characteristics of taurine cattle, while retaining standard indicine lineages for all other markers. Geographical distribution found to be an unreliable predictor of parental variation, and Y-lineages seemed closely related to Indian breed function/utility. The comprehensive Y-chromosome information will be useful to examine the demographic expansion/spread of Bos indicus lineages from close proximity to the domestication centre across different countries worldwide and such diversity should be preserved through effective management and conservation programs.Y-chromosome genetic diversity in and around its domestication origin and a better understanding of indicine-specific microsatellite alleles are imperative concerns but less -targeted. We analysed Y-chromosome markers in 301 bulls representing 19 native Indian cattle ( B.p. primigenius in Southwest Asia and B.p. namadicus in India are the ancestors of taurine (Bos taurus) and zebu (Bos indicus) cattle, respectively1. Around 2000 years after the taurine cattle domestication, zebu cattle were domesticated in the Indus Valley at the edge of the Indian Desert3. Later, pastoralist migrations from the centre of domestication introduced zebu cattle across the East Asia, southwestern Asia, Europe and African countries between 4000 and 1300 YBP, leading to admixture between zebu and taurine populations4. Over the past 150 years zebu cattle have reached America and Oceania, contributing to the growth of the developing economies6.The wild aurochs subspecies 7. The Y chromosome diversity found in indicus cattle is represented by a single haplogroup (Y3) and variability within this haplogroup makes it possible to differentiate paternal lineages between Indian and Chinese cattle8 and to recognize the presence of Y3b haplotype family of Indian origin exclusively in West African Zebu animal9. The Y-specific microsatellite loci have been studied in several cattle breeds from different geographical areas13 or local breeds such as Ethiopian cattle14, Portuguese cattle15, Spanish cattle16 and Polish cattle17. The in-depth diversity analyses based on paternal lineages have allowed the identification of several Y-haplotypes within the major haplogroups. Generally, Y chromosome phylogenetic surveys of Indicine cattle include only a few breeds and have focused on taurine and zebuine crosses20. In this context, Bos indicus Y-chromosome diversity has not yet been extensively analyzed particularly close to their domestication centre.The identification of five single nucleotide polymorphisms (SNPs) has permitted the classification of worldwide extant cattle breeds into three Y-chromosome haplogroups, named Y1, Y2 and Y3Bos indicus were domesticated, has rich cattle genetic resources with 50 well defined breeds (http://www.nbagr.res.in) classified according to their utility as dairy, draft and dual. Analysis of autosomal microsatellites (STRs) and mitochondrial DNA (mtDNA) sequences revealed a considerably high genetic diversity within Indian native cattle breeds24. However, Y-chromosome diversity of these native cattle breeds is yet to be explored. The analysis of Y chromosome variations will help to infer the origin and genetic relationships of Indian cattle breeds, generate comprehensive information about Bos indicus Y-chromosome haplotype diversity in close proximity to the centre of domestication and shed light over demographic expansion/spread of B. indicus lineages worldwide. Moreover, investigation of Y-chromosome haplotypes and patriline diversity could complement previous studies based on autosomal markers and help in defining conservation priorities. The aim of this study was therefore to investigate haplotype diversity and relationships among Indian native cattle breeds using a combination of SNPs and STRs specific to the non-recombinant region of the Y chromosome.India, where B. indicus bulls were found to be restricted to the Y3 haplogroup. PCR specific to USP9Y resulted a 362 bp fragment for Y1 and a 443 bp fragment for Y2 and Y3. The Ssp I enzyme cleaved the Y3 into two distinct fragments of 337 bp and 107 bp, allowing to differentiate Y3 from Y2  in the same sequencing run. In the combine run of B. indicus and B. taurus samples, GeneMapper profile of present study revealed INRA189-100 and INRA189-106 bp alleles in Holstein Friesian and Jersey, respectively. On the other hand, DDX3Y1 amplification revealed a 249 bp allele in B. indicus and 253 bp allele in all the Holstein Friesian, Jersey and HF crossbred bulls. The GeneMapper profile above showed that the allele sizing was correct, but in the present study, the allele codes for INRA189 and DDX3Y1 STRs were off by 2 and 4 bp, respectively. This means that INRA189-90 allele in fact corresponds to previously published allele 8826, making it the most frequent allele for this marker in B. indicus cattle. Likewise, DDX3Y1 corresponds to previously published allele 245 and found to be monomorphic in all the indicine breeds. For the remaining loci, allele codes appeared to be matching published data. A total of 18 alleles were observed   in 301 bulls representing 19 Indian cattle breeds  and five polymorphic Y-chromosome microsatellites  identified 17 ST values are presented in the Supplementary Table ST value is the measure of potential frequency of heterozygotes if all members in a population are randomly mixed. Kangayam was the most and Tharparkar was the least differentiated breeds under study with average FST values (±standard deviation) of 0.735 ± 0.243 and 0.302 ± 0.183, respectively. Among dairy breeds, Sahiwal (0.646 ± 0.374) was the most differentiated and Thaparkar (0.302 ± 0.183) was the least. The most and least differentiated draft breeds were Kangayam (0.735 ± 0.243) and Malnad Gidda (0.347 ± 0.173), respectively. Among dual-purpose breeds, Hariana (0.640 ± 0.327) was the most diverse and Mewati (0.502 ± 0.276) was the least.Pairwise FST represented in two dimensional space with multi-dimensional scaling resulted in 3 distinct clusters as shown in Fig. Group 1: Gir, Sahiwal, Ongole, Punganur, Rathi, Vechur and Tharparkar; Group 2: Kankrej, Mewati, Nagori and Hariana; Group 3: Malnad Gidda, Krishna Valley, Red Sindhi, Kherigarh, Dangi, Khillar, Nimari and Kangayam. The dairy breeds clustered together along with dual purpose breed Ongole and miniature dairy breeds Vechur and Punganur. On the other hand, the draft breeds were clustered together along with Red Sindhi , Kankrej (Guzerat) and Gir (Gyr) bulls reached the Americas 150 YBP2 and had been extensively used in European B. taurus cows, especially Creole breeds, followed by repeated backcrossing to B. indicus bulls. Therefore, as observed in our study, the most common Y-haplotypes (H18Y3 and H9Y3) are now widely present in international transboundary indicine breeds, Creole cattle and indigenous African cattle27 , commonly seen in many Bos indicus29. Khillar appears to be a typical breed with very high frequency BM861-158 bp allele, a clear case of size homoplasy30. In addition, it does not even seem to be related to taurine animals, as all haplotypes are Y3.The new alleles identified in indicine bulls of Indian origin are BM861-158 bp; UMN0307-155 bp; UMN0504-148; UMN0504-150 bp; UMN0103-114/116 and UMN0103-124/124. The UMN0103 STR consistently revealed two alleles in all the bull samples, comparable to previous studies on 17, Lidia bovine breeds (0.42)16, Continental Europe (0.543 ± 0.026) and Zebu cattle (0.389 ± 0.084)27; but relatively lower than Ethiopian cattle (0.751 ± 0.015)14, Creoles (0.779 ± 0.019), Iberian breeds (0.712 ± 0.016) and cattle of Atlantic Islands (0.677 ± 0.037)27. Haplotype diversity within lineages as well as retention of allelic variants by various breeds act as a genetic reservoir. Nevertheless, Bos indicus Y-chromosome diversity is observed to be far less than that of humans (>0.89)31. This disparity is possibly due to the comparatively reduced effective male population size of cattle breeds. Use of artificial insemination (AI) in breeds like Sahiwal, Gir and Ongole as well as use of limited number of bulls in other breeds (Vechur and Hariana) may be associated with their reduced Y-haplotype diversity. Restricted use of males32 as well as extensive use of AI15 have been reported to be the key reasons for reducing the number of patrilines in horse and cattle, respectively, based on Y-chromosome polymorphism.The overall Y haplotype diversity (0.630 ± 0.027) observed in Indian cattle is reasonably higher than previously reported estimates in Polish cattle (0.037 ± 0.019)Bos indicus. It supports the expectation of greater genetic diversity surrounding centres of origin which declines with distance from the primary site of domestication33. Comparatively lower Y-chromosome diversity has recently been reported in transboundary indicine cattle13. The autosomal microsatellite based diversity analyses have also revealed high allelic diversity and heterozygosity in Indian native cattle breeds34. Overall, the diversity in Indian cattle was found to be higher than that of their European counterparts35, and might be attributed to lack of artificial selection pressure, relatively larger effective population size, as well as proximity to the domestication centre especially in the case of Bos indicus. Genome-wide survey of SNPs also revealed a much larger ancestral population (pre-domestication Ne) of indicine breeds originated in Southern Asia compared to modern taurine cattle35.The present study demonstrates the existence of high Y-chromosome diversity in ST value over all B. indicus breeds (0.490 ± 0.106) shows that a considerable amount of variation is explained by breed differences. The present analysis finds no geographical grouping of Indian cattle breeds. Similar trends have also been recorded in Portuguese cattle breeds15, with contrasting results in dog36, sheep28 and human31. However, contrary to the present findings, geographical clustering of Y chromosome variability has been described for several species showing male-mediated dispersal where differences among population groups are further augmented by genetic drift37. Grouping of breeds in the present study tends to follow the division of dairy, draft or dual. An explanation for the clustering would be their common paternal roots. The presence of haplotype H5Y3 with a comparatively higher frequency in Kangayam and Nimari positions them far apart from other breeds in the draft category  of India were included  in a 2.0 ml eppendorf tube after thawing. Sample was washed with 70% ethanol and centrifuged at 13000 rpm for 5 min. Supernatant was discarded without disturbing the cell pellet.750 µl of lysis buffer  was added to the cell pellet followed by addition of 3.75 µl Triton X-100, 31.5 µl of DTT and 15 µl proteinase-K (20 mg/ml). The contents were mixed well for 5 min and kept at 50 °C in a water bath overnight. Later, digested content was cooled down to room temperature and approximately an equal volume of Phenol: Chloroform: Isoamyl alcohol (650 µl of PCI per 500 µl of sample), was added. The content was mixed gently and centrifuged at 13000 rpm for 5 min. Aqueous phase was separated and transferred to a clean eppendorf tube (2.0 ml) followed by addition of 1.0 ml Chloroform and Isoamyl alcohol (24:1). The contents were mixed gently and centrifuged at 13000 rpm for 5 min. The uppermost aqueous phase containing DNA was separated without disturbing the middle and lower phases.th volume of sodium acetate was added followed by addition of equal volume of chilled absolute ethanol, to precipitate the DNA. The precipitated DNA pellet was washed 2–3 times with 70% ethanol by centrifugation at 5000 rpm for 5 min. Discarded the supernatant and air dried the pellet carefully avoiding over dryness while leaving no traces of ethanol. Later, the pellet was dissolved in 100 µl Milli-Q water. The concentration of DNA was checked with Nanodrop spectrophotometer . The quality of DNA was verified on 0.8% agarose gels.1/107, were analysed by allele-specific PCR (AS-PCR) protocols40. ZFY9 (C/T) and DDX3Y1 (C/T) were genotyped to differentiate zebu (Y3) and taurine specific (Y1 and Y2) Y-haplogroups; whereas, UTY19 (C/A) was analysed to distinguish between Y1 and Y2. Y-haplogroups were further confirmed by PCR-RFLP of USP9Y marker41 using newly designed primers and sequencing representative samples. Based on GeneBank sequences , new primers were designed. (a) Forward primer USP9YF: 5′ GGG GCT TAG AGT GCT CCA GT 3′, (b) Reverse primer USP9YR: 5′ ACA GCT CCT CAA AAC CAG AAT 3′. The standardized PCR protocol was as follows: Initial denaturation at 95 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s, extension at 72 °C for 30 s and the final extension at 72 °C for 10 min. PCR products of 10 µL were subjected to restriction endonuclease enzyme digestion by Ssp I enzyme (5U per reaction) at 37 °C for 3 hours. The products (5 µL) were later subjected to agarose gel (2%) electrophoresis and the samples were grouped based on the bands obtained.Three single nucleotide polymorphisms 2, 200 µM dNTPs, 0.25 µM of each forward and reverse primer, 1U of Taq DNA polymerase and approximately 50 ng of genomic DNA in a total volume of 15 µl by adding molecular grade water. The standardized thermocycling protocol for each microsatellite used to screen the haplotypes was as follows: initial denaturation at 95 °C for 5 min, following 35 cycles of denaturation at 94 °C for 30 s, annealing at 58/62 °C , Jersey (n = 25) and HF crossbred (n = 10) bulls were genotyped to verify allele sizing and correct allele code assignment.Six STRs , specific to the non-recombining region of Y-chromosome, were genotyped. Detailed information of primers is presented in Supplementary Table 42. Frequencies of Y chromosome haplotypes were then used to compute the haplotype diversity (H) for each breed and pairwise FST genetic distances with 10,000 permutations at 5% level of significance using ARLEQUIN v3.543. The pairwise FST values were represented in two-dimensional space with multi-dimensional scaling in the SPSS version 24.0 software package. Analysis of Molecular Variance (AMOVA) was done with ARLEQUIN v3.5 without subdivision into groups, as well as partitioning the total genetic variation through grouping the native breeds as follows: by geographic region as Northern , Southern  and Western ; by breed utility characteristics such as dairy , draft  and dual ; and by MDS grouping . Vechur and Punganur are miniature dairy cattle breeds of India (http://www.nbagr.res.in). Significance levels for the estimated fixation indices were obtained by comparison of the actual values with 10,000 permutations. Phylogenetic relationships among haplotypes were investigated using a median-joining network (M-J) implemented in NETWORK v4.2.0.1 44. Reduced-median joining analysis was performed first and the output was then used as input for the Median-Joining calculations. As in previously published studies, UMN0103 was divided into A & B because of having two alleles/loci with variable sizes specific to Bos indicus. Locus specific weights were given proportional to their within-breed variance component. Accordingly, polymorphic loci with highest variance were given the lowest weight14. Such weights were intended to account for the differences in genetic variation of different microsatellite loci12. Hence, STRs BM861, INRA189, UMN0103A, UMN0103B, UMN0307, UMN0504 were given weights of 7, 9, 5, 4, 6 and 8, respectively. However, for major haplotypes the proportional change in weights allocated to the loci showed no noticeable change in the network structure. The principal components analysis was performed by using Y-haplotype frequencies. The R package “factoextra” was used to compute the eigenvalues and contribution of the PCs to the total genetic variation. The different graphs and plots were generated representing the contribution of the loci and individuals to the total genetic variation.Microsatellite data was analysed for estimation of haplotype frequencies in different breeds using GenAlEx 6.5http://cpcsea.nic.in/WriteReadData/userfiles/file/Compendium%20of%20CPCSEA.pdf) and approved by the Institutional Animal Ethics Committee (IAEC) of ICAR-National Bureau of Animal Genetics Resources (ICAR-NBAGR), Karnal.Semen samples, routinely collected by various semen stations in the country following standard procedures were incorporated. The frozen semen straws thus obtained were not directly related to our research project. Cow blood samples were obtained with written informed consent from the owner during veterinary inspections under the official health care program (fertility camp) by a qualified veterinarian in compliance with the relevant guidelines issued by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA; Supplementary information.Supplementary information 2.


Aphelocheirus aestivalis (Aphelocheiridae) is widely distributed in Europe but occurs rather locally and often in isolated populations. Moreover, it is threatened with extinction in parts of its range. Unfortunately, little is known about the genetic diversity and population structure due to the lack of molecular tools for this species. Thus, to overcome the limitations, a whole-genome sequencing has been performed to identify polymorphic microsatellite markers for A. aestivalis. The whole-genome sequencing has been performed with the Illumina MiSeq platform. Obtained paired-end reads were processed and overlapped into 2,378,426 sequences, and the subset of 267 sequences containing microsatellite motifs were then used for in silico primer designing. Finally, 56 microsatellite markers were determined and 34 of them were polymorphic. Analyses performed in two samples  showed that the number of alleles per locus ranged from 2 to 21, and the observed and expected heterozygosity varied from 0 to 0.933 and 0.064 to 0.931, respectively. The microsatellite markers developed in the present study provide new suitable tools available for the scientific community to study A. aestivalis population dynamics. The assessment of its genetic diversity and population structure will provide important data, that can be used in population management and conservation efforts, elucidating the broad- and fine-scale population genetic structure of A. aestivalis.The freshwater true bug  Aphelocheirus aestivalis (Fabricius 1794) and two endemic congeneric species from the Iberian Peninsula, A. murcius (Nieser and Millán 1989) and A. occidentalis (Nieser and Millán 1989) [A. aestivalis is distributed throughout Europe [A. aestivalis occurring in Europe are predominantly micropterous [A. aestivalis disperses mainly through the watercourses [The benthic freshwater true bug t Europe , where lt Europe , 4. The opterous . Due to opterous , it has rcourses . The remrcourses  and the rcourses .A. aestivalis is relatively tolerant of changes in some physical and chemical characteristics of water, it is mainly threatened by industrial and agricultural pollution and the regulation of watercourses [A. aestivalis is considered an endangered species and is included in local and national Red Lists or Red Data Books [A. aestivalis and its environmental requirements  according to the manufacturer’s protocol. Genetic material was extracted from the abdominal tissue of all collected individuals.Total genomic DNA isolated from four individuals collected from different populations  was pooled and used for library preparation using the TNEBNext® DNA Library Prep Master Mix Set for Illumina® (New England BioLabs Inc.). The sequencing was performed with an Illumina MiSeq sequencer at Genomed . A 2 × 250 base pair (bp) read mode was used during sequencing. The obtained sequences were trimmed and filtered with the Cutadapt v.1.12 software package .The obtained sequences (2.4 Mega reads) were used for microsatellites detection with QDD v.3.1.2 . PrimersTaq ReadyMix , 0.4 µM of forward and reverse primers, 0.3 µM of fluorescently labeled  M13 primer and ~100 ng of DNA. The following PCR conditions were used: 1 min initial denaturation at 94°C, followed by 30 cycles of 94 °C for 30 s, gradient temperature for 30 s (range from 52 to 65 °C), and 72 °C for 1 min. Since the M13 primer needs a 53 °C annealing temperature, eight final cycles were added at the end of the PCR cycles to allow the annealing of the M13 primer with the previously formed amplicons . At the end was 7 min final extension at 72 °C. The amplification products were separated by 2% agarose gel electrophoresis in a 1 × SB buffer and visualized with SimplySafeTM  in UV light.All forward primers were tagged with M13(-21) (5′-TGTAAAACGACGGCCAGT-3′) tails at the 5′ end . PCR reaA. aestivalis individuals collected from seven sampling sites from Central and Western Europe (Poland: specimens from Brda (BR), Słopica (DS), Drawa (DR), Stary Potok (SP) rivers, and two sampling sites from Krąpiel river , and Portugal: specimens from Corgo river (CR); two individuals per sample;) was used as templates for amplification. Then, microsatellite loci with amplification success were chosen for routine genotyping. Amplification products were run on an ABI Prism 310 DNA Sequencer  and analyzed with PeakScanner v.2.0 (Applied Biosystems) using GS-500 (ROX) as a size standard. Routine genotyping was performed using template DNA extracted from specimens collected from Drawa  and Gowienica  rivers. Details for developed microsatellite loci are given in Table After the suitable annealing temperatures were determined, the screening for polymorphic and consistently amplifiable loci was performed. In the first step, DNA extracted from 14 O) and expected (HE) heterozygosities, as well as tests for Hardy-Weinberg equilibrium (HWE) were calculated in GenAlEx v. 6.503 [Basic summary statistics for each locus , number of private alleles, observed . Observed heterozygosity ranged from 0.000  to 0.933 across loci. In turn, expected heterozygosity ranged from 0.064 (Aaesti5 in DR sample and Aaesti21 in both DR and GOW samples) to 0.925 (Aaesti39 in GOW sample). Within the overall sample, the same parameters ranged from 0.000 to 0.898 and from 0.065 to 0.935, respectively. Null alleles were identified in 26 microsatellite loci and that in turn may resulted in a heterozygote deficiency . This may be a result of physical linkage  or other phenomena . Consequently, one of the two associated markers should be excluded from further population genetic analyses in order to avoid the overstating of population structure [Polymorphism of 56 selected microsatellite loci was then analyzed among specimens collected from two populations inhabiting unconnected rivers . Analyses revealed 22 monomorphic loci and 34 loci which were polymorphic and consistently amplifiable. Further analyses of these informative loci showed that the total number of observed alleles per locus ranged from 2 to 21 in separated samples, and from 2 to 22 in the overall sample  are relatively high, indicating that tested A. aestivalis populations are not devoid of the intra-population genetic variation. In turn, analyses for connected samples revealed the departures out of Hardy-Weinberg equilibrium after Bonferroni correction only in two identified loci.The departures out of Hardy-Weinberg equilibrium after Bonferroni correction were found in all but thirteen microsatellite loci. These deviations were mainly due to a deficit of heterozygotes. Low heterozygosity estimated for most identified polymorphic microsatellite markers may be the effect of small size of selected A. aestivalis has a potential as a model organism for the study on the impact of past demographic phenomena, phylogeographic relationships, and infection with endosymbiotic Wolbachia on the genetic diversity of slowly dispersing freshwater insects. In the present study, a set of 56 microsatellite markers have been identified for freshwater A. aestivalis and 34 of them were polymorphic. These markers provide new suitable tools available for the scientific community to study A. aestivalis population dynamics. Finally, the assessment of its genetic diversity and population structure will provide important data, that can be used in population management and conservation efforts, elucidating the broad- and fine-scale population genetic structure of A. aestivalis.Microsatellite markers have proven to be crucial in understanding population genetics and ecology of many species, including freshwater insects (e.g. ). Within


Mastoidectomy is considered an aerosol-generating procedure. This study examined the effect of wearing personal protective equipment on the view achieved using the operating microscope.ENT surgeons assessed the area of a calibrated target visible through an operating microscope whilst wearing a range of personal protective equipment, with prescription glasses when required. The distance between the surgeon's eye and the microscope was measured in each personal protective equipment condition.p < 0.001). The median area visible while wearing the filtering facepiece code 3 mask and full-face visor was 4 per cent .Eleven surgeons participated. The distance from the eye to the microscope inversely correlated with the diameter and area visible (The full-face visor is incompatible with the operating microscope. Solutions offering adequate eye protection for aerosol-generating procedures that require the microscope, including mastoidectomy, are urgently needed. Low-profile safety goggles should have a working distance of less than 20 mm and be compatible with prescription lenses. The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)/coronavirus disease 2019 (Covid-19) pandemic presents several previously unconsidered challenges for otological surgeons, including how to minimise the risk of coronavirus transmission to the surgeon and operating theatre staff during aerosol-generating mastoid surgery.1 Mastoidectomy, which utilises high-speed drills within the mastoid air cells, is therefore considered to be an aerosol-generating procedure; the plume of potentially virus-containing aerosol generated by the drill may pose a risk to the surgeon and operating theatre staff.4The mastoid air-cell system is lined with respiratory mucosa, and is continuous with the middle ear and nasopharynx via the Eustachian tube. The middle ear has been demonstrated to harbour pathogens including coronavirus.6 This includes, as a minimum, fluid-resistant long-sleeved gowns, gloves, filtering facepiece code 3 (FFP3) respirator masks, and eye and face protection. Whilst some degree of eye protection is conferred by surgical masks with integrated visors or by polycarbonate safety spectacles, current Public Health England guidance specifically recommends a full-face shield or visor for aerosol-generating procedures, to fully protect the eyes from potentially hazardous droplets.5The UK government and specialist healthcare bodies currently recommend the use of personal protective equipment (PPE) for all surgery involving the use of high-speed drills.The challenge specific to the otological surgeon is the need to use an operating microscope when performing mastoidectomy. The full-face shield recommended for aerosol-generating procedures introduces a physical barrier, increasing the distance between the surgeon's eyes and the operating microscope eyepieces. This may reduce the microscopic view of the surgical field, making surgery more difficult and potentially increasing the risk of surgical error.In order to quantify this concern, this study examined the effect of different forms of PPE on the operator–microscope distance and the surgical view obtained by the operator.This study did not involve clinical care, active interventions, patients or members of the public. As such, formal ethic approval was not required. All surgeons participating in the study were consenting volunteers.This study utilised a Zeiss OPMI Vario S88 microscope  in the normal position for ear surgery, which was focused on a scaled archery target with 10 equally spaced concentric circles across 5 coloured zones . The micENT surgeons positioned themselves for using the microscope, adopting a comfortable posture appropriate for an extended period of operating . Each suSurgeons stated how much of the target they could see in their peripheral vision, whilst staring at the centre point with their head in the usual neutral position. A score of 1 (outer white ring) represented a full view, with progressively higher scores indicating a more restricted view visualising only a central portion of the target. The percentage of target visible was calculated from these scores (diameter and area). The distance between the most anterior aspect of the surgeon's cornea and the edge of the microscope eyepiece was measured to the nearest millimetre using a ruler secured to the side of the microscope, taking care to avoid parallax error .This process was repeated with the surgeon wearing an FFP3 mask, and various forms of eye and face protection. Scores for target view (outer-most complete ring visible) and eye–microscope distance were recorded for each PPE condition. Combinations of PPE were categorised from A to E, with A representing no PPE and E representing a full-face visor . SurgeonEleven ENT surgeons took part, all of whom were experienced in using the operating microscope. The grade of surgeon ranged from year three specialty trainee registrar to senior consultant. Inter-pupillary distance ranged from 58 mm to 75 mm . Four of the 11 surgeons wore prescription glasses for operating, and as such could not be tested at PPE level B (FFP3 mask with no eye protection or glasses). A total of 51 measures of eye–microscope distance and target scores were collected, covering all of the defined PPE categories (A to E).p < 0.001).The diameter and area of the visible target were calculated for each surgeon-reported visibility score and expressed as percentages of the overall target. The results are demonstrated in The effect of PPE on eye–microscope distance and target visibility was assessed. The median and range of eye–microscope measurements and target visibility calculations are shown in This workplace study of practising ENT surgeons supports and quantifies our concern that using PPE reduces the view of the surgical microscopic operating field, with an inverse correlation between eye–microscope distance and target view.5 However, the reduction in surgical view when wearing the visor in this study was severe, with less than 10 per cent of the microscopic view visible in most cases. This makes the recommended aerosol-generating procedure PPE incompatible with the use of an operating microscope at this time.The use of a full-face visor is required to satisfy the level of PPE recommended for aerosol-generating procedures such as mastoid drilling.The use of FFP3 masks with the surgeon's own prescription glasses and slim-line polycarbonate safety glasses (PPE level C) or over-glasses (PPE level D) produced a highly variable effect on view, depending on the fit of the PPE to the surgeon's face and resultant increase in eye–microscope distance. Importantly, neither of these PPE levels provides the droplet protection recommended by current UK Covid-19 PPE guidelines, as the glasses do not fully protect the eyes from droplet ingress.8 In addition, bespoke surgical shields that attach to the microscope may afford some protection to the surgeon.This study highlights the challenges facing otological surgeons in the Covid-19 era. Urgent action is needed to address the need to provide a safe operating environment for otological surgeons, whilst preserving the ability to deliver effective, safe surgical care. A number of adaptations have been suggested in order to address this risk. These include variations in surgical technique, which may obviate the need for simultaneous high-speed mastoid drilling and an operating microscope . Novel draping techniques, such as the ‘double-drape’ system, have also been suggested in order to reduce droplet spray.Sealed, low-profile goggles could potentially provide adequate eye protection with a minimal increase in eye–microscope distance. Based on the data obtained in this study, a maximum working distance of around 20 mm is likely to preserve a view of 80 per cent diameter and 65 per cent area of the microscopic field, with an ideal working distance of 15 mm or less preserving an almost complete view of the surgical field . Any such goggles should ensure complete eye protection against droplets.Specific legislation and testing criteria are defined by the European standard EN166:2001 and American National Standards Institute Z87.1 2020. In both systems, safety glasses or goggles are tested against splash, droplet, dust and aerosol protection, with results ranging from no protection to level 5 protection. Level 4 protection corresponds to protection against fine dust and droplets over 5 μm; the authors suggest this is the minimum level of protection required for mastoidectomy. Such goggles must also be able to accommodate prescription lenses, required by around one-third of surgeons in this study.Any modification or device designed to shield the otological surgeon's eyes and face from the aerosol generated by mastoid drilling must fulfil strict PPE requirements (preventing droplet contact with the surgeon's eyes), whilst also allowing relatively unhindered access to the microscope eyepieces. Individual surgeons may need to judge their level of confidence in performing safe otological surgery with even minor reductions in view. The data obtained in this study may be specific to the microscope and PPE used in our institute, and should be interpreted with this in mind.This study demonstrates that PPE including an FFP3 mask and full-face visor is incompatible with the use of an operating microscope; the view of the surgical field was reduced to below 10 per cent in most cases. Urgent consideration needs to be given to solutions that allow for concurrent use of the operating microscope and drill, which are required for mastoid surgery, whilst affording the surgeon adequate protection from viral transmission in the Covid-19 era. Low-profile safety goggles provide one possible solution, but a working distance of 20 mm or less should be achieved, with fully PPE-compliant sealed frames and the ability to include prescription lenses.