The genetic diversity of orthohepadnaviruses is not yet fully understood. This study was conducted to investigate the role of structural variations (SVs) in their diversity. Genetic sequences of orthohepadnaviruses were retrieved from databases. The positions of sequence gaps were investigated, since they were found to be related to SVs, and they were further used to search for SVs. Then, a combination of pair-wise and multiple alignment analyses was performed to analyze the genomic structure. Unique patterns of SVs were observed; genetic sequences at certain genomic positions could be separated into multiple patterns, such as no SV, SV pattern 1, SV pattern 2, and SV pattern 3, which were observed as polymorphic changes. We provisionally referred to these genetic changes as SV polymorphisms. Our data showed that higher frequency of sequence gaps and lower genetic identity were observed in the pre-S1-S2 region of various types of HBVs. Detailed examination of the genetic structure in the pre-S region by a combination of pair-wise and multiple alignment analyses showed that the genetic diversity of orthohepadnaviruses in the pre-S1 region could have been also induced by SV polymorphisms. Our data showed that novel genetic rearrangements provisionally termed SV polymorphisms were observed in various orthohepadnaviruses.  Hepatitis B virus (HBV) infection is an important global health issue and can cause acute and chronic liver diseases. An estimated 257 million people are deemed to be infected with HBV as they are positive for hepatitis B surface antigen (HBs-Ag), and HBV infection causes approximately 900,000 deaths per year from complications such as liver cirrhosis and hepatocellular carcinoma . In addiHBV is a member of the hepadnaviridae family, which is composed of two genera—the genus Orthohepadnavirus infects mammals, and the genus Avihepadnavirus infects birds . PreviouPrevious reports have suggested that the complexity of genetic diversity of HBVs is related to their mosaic genome structure ,16,17, aConsensus reference sequences and original datasets of human and non-human primate HBVs are described in Fujiwara et al. . InformaPhylogenetic analysis was performed with the MEGA software version 6 , using nHuman HBV genotypes A to H, non-human primate HBVs , bat HBVs, and rodent HBVs listed in The sequence identity (%) among the complete genomes and each ORF of WMHBV, bat HBVs, and rodent HBVs were determined by MAFFT . Three sMultiple alignment comparisons were performed using MAFFT , bat HBVs  and species of primate HBVs were analyzed in this study . PhylogeIn previous studies, we found that complex SVs tended to accompany sequence gaps ,22,23, tIn order to identify genetic variations that could have been caused by SVs in the genetic sequences of these orthohepadnaviruses, the areas with frequent sequence gaps as shown in section (Sec) 1 to 11 in Sec 8 also showed unique variation among the different species of orthohepadnaviruses. A previous study has shown that HBV/A has a 6-bp insertion in its Core ORF . We showWe next analyzed Sec 10, which is located around the pre-S1 promoter to pre-S1 ORF start site in our repertoire of orthohepadnaviruses. Different SVs in human and non-human primate HBVs have been previously reported in this area . Our datThus far, very unique SV polymorphisms were observed in the Core and pre-S1 promoter to pre-S1 ORF start site regions, as shown in Previous studies that analyzed human and non-human primate HBVs did not focus on sequence gaps since they are not frequent in these HBVs. Nucleotide substitutions and recombinations have been considered as main causes of genetic diversity of primate hepadnaviruses ,30,31. HIn this report, we discovered unique arrangements of SVs that were composed of multiple SVs at certain positions of the genome as shown in Combinations of repeated pair-wise and multiple alignment analysis revealed highly complicated covert SV polymorphisms in the pre-S1 region. Previous reports have suggested that the complexity of genetic diversity of HBVs is related to their mosaic genome structure ,16,17, aLauber et al.  proposedRegarding the correlation of SVs in HBV genome with virological and clinical characteristics, previous studies reported that complex forms of SVs in human HBV changed the transcription efficiency of pregenomic and preS/S RNA levels, and excessive expression of HBcAg in nucleus and perinucleus was observed. In addition, the SVs in HBV were observed in patients with HCC, fulminant hepatitis, and severe liver disease clinically ,22,23. AAlthough our analyses of SV polymorphisms in the pre-S1 region may have some limitations, the important findings that we have discovered in this study is an alternative and more plausible explanation for the genetic diversity of orthohepadnaviruses; rather than simple accumulations of nucleotide substitutions and recombinations, we propose that SV polymorphisms contributed to the viral genome diversity. Conventional concepts and methodologies have elucidated the genetic diversity of primate HBVs to a certain extent, but we believe that, due to the high complexity of the genetic diversity in other mammalian HBVs, our findings on complicated SVs in HBVs and the methods of analyses that we present here would be useful to further elucidate genetic changes in orthohepadnaviruses. Our data and further research on SVs in HBVs may help clarify the pathogenesis of these viruses in addition to understanding the genetic diversity and evolution of hepadnaviruses.We have discovered unique polymorphic SVs, which we referred to as SV polymorphisms, by the analysis of multiple mammalian HBVs. Further, using a combination of pair-wise and multiple alignment analysis, we found very complicated SVs in the pre-S1 region, which were considered to be composed of SV polymorphisms.


In this study we investigate the correlation between spontaneous blastocyst collapse and pregnancy outcome.2, 5.0% O2, 89.0% N2, using single step medium  and selected for transfer using standard morphological criteria. Using the EmbryoScope™ time-lapse monitoring (TLM), blastocysts collapse was analyzed by measuring the maximum volume reduction and defined as having collapsed if there was >50% volume reduction. Couples undergoing IVF/ICSI treatment and having an elective single embryo transfer (eSET) at blastocyst stage were included in this study. After the embryo transfer, retrospectively, each blastocyst was allocated to one of two groups (collapsed or not collapsed). 62 blastocysts collapsed once or more during development (17.4%), the remaining 294 showed no collapse (82.6%).This is a retrospective study performed at Edinburgh Assisted Conception Programme, EFREC, Royal Infirmary of Edinburgh, UK. Embryos were cultured individually in 6.0% COA significantly higher implantation rate (IR) of 61.2% and ongoing pregnancy rate (OPR) of 53.7% was observed when blastocysts which had not collapsed were replaced compared to cycles in which collapsed blastocysts were replaced (IR rate 22.6% and OPR 17.7%).in vitro development are less likely to implant and generate a pregnancy compared with embryos which do not. Although this is a retrospective study, the results establish the utility of collapse episodes as new marker of embryo selection following eSET at blastocyst stage.This study demonstrated that human blastocysts which collapse spontaneously during  Over the past forty years assisted reproductive technologies (ART) have evolved considerably from and ambitious and experimental procedure to a mainstream medicine which has brought more than 7 million children to the world . However+/K+-ATPase enzyme, which raises the salt concentration within the embryo, attracting water through osmosis  incubator .This is a retrospective consecutive cohort study including 356 IVF/ICSI cycles carried out at Edinburgh Assisted Conception Programme, Royal Infirmary of Edinburgh, between January 2016 and July 2018. All cycles were with patient’s own oocytes and fresh eSET at blastocyst stage. For the purposes of this study, the blastocyst volume reduction was automatically measured using EmbryoViewer™ drawing tools and we have defined collapse if there was a volume reduction >50%, from the expanded blastocyst and the collapse event. If the volume reduction was <50% the event was considered as a contraction only. Blastocyst transfer was scheduled on the morning of day-5, approximately 116 to 120 hours after insemination. In this study we included only eSET at blastocyst stage, and embryo implantation was confirmed by an ultrasound scan for gestational sac with fetal heartbeat after seven weeks of pregnancy. The retrospective observational study design did not require ethical approval for the use of human subjects.2 in atmospheric air using the Hera cell 240 incubator (Thermo Scientific). Sperm used for either routine IVF insemination or ICSI procedure was collected by masturbation and processed using a standard method as described by A detail of ovarian stimulation and egg retrieval has been previously described by 2 in atmospheric air in G-IVF™ medium until ICSI was performed. ICSI was carried out using G-IVF™ medium, polyvinylpyrrolidone  and a Narishigue  or Research Instruments  micromanipulator system at 38-42 hours post hCG injection. Only MII oocytes were injected. After ICSI, oocytes were placed in independent drops of 10µl of culture medium  covered with mineral oil  and incubated at 37ºC in 6.0% CO2.For IVF procedures, oocytes were exposed to 150,000 motile sperm/ml, approximately 38-42 hours post hCG injection. For ICSI cycles, COCs were exposed to 80 IU/mL of hyaluronidase  and mechanically removed by gentle pipetting in buffered medium  using denudation pipettes of progressively smaller diameter . MII oocytes were incubated at 37ºC in 6.0% COin vitro culture was performed in an atmosphere of 6.0% CO2, 5.0% O2 and 89.0% N2 at 37ºC in single step medium (GTL™ Vitrolife) which is designed for TLM allowing undisturbed culture.Fertilization was identified by the presence of two pronuclei approximately 16-19 hours after insemination or microinjection. At this stage, normally fertilized pronuclear stage embryos were allocated to the EmbryoScope time-lapse incubator for culture in a 12-well EmbryoSlide™, which is a specifically designed dish for the EmbryoScope™ imaging system . This slide has 12 individual wells for embryo culture, each well containing 20µl of culture media with 1.4 mls overlay of mineral oil to prevent evaporation. The In the EmbryoScope embryo culture, images were acquired every 10 minutes in 7 focal planes and morphological assessment was made by examining a video of development using the associated EmbryoViewer™ software, without moving embryos from the incubation. Embryos were assessed morphologically at cleavage stage according to British Fertility Society and Association of Clinical Embryologists guidelines, published by To evaluate the collapse event, we used the EmbryoViewer™ workstation drawing tools in order to automatically measure the entire surface occupied by an embryo. Two measurements were performed: the first when the blastocyst was at the maximum expansion and the second, if the blastocyst showed a collapse episode, was carried out when the embryo occupied the smallest surface. Therefore, according to the maximum volume reduction we described two distinct episodes: “collapse” when the volume reduction was 50% or more , 1c-1d. Chemical pregnancy was assessed based on serum βHCG levels 9 days after the embryo transfer. At seven weeks, a scan was performed to confirm the presence of fetal heart activity or gestational sac formation. The implantation rate (IR) was defined as the number of gestational sacs at the seven-week scan. A clinical ongoing pregnancy rate (OPR) was defined as a pregnancy with a fetal heart.p<0.05.A Chi-square test was performed to examine the pregnancy outcomes between blastocyst which had a collapse event during the development and those which not. Differences were considered statistically significance at the level of p>0.05; A total of 356 consecutive cycles was analyzed and included in this study, all of which were eSET at blastocyst stage. A total of 62 blastocysts presented collapse episode (17.4%), of these twelve presented two, and one blastocyst presented three collapse events. The remaining 294 either contracted minimally or show no sign of collapse at any time (82.6%). There were no differences in the characteristics of patients who had a collapsed blastocyst transferred compared to those that had a transfer of a non-collapsed blastocyst. None of the parameters, including age, body mass index (BMI), year of infertility, days of stimulation, gonadotropin used and endometrial thickness were significant p>0.05; .p>0.05; Summarized in versus 30/62 (48.4%) in ICSI].Blastocysts were classified according to degree of expansion of the blastocoel cavity and quality of the inner cell mass and trophectoderm cells. On the morning of day-5, the top quality blastocyst was selected for transfer by considering the morphology only, regardless the collapse episode. Following embryo replacement, the analysis was performed and each blastocyst was allocated to one of two groups (collapsed or not collapsed). No difference has been noted on blastocyst collapse event in relation to the insemination technique adopted [32/62 (51.6%) in IVF versus a non-collapsed blastocyst. A significantly higher positive pregnancy test rate of 69.4% was observed when blastocysts which had not collapsed during development were replaced compared to cycles in which collapsed blastocyst were transferred 40.3%. In addition, a significantly higher ongoing pregnancy rate of 53.7% was noticed when blastocysts without any collapse were transferred compared to blastocysts which exhibited a collapse event during their development 17.7%. The miscarriage rate was higher in the collapsed group 24.2% versus non-collapsed 15.6% but this trend was not statistically significant  could be clearly recognized, additionally, if any reproductive effect is caused by this behaviour it could be more perceptible. Our data are in agreement with a study presented by In the present study we observed that a small number of blastocyst presented multi-collapsed events (twelve blastocysts had two collapses and one blastocyst three). Due to the small number of those embryos we did not perform any analysis comparing clinical outcome between embryo showing one collapse and those showing multiple collapses. The same was also related to blastocysts which show a weak contraction (<50%), this was not considered as a collapse event. The results presented in this study were only related to the two groups analyzed (collapse and not-collapse).versus 48.4% in ICSI).Another aspect analyzed was the relationship between the fertilization method and the collapse episode. Since at the ICSI time an injection needle needs an opening through the ZP, this manipulation might have an influence on the collapse episode. Indeed the analysis performed has shown no difference in the blastocyst collapse event in relation to the insemination technique used . Although this is the first study to report such data, it might be clear that spontaneously blastocyst collapse, despite being a physiological feature during blastulation, is conditioned by the ploidy status of the embryo . A recent study published by Human and animal rights. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and with the 1964 Helsinki declaration and its later amendments. For this type of study, formal consent is not required.


Sambucus palmensis is a critically endangered endemic to the Canary Islands. During the past 30 years, the Garajonay National Park (La Gomera) has carried out an intensive program of translocations using cuttings, due to the low germination rates of seeds. To assess the effect of the restorations on the population genetics of S. palmensis in La Gomera, we collected 402 samples from all the restored sites and all known natural individuals, which were genotyped with seven microsatellite markers. In addition, we conducted a species distribution modeling approach to assess how restorations fit the ecological niche of the species. Results show that there is a high proportion of clone specimens due to the propagation method, and the natural clonal reproduction of the species. Nonetheless, the observed heterozygosity has increased with the restorations and there still are private alleles and unique genotypes in the natural populations that have not been considered in the restorations. The population of Liria constitutes a very important genetic reservoir for the species. To optimize future reintroductions, we have proposed a list of specimens that are suitable for the extraction of seeds or cuttings in a greenhouse, as well as new suitable areas obtained by the species distribution models.The translocation of individuals or the reinforcement of populations are measures in the genetic rescue of endangered species. Although it can be controversial to decide which and how many individuals must be reintroduced, populations can benefit from reinforcements.  The preservation of endangered plant species usually involves population restorations, either by reinforcement of the extant populations or the reintroduction of new populations. Before starting restoration programs, biological information on the species must be first gathered in order to determine the most important factors limiting the growth of the founding population . The bioOceanic islands ecosystems are generally threatened either by biological (inherent to islands) or socio-economic factors. The biological causes are mainly habitat loss or degradation, small populations sizes and fragmentation, and the introduction of alien species or direct predation. The socioeconomic factors are mainly due to demographic and economic growth accompanied by a lack of natural resources management capacity, and with special relevance in the Canary Islands, the high touristic pressure . ClimateIn Macaronesia, the laurel forest is one of the best representations of this insular vulnerability as it has experienced a major reduction since human colonization. In the Canaries, most of the laurel forest disappeared after the arrival of Castilians (15th century), affecting the distribution and abundance of many species . DespiteSambucus palmensis as a model species to evaluate the effectiveness of long-term conservation programs in clonal perennial species. Saúco or Canary elderberry is a rare endemic of the Canarian Archipelago and is present in four of the seven main islands  to provide a background for future conservation programs in the Garajonay National Park.The aims of this study were (1) to estimate changes in genetic diversity after the restoration programs in La Gomera, (2) to identify the current genetic structure and the number of identical genotypes in the populations, (3) estimate the topoclimatic suitability of Sambucus palmensis Link,  R. Bolli) (Sambucaceae), It grows in the laurel forest vegetation zone between 600–1,000 m a.s.l., with a preference for shady and humid places. It can also be found on the margins of agricultural fields being grown by locals due to its historical use for medicinal purposes , present in the sites of Acebiños, Ancón de Candelaria, Liria, Meriga and Presa de Las Rosas, and a significant representation of the reintroduced individuals from the whole distribution in La Gomera (355) were collected during 2012 and 2013. The categories of natural or reintroduced were considered according to the Garajonay National Park records and annotations. The individuals from Liria and Presa de Las Rosas are all natural, with no reintroduced individuals. In total, 402 samples were collected and genotyped for the study. They are distributed in 15 sites according to their geographical distribution or management area for the National Park. The distribution of the analyzed individuals and the sampling sites are represented in Sambucus palmensis individuals in La Gomera. Five markers that were developed for Sambucus nigra . Amplifications were performed using the following conditions: 3 min denaturation at 95 °C, 35 cycles of 30 s denaturation at 95 °C, 30 sannealing at 50–62 °C, and 72 °C for 1.5 min; followed by 5 min elongation at 72 °C. Reverse primers were color-labelled at the 5′-end with 6-FAM, PET, NED or VIC.Microsatellite loci were selected from an Illumina paired-end shotgun library developed by the company AllGenetics (University of A Coruña) using three probe mixes of 20. Multiplexing was carried out in two primer groups as indicated in Once the new markers where characterized, we conducted the subsequent multiplex amplifications for the seven primer pairs using the QIAGEN Multiplex Kit (QIAGEN). PCR were performed in 15 µL reaction volumes: 7.5 µL of Multiplex PCR Master Mix, 1.5 µL primer mix (containing 0.2 µM of each primer in TE), 1.5 µl of Q-solution, 20–40 ng of DNA and dHS. palmensis in La Gomera, multilocus matches were identified with GenAlex version 6.5 (Na); number of private alleles (Pa), rare alleles , observed (Ho), and unbiased expected (He) heterozygosities for each locus and locality were estimated. Measures of allelic (Ar) and private allelic richness (Par) were calculated using HP-RARE 1.0   with ARLK (from 1 to 15) and analysis consisted of a 105 burn-in period replicated and a run length of 106 replicates. The optimal number of clusters was found by the ΔK method . Stations with 10 or more years of climate records were filtered for the models. For the monthly variables of minimum and maximum temperature and precipitation, a generalized additive model (GAM) with altitude, northness, latitude and longitude as predictor variables was developed. The best model was selected based on the AIC scores and then projected to La Gomera island. To account for spatial biases of the models, residuals of each model in each meteorological station were used to develop an interpolated map of residuals for each variable by ordinary kriging. This interpolated layer was added to the predicted value of the GAM model to obtain the final layers of each monthly variable. The final dataset of monthly variables was used to develop the bioclimatic variables described by The explanatory power of SDMs (Species Distribution Models) is often improved when accurate resolutions of spatial predictors are used . We therA total of 441 reintroduced presence cells were recorded in total at 50 meter working resolution, 47 were natural presences in La Gomera and nine recorded in Tenerife and La Palma. A total of 51 presences were discarded as they could not be unambiguously assigned to natural or reintroduced populations in Tenerife and La Palma. To select climatic predictor variables for the modeling procedure first a correlation analysis was conducted for the values of the variables in the cells where the species was present with the R package ‘ecospat’ , which rThree different niche modeling procedures were conducted with biomod2 package . The firSix algorithms available in biomod2 were used: generalised linear models (GLM), generalised additive models (GAM), both with stepwise selection, boosting regression (GBM), multiadaptive regression splines (MARS), annual neural networks (ANN), and random forest (RF). 10 runs of each presence-pseudoabsence dataset were run for the first two modeling approaches. In each run, 85% of data was randomly selected for calibration and the rest for model evaluation. The third procedure was evaluated using the natural occurrences of the species. Models were evaluated by means of TSS and ROC scores. Any model with a score below 0.8 for any of the two metrics was excluded for the ensemble model. The remaining models were retained to build an ensemble model based on the contribution of each individual model weighted according to the TSS score.All microsatellite markers used for this study yielded enough polymorphism to identify the possible number of genotypes in the sampling sites analyzed and to detect possible identical genotypes. Out of the 402 individuals sampled, 147 different genotypes were found. 84 of these were unique genotypes , and 63 were shared genotypes  . We founBasic genetic diversity indices, such as allelic richness and expected heterozygosity values were similar across sites. The expected heterozygosity ranged from 0.357 (Presa de las Rosas) to 0.495 (Raso de la Bruma). The observed heterozygosity values did show higher differences across sites, ranging from 0.366 (El Cedro) to 0.714 (Cañada Jorge). Liria and Meriga were the only sites with private alleles, and Liria presented the highest rarefied private allelic richness at 0.26 . MoreoveAMOVA results were similar in the two approaches tested . In bothK according to the ΔK and the mean of log-likelihood values (K = 2 and K = 5)  . Therefogenotype .Finally, we proposed a list with the 25 most suitable individuals with which to build a conservation genetic program to enhance the genetic variability of the populations. Individuals with unique or rare genotypes, which also presented a high individual heterozygosity and private or rare alleles were considered .The PCA analysis conducted with all spatial predictors considered accounted for a 96.35% of the variance and showed patent differences between the niche of introduced and natural populations , correspF2,317 = 20.73, P ± 3.575 × 10−9, pseudoabsence dataset and model run as random effects) and models , with the introduced model showing better performance . Only 17 cells did fall within the limits of Garajonay National Park.The suitable areas declared by the three modeling procedures projected suitable areas in the northern parts of the island in areas exposed to humid trade winds, but were conspicuously different : the suiSambucus palmensis in La Gomera have greatly improved the genetic status and distribution range of this species in the island. Although there is a high proportion of clonal specimens, natural regeneration has occurred in the restored sites, generating new genotypes and alleles that were not present in the original populations. Moreover, the restored sites are occupying new climatic suitable areas, which has led to an expansion of the distribution, compared to the natural populations. Nonetheless, there are still some major concerns in the conservation of S. palmensis, such as the difficulties in sexual reproduction, or the high mortality rates of young plants.An integrative approach towards the conservation of endangered species, with the combination of molecular and modeling tools, is starting to be more considered, although few examples can still be found on islands . Our resThe genetic diversity estimates for La Gomera are higher than those reported by S. palmensis presents moderate levels of genetic diversity for a rare endemic . Although we did not find a sign of recent bottleneck events in the natural source, most of the individuals came from Liria, which has naturally increased its population size since the monitoring programs started. Thus, bottleneck events in the other natural sites are difficult to infer, as only a few individuals have remained.The percentages of variation found between populations are low for an outcrossing species , and theoduction . TherefoS. palmensis, hindering high population differentiation or local adaptation. Even if the translocations of genotypes have led to outbreeding depression, the advantages of outcrossing can be greater, especially for self-incompatible species, as it increases the chances of finding available mates , (B) Ad hoc statistic based on the rate of change in the log probability of data between successive K values  The mean of log-likelihood values for each value of Click here for additional data file.10.7717/peerj.4985/supp-3Supplemental Information 3Unique genotypes are assigned a numeric code and shared genotypes are coded with letters from A to ZZ.Click here for additional data file.10.7717/peerj.4985/supp-4Supplemental Information 4Private alleles per locality are in bold and italics. Rare alleles  are in bold.Click here for additional data file.10.7717/peerj.4985/supp-5Supplemental Information 5Individuals with unique or rare genotypes, which also presented a high individual heterozygosity and private or rare alleles were considered. ID = Code assigned to each individual, Genotype code = Identification code of the genotypes assigned to each individual, H-ind = Individual heterozygosity.Click here for additional data file.10.7717/peerj.4985/supp-6Supplemental Information 6Click here for additional data file.10.7717/peerj.4985/supp-7Supplemental Information 7(A) Boxplots temperature seasonality and annual precipitation of natural occurrences and reintroduced occurrences. (B) Response curves for temperature seasonality and annual precipitation for the ensemble modelling calibrated with natural or reintroduced occurrences.Click here for additional data file.10.7717/peerj.4985/supp-8Supplemental Information 8Click here for additional data file.10.7717/peerj.4985/supp-9Supplemental Information 9Click here for additional data file.


Their microbiota were investigated using high-throughput sequencing targeting the V3–V4 regions of the 16S rRNA gene. The genus level phylotypes was increased from 649 to 767 in the post-smog pharyngeal microbiota, of which 142 were new and detected only in the post-smog microbiota. The 142 new genera were traced to sources such as soil, marine, feces, sewage sludge, freshwater, hot springs, and saline lakes. The abundance of the genera Streptococcus, Haemophilus, Moraxella, and Staphylococcus increased in the post-smog pharyngeal microbiota. All six alpha diversity indices and principal component analysis showed that the taxonomic composition of the post-smog pharyngeal microbiota was significantly different to that of the pre-smog pharyngeal microbiota. Redundancy analysis showed that the influences of PM2.5/PM10 exposure and smoking on the taxonomic composition of the pharyngeal microbiota were statistically significant (p < 0.001). Two days of exposure to high concentrations of PM2.5/PM10 changed the pharyngeal microbiota profiles, which may lead to an increase in respiratory diseases. Wearing masks could reduce the effect of high-level PM2.5/PM10 exposure on the pharyngeal microbiota.Previous studies showed that high concentration of particulate matter (PM) 2.5 and PM10 carried a large number of bacterial and archaeal species, including pathogens and opportunistic pathogens. In this study, pharyngeal swabs from 83 subjects working in an open air farmer’s market were sampled before and after exposure to smog with PM2.5 and PM10 levels up to 200 and 300 μg/m Short-tobserved , and a d35 years . Startinortality . An epidortality .Streptococcus pneumoniae and Aspergillus fumigatus  and on January 19, 2017 (after the episode), to explore the possible difference in the composition of the microbiota. If the number of bacterial species or the number of a given bacterial species detected in the post-smog sample were statistically significantly higher than that in the pre-smog sample, the increased number was considered to be caused by exposure to the severe smog episode.2, with approximately 300 small vendors. The market is open daily from 6 o’clock a.m. to 12 o’clock a.m. After the market was closed at noon, some of the vendors stayed for additional business hours in the afternoon. Generally, approximately 3,000 customers visited the market each day. Most of them were nearby residents.We selected the pharyngeal microbiota to study inhalable PM-associated bacteria, which play a significant role in the development of respiratory tract diseases. Vendors in an open-air farmer’s market were selected for this study because they represented a group of people who worked in the same place and were exposed to smog over the same business hours. The outdoor farmer’s market in Liaocheng City of Shangdong Province was selected for this study. The market is approximately 10,000 mThe timing of study initiation was carefully calculated using information from the official air quality forecast, which is released to the public every day. Sampling was conducted by scientists from the local Center for Disease Control and Prevention (CDC), who are authorized for such activity. The vendors sampled were fully informed and agreed to participate in the study.3 in a single day. We decided to sample the pre-smog pharyngeal swab on January 16, 2017, when the PM2.5 concentration was reduced from 128 μg/m3 to about 80 μg/m3 for three consecutive days and was forecasted to increase to 220 μg/m3 for the next three consecutive days. The PM2.5 indeed reached a peak of 287 μg/m3 on January 18 and maintained a level of 217 μg/m3 on January 19, when we sampled the post-smog pharyngeal swab  containing 3 ml of phosphate-buffered saline (PBS). The tubes with pharyngeal specimens were immediately place in an icebox and transported to the laboratory of the local CDC where the DNA extraction was performed.From the pharyngeal samples, total genomic DNA was extracted using a QIAamp DNA mini-kit . The V3–V4 region of the 16S rRNA gene was amplified by PCR using universal primers targeting most bacteria  with a 6-bp barcode unique to each sample. The PCR conditions were 94°C for 4 min; followed by 30 cycles of 94°C for 30 s, 54°C for 30 s, and 72°C for 30 s; and then 72°C for 5 min. The single amplifications were performed in 25 μL reactions with 50 ng of template DNA, which was quantified using a Qubit 2.0 Fluorometer . The expected size of the amplicon amplified by the primers used in this study was 485 bp. The resulting amplicons were purified, quantified, pooled, and sequenced on an Illumina HiSeq 2500 PE-250 platform  using pair-end sequencing (2 × 250 bp).1), and quality filtering was performed under specific filtering conditions (-q 19) using QIIME (2). After processing, sequence analysis was performed using the Uparse software (Uparse v7.0.10013). Sequences with ≥97% similarity were assigned to the same operational taxonomic units (OTUs). Representative sequences for each OTU were screened for further annotation using the RDP-classifier (Version 2.24) against the SILVA_123 database with an 80% confidence level. OTU representative sequences were identified using MUSCLE software (Version 3.8.31), and a phylogenetic tree was built using the FastTree algorithm.Barcodes and sequencing primers were trimmed before the paired end reads were merged using FLASH , permutational analysis of variance (PERMANOVA), and redundancy analysis (RDA) were performed in the R software (Version 2.15.3). Linear discriminant analysis effect size (LEfSe) was used to detect unique biomarkers by determinations of the relative abundances of the members of the bacterial taxonomies. The differences between alpha diversity indices and the profiles of specific respiratory pathogens on the pre- and post-smog pharynx swabs were tested using paired There were no unexpected, new, and/or significant hazards or risks associated with the reported work.Ethical approval for this study was obtained from the Ethical committee of National Institute for Communicable Disease Control and Prevention Chinese Center for Disease Control and Prevention (ICDC-2018001). All participants provided written informed consent.A total of 166 pharyngeal swabs were sampled from 83 vendors, two for each vendor. We obtained a total of 10,585,553 high-quality bacterial 16S rRNA sequences . The seqProteobacteria (24.1% of the reads), Firmicutes (23.8%), Bacteroidetes (22.6%), Fusobacteria (14.1%), and Actinobacteria (12.3%). The number of phyla in the post-smog swabs increased from 36 to 46 compared with that of the pre-smog swabs. There were 11 new phyla: SHA-109, Woesearchaeota_DHVEG-6, Caldiserica, Chlamydiae, Parcubacteria, MEG, WD272, TA06, Atribacteria, Microgenomates, and WCHB1-60.The 47 annotated phyla accounted for 99.33% of the total reads. The top five most abundant phyla accounted for the majority of total reads, at 97.6% and 96.3% for the pre-smog and post-smog swabs, respectively , and incFirmicutes, Fusobacteria, and Actinobacteria were increased in the post-smog samples. The increased read numbers for Firmicutes, Fusobacteria, and Actinobacteria were 343,931, 169,013, and 160,158, respectively.The relative abundance of 38 phyla on the post-smog swabs were increased compared with those of the pre-smog swabs . Among tPrevotella, Neisseria, Fusobacterium, Streptococcus, Leptotrichia, Rothia, Veillonella, Alloprevotella, Haemophilus, and Actinomyces. The profiles of the top 100 most abundant genera are shown in Leptotrichia, Corynebacterium, Veillonella, Dolosigranulum, unidentified_Chloroplast, Moraxella, Gemella, Actinomyces, Granulicatella, and Haemophilus, with more than 20,000 reads for each.A total of 791 genera were annotated, accounting for 97.82% of total reads. The top ten most abundant genera accounted for 69.0% of total sequences, and included There were 142 new genera detected only in the post-smog swabs . The oriP < 0.001), chao1 (P < 0.001), and ACE (P < 0.001). Furthermore, the evolutionary distance index and whole-tree phylogenetic diversity showed significant differences  between the pre- and post-smog data. However, the differences in evenness between the pre-smog and post-smog data were not significant   .P value = 0.001).The principal component analysis (PCA) showed that the composition of the pharyngeal microbiota changed significantly after exposure to high levels of PM2.5, as revealed by separable clusters in the pre- and post-smog samples . This fip < 0.001). No significant effects were observed for working hours, history of using antibiotics, or having respiratory symptoms.The redundancy analysis (RDA) results  showed tFurthermore, Spearman correlation analysis was carried out to test the correlation between the significant influential factors and alpha diversity . The ricP value = 0.01) among people not wearing masks   .Streptococcus, Haemophilus, Moraxella, Neisseria, and Staphylococcus. These five genera were detected from both pre- and post-smog samples. The relative abundance of Streptococcus, Haemophilus, Moraxella, and Staphylococcus increased in the post-smog swabs. Among them, the relative abundance of Staphylococcus increased by up to 300%. Interestingly, the pathogens Haemophilus influenza and Moraxella catarrhalis were annotated, showing 240% and 150% increases in relative abundance, respectively, compared with that of the pre-smog samples (P > 0.05) (Neisseria between the post- and pre-smog data (P < 0.05).The profiles of the main recognized respiratory pathogens on the swabs were also analyzed, including  samples . However > 0.05) . Among tSeveral studies showed that exposure to PM2.5/PM10 harmed the human respiratory system and caused respiratory diseases . In addiWe hypothesized that high-level PM2.5/PM10 exposure might lead to alterations in the human pharyngeal microbiota composition. Therefore, we compared the profiles of bacterial phylotypes and the abundance of the pharyngeal microbiota sampled pre- and post-smog in a cohort of 83 subjects. All six alpha diversity indices between the pre- and post-smog pharyngeal microbiota were different. Among them, the indices of richness, the evolutionary distance index, and the whole-tree phylogenetic diversity were significantly different, including the number of observed species. PCA analysis showed that the composition of the post-smog pharyngeal microbiota was significantly diversified compared with that of the pre-smog microbiota , which wFirmicutes, Fusobacteria, and Actinobacteria. In addition, 11 new phyla were detected, including Caldiserica, Chlamydiae, Parcubacteria, Atribacteria, and Microgenomates. At the genus level, the number of phylotypes increased from 649 to 767 in the post-smog pharyngeal microbiota. The relative abundance of 559 genera increased, such as Leptotrichia, Corynebacterium, Veillonella, Dolosigranulum, unidentified_Chloroplast, Moraxella, Gemella, Actinomyces, Granulicatella, and Haemophilus. The 142 new genera in post-smog microbiota originated from soil, marine, feces, sewage sludge, freshwater, hot springs, and saline lakes   . Smoking< 0.001) ; therefo< 0.001) . The pre of mice , and expn humans . Variousinomyces , Gemella(Gemella , Actinobbacillus , and Stalococcus .In summary, we reported that exposure to two days of smog altered the composition of the pharyngeal microbiota, which may introduce unexpected health risks, especially respiratory infections. The concentrations of PM2.5/PM10 in Liaocheng City of Shandong Province in 2017 were far in excess of the level recommended by the World Health Organization’s guidelines on air quality and this may be the case due to chronic exposure to levels above those recommended by the WHO. The effect of high concentrations of PM2.5/PM10 on the composition of pharyngeal microbiota might be more severe than that represented by the data reported in the present study.JX conceived the study. TQ, FZ, and HZ performed the literature search and wrote the protocols. FZ, YD, SL, FW, LC, and XX recruited the vendors and collected the specimens. TQ, HZ, YD, HR, and SL performed the laboratory analyses. TQ, HZ, and AJ analyzed the data. TQ, HZ, AJ, YW, JZ, and JX interpreted the data. All authors contributed to the writing and editing of the report and approved the final 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.


Salix purpurea and S. helvetica that has emerged within the last 30 years on a glacier forefield in the European Alps due to secondary contact of the parental species. We used 5758 biallelic SNPs produced by RAD sequencing with the aim to ascertain the predominance of backcrosses (F1 hybrid x parent) or F2 hybrids (F1 hybrid x F1 hybrid) among hybrid offspring. Further, the SNPs were used to study segregation distortion in the second hybrid generation.Hybridization of species with porous genomes can eventually lead to introgression via repeated backcrossing. The potential for introgression between species is reflected by the extent of segregation distortion in later generation hybrids. Here we studied a population of hybrids between structure and NewHybrids revealed that the population consisted of parents and F1 hybrids, whereas hybrid offspring consisted mainly of backcrosses to either parental species, but also some F2 hybrids. Although there was a clear genetic differentiation between S. purpurea and S. helvetica (FST = 0.24), there was no significant segregation distortion in the backcrosses or the F2 hybrids. Plant height of the backcrosses resembled the respective parental species, whereas F2 hybrids were more similar to the subalpine S. helvetica.The analyses in The co-occurrence of the parental species and the hybrids on the glacier forefield, the high frequency of backcrossing, and the low resistance to gene flow via backcrossing make a scenario of introgression in this young hybrid population highly likely, potentially leading to the transfer of adaptive traits. We further suggest that this willow hybrid population may serve as a model for the evolutionary processes initiated by recent global warming.The online version of this article (10.1186/s12870-018-1552-6) contains supplementary material, which is available to authorized users. Natural hybridization due to secondary contact has been observed in many plant and animal species. Especially in North America and Northern and Central Europe, a major driving force for secondary contact is the ongoing recolonization after the retreat of glaciers . This prMany studies have investigated the evolutionary relevance of hybridization. Although there are some documented cases of homoploid hybrid speciation –9, speciTo assess the evolutionary impact of a hybridization event, it is crucial to know the extent to which a genome is susceptible to the introgression of heterospecific alleles. Segregation distortion, the deviation from expected Mendelian segregation ratios, can be used as a measure of the resistance of the hybridizing species’ genomes to introgression , 25. FurSalix purpurea L. and S. helvetica Vill., which are situated on the forefield of the Rhône Glacier in central Switzerland. Salix helvetica is a shrub that occurs naturally in the subalpine to alpine zone. Salix purpurea, on the other hand, is a widespread lowland species that was recently able to colonize higher altitudes due to global warming and subsequent glacier retreat [Salix [S. purpurea and S. helvetica belong to different sections of the genus [2 hybrids (i.e. F1 hybrid x F1 hybrid) might be an indication of the potential for hybrid swarm formation and further hybrid evolution. In an earlier study on the willow population at the Rhône Glacier, an attempt has been made to determine the exact class of the hybrids  on the glacier forefield based on genotyping with microsatellite markers [S. purpurea and S. helvetica are fertile and produce viable seeds in the natural population, and thus confirmed that hybridization can proceed beyond the F1 hybrid generation [In this study, we investigate hybridization in a zone of secondary contact between two willow species,  retreat . Seconda retreat –41. Hybrt [Salix , 43. Althe genus , they fohe genus . The com markers . These m markers . Thus, tneration . The off1 hybrids in order to predict the consequences of this hybridization event. Further, (ii) we were looking for deviations from expected segregation patterns at individual loci in F2 hybrids and backcrosses to determine if alleles of one parental species were favoured over the other. Population genomic analyses were accompanied by morphometric measurements to (iii) get insights into the variation of a selected, potentially adaptive phenotypic character (plant height) in the respective second generation hybrid classes.In order to overcome the limitations caused by a low number of markers, we used restriction-site associated DNA sequencing (RAD-seq) to generate a genome-wide set of thousands of single-nucleotide polymorphisms in this nonmodel species. High-quality biallelic SNPs were used to (i) determine the class of the hybrids on the glacier forefield and of offspring produced by FS. purpurea, S. helvetica and their hybrids yielded an average of 7.5 × 106 reads per individual (SD 2.4 × 106). The average per base sequence quality was very high with a Phred score of 40 for all positions in the reads in all samples. The average depth of read coverage was 59x (SD 18). The stacks-pipeline initially generated 49,081 loci. After the application of all filters, 5758 nuclear loci remained. After mapping the loci to the plastid genome no match was observed. However, of the filtered loci 933 reads aligned to coding regions in the genome of P. trichocarpa. The results of population genetic and progeny analyses did not change when the SNPs lying in coding regions were excluded, and thus we performed all analyses with all 5758 SNPs.RAD-seq of structure analysis confirmed that the most likely number of populations (K) in the sample was two . F2 hybrids were only observed among the offspring of one of the five mother-plants  than to S. helvetica (n = 16) were detected . The different hybrid classes also formed well-separated clusters in the PCoA analysis  and were significantly taller than backcrosses to S. helvetica  or F2 hybrids .The one-way ANOVA revealed significant differences in plant height between the three groups (FS. helvetica and S. purpurea on the Rhône glacier forefield. RAD-seq data enabled a much better resolution of the hybrid classes than the microsatellite markers applied in our previous study [2 hybrids or backcrosses), the analysis of the same hybrids with RAD-seq data showed with maximum statistical support that all hybrid individuals sampled on the forefield of the Rhône Glacier were F1 hybrids. This discrepancy in the hybrid classification is probably due to the low number of microsatellite loci used in the previous study that had to be restricted to primers amplifying in both species. Further, the lack of species-specific alleles at the microsatellite loci made it difficult to determine from which species an allele was inherited in the admixed genotypes. Similar discrepancies in the classification of hybrids were also observed in studies on Populus hybrid zones. While no hybrids were classified as F1 hybrids based on genotyping with microsatellites [1 generation [Our study confirms the existence of a natural secondary contact hybrid zone of us study . While ttellites , subsequneration . The autneration . We thinneration . Further1 hybrids at the forefield of the Rhône Glacier [2 offspring can be regularly formed in this population. However, the results revealed only F1 generation hybrids. We assume that this lack of second generation hybrids is due to the sampling strategy. We included only material from adult individuals because the leaves, flowers and fruits of juvenile plants and seedlings did not yet show the typical phenotypic characteristics of adult willows. In a previous study, we already concluded that the hybrid population on this recently emerged glacier forefield is probably not more than 20–30 years old [2 hybrids have meanwhile grown up in the natural population, an extended sampling strategy that includes juvenile plants and a broad representation of phenotypes would have to be applied.We expected to find some later generation hybrids on the glacier forefield because the analysis of offspring grown from seeds that had been collected from naturally pollinated F Glacier  suggesteears old . We thus2 hybrids cannot establish on the glacier forefield. In a study on hybrid fertility, we found that the seed output of hybrids was reduced compared to the parents, but that seeds showed high germination ability, and seedlings developed well [2 hybrids on the glacier forefield, although their numbers may probably be still lower compared to F1 hybrids. Alternatively, habitat mediated selection may act against the establishment of later generation hybrids on the parental sites, as it has been observed in Rhododendron hybrid zones [1 hybrids were formed, the glacier forefield was still in an early state of succession with less vegetation cover so that the conditions were more favourable for the establishment of willows, which are pioneer species. Later in time, when the backcrosses and F2 hybrids were produced, the vegetation may have been denser so that the conditions for the establishment may have become more difficult. However, it should also be kept in mind that glacier retreat is ongoing and that open pioneer sites for colonization will be continuously available. Although we did not find later generation hybrids in our present sampling, it may still be concluded that the hybrid population on the glacier forefield is able to develop beyond the F1 generation so that hybridization may have further consequences as discussed in the next section.We have no reason to assume that backcrosses to either parent as well as Fped well . Thus itid zones . At the 2 hybrids (F1 crossed with F1) and backcrosses to both parental species. Interestingly, F2 hybrids were only found among the offspring of one of the five mother-plants. On the glacier forefield, this female plant stands less than three metres away from a male F1 hybrid . It thus seems that F2 hybrids are only produced in high numbers, when male and female F1 hybrids stand close together. This arrangement is quite rare on the glacier forefield so that female F1 hybrids are closer to male individuals of S. purpurea or S. helvetica in most cases . The parental species and the hybrids occur evenly dispersed over the whole area so that there is no spatial structure like a clumped distribution or a cline from one parental species to the other [1 hybrids are pollinated by one of the purebred species so that they will produce backcrosses. Accordingly, our results showed that the offspring of the sampled F1 hybrids consisted mainly of backcrosses to the parental species and only few F2 hybrids. Similar findings were made in poplars where purebred female plants produced exceptionally large amounts of backcross seedlings when they were surrounded by F1 hybrids [S. purpurea than to S. helvetica in the sample, yet the reason for this result is unclear. Possible causes could be a greater overlap of flowering time between S. purpurea and the hybrids, postzygotic selection against backcrosses to S. helvetica, stochastic factors like a closer proximity between male S. purpurea and female F1 hybrids than between S. helvetica males and F1 hybrids, or sampling bias due to the choice of mother plants in the analysis, the limited number of progenies, or conditions for pollination in the year of sampling. Another possible interpretation could be that pollen limitation is stronger for pollen of S. helvetica than for pollen of S. purpurea. Accordingly, a reduced seed set was found in purebred S. helvetica compared to purebred S. purpurea [S. helvetica could also be transported less efficiently. Pollen-pistil incongruences also represent a strong prezygotic crossing barrier in willows [S. purpurea and S. helvetica, as the former species has a much shorter style and a capsule without a beak, while the latter has long styles and beaked capsules. However, the determination of the exact causes of the observed pattern requires further research. Irrespective of the direction of backcrossing it can be concluded that, at least in this early stage of secondary contact, a higher production of backcrosses than of F2 hybrids renders a future trajectory of introgression more likely than hybrid speciation.In contrast to the findings made in the natural population on the glacier forefield, the offspring raised from seeds formed in the wild consisted of Fhe other . Therefo hybrids . Overallpurpurea . The seepurpurea , and thu willows . Pollen Salix genome.This is one of the first studies on nonmodel plant species where segregation distortion was analyzed with RAD-seq data. The power of this marker system for detecting segregation distortion and linkage groups was demonstrated e.g. on hybrid fish  and on w2 hybrids or backcrosses. It is expected that the magnitude of distorted loci correlates with the level of divergence between the parental species [S. purpurea and S. helvetica turned out to be quite low with a FST value of 0.3 in our study based on microsatellite makers [ST value of 0.24 for completely unfiltered loci, but moderate for the filtered loci (FST = 0.53). This increase of FST is thought to be due to the removal of loci that do not discriminate the parental species. Thus we think that the low FST value based on the unfiltered loci gives a more realistic estimate of the population divergence. Another hint for the low divergence between S. purpurea and S. helvetica is that they hybridize easily although they belong to different, unrelated sections or clades of the genus Salix [Salix, especially in the shrub species [We did not detect significant deviations from the expected Mendelian segregation ratios in the F species . The dive makers . Divergeus Salix , 54. How species –57. Rece1 hybrids produced less seeds than S. purpurea or S. helvetica but that the seeds they produced were viable and developed equally well as seedlings from the purebred species [Iris [Genetic incompatibilities that cause hybrid sterility or inviability and thus act as postzygotic reproductive barriers accumulate with evolutionary divergence of the parental species , 59. We  species . Due to es [Iris .Salix [Salix, as well as in Populus, no heteromorphic sex chromosomes have been discovered yet. Stölting et al. [Populus species on chromosome XIX that carries the sex determining locus in Populus [Populus is not resistant to gene flow and introgression. Accordingly, Macaya-Sanz et al. [Populus. This finding seems to be reflected in Salix due to the low number of species-specific SNPs on chromosome XV detected in this study.Another interesting finding is that only two species-specific loci are located on chromosome XV that carries the sex determination locus in Salix . Other sSalix . Howeverg et al.  did also Populus –64. Theyz et al.  also detSalix helvetica is a shrub that reaches a height of ca. 50–80 cm [Salix purpurea can reach up to 6 m in the lowland [2 hybrids adopted the lower height of S. helvetica, even in the absence of external selection under equal garden conditions. This is striking because a typical feature for alpine shrubs is the reduction of plant height  as the plants are better protected by snow cover during freezing periods [In contrast to the genomic data, segregation became obvious in phenotypic traits in one-year old juvenile plants. 50–80 cm . Salix p lowland  but on t periods , 67. GroS. purpurea and S. helvetica are low. Further, hybrids and the purebred species occur in a mixed stand on the glacier forefield leading to a continuing formation of F1 hybrids and backcrosses. Introgression might be asymmetric because there were more backcrosses to S. purpurea in the sampling. Due to the isolated location, introgression might be highly localized affecting mainly the gene pools of S. purpurea and S. helvetica individuals on the glacier forefield and the surrounding slopes. On the other hand, we already discovered another population of hybrids between S. purpurea and S. helvetica at a higher altitude on the Morteratsch glacier [Range shifts initiated by climate change will increase the likelihood of secondary contact hybridization in some species . Compari glacier , and thuIn general, hybridization appears to increase genetic and phenotypic variability in the offspring population. Interspecific exchange of genes via introgression is considered to be an important evolutionary force because it may lead to the transfer of adaptations . AdaptatWillow hybrids may also serve as models for the evolutionary processes initiated by global warming. Due to their properties as pioneer species, range shift and establishment of willows in novel habitats may be more rapid than in other species. The observations made in this model system may thus help to anticipate evolutionary processes that might affect species with lower dispersal rates much later in time.S. purpurea, S. helvetica, and their hybrids. All plant samples were collected with the permission of the Canton du Valais, Service des forêts et du paysage. All individuals have already been genotyped at nine microsatellite loci in a previous study, which also included some reference populations sampled outside the glacier forefield [Salix purpurea and nine individuals of S. helvetica from the glacier forefield. To extend the data of the purebred species, we also included four individuals of S. purpurea from three additional locations in Germany  and one S. helvetica individual from Austria .Leaf samples were collected at the forefield of the Rhône Glacier in central Switzerland  from a mixed stand of orefield . For theWe included a comprehensive sampling of 45 hybrids from the glacier forefield. These plants were classified as hybrids by both an intermediate phenotype between the parental species and genetic analysis that had been conducted in a previous study . The ide1 hybrid in the NewHybrids analysis of our previous study [1 hybrids at the forefield of the Rhône Glacier were germinated under controlled conditions  were finally sampled for RAD-seq analysis. The five mother plants from the natural population at the Rhône Glacier were also included in the sampling. The final dataset for RAD-seq analysis comprised 133 individuals.Among these 45 hybrids, we selected five hybrids that had a > 95% probability of being a Fus study . To invePstI. The size selection of 300 bp – 500 bp with a Pippin Prep  was followed by the ligation of sequencing adaptors and unique 10 bp barcodes for each sample. The samples were sequenced on an Illumina HiSeq 2500 Instrument  and raw reads were delivered in FASTQ format trimmed to 100 bp.DNA was extracted from silica-dried leaves using the DNeasy Plant Mini Kit  following the manufacturer’s protocol. The DNA concentration was assessed with a Qubit 3.0 fluorometer  and the samples were normalized to a concentration of 30 ng/μl. Aliquots of 3 μg DNA were then submitted to Floragenex Inc.  for library preparation and single-end RAD sequencing (following the protocol of ). The tostacks v. 1.44 [process_radtags program implemented in stacks with the default parameters. In this step, the 10 bp barcodes were removed from the reads so that the final length of the reads was shortened to 90 bp. Afterwards, the quality of each sample was assessed with FastQC v. 0.11.4 [denovo_map pipeline that merges RAD-tags into loci in each sample (ustacks), creates a catalog containing the merged loci from multiple samples (cstacks), and finally matches the loci from each sample against the catalog (sstacks). The first step within the pipeline is the creation of so called stacks (matching reads) out of the raw reads of each individual. The stacks provide the basis for building loci [m) was set to 10. Thus, calling of heterozygotes requires at least 10 reads of each allele. The maximum number of nucleotide mismatches allowed between two stacks was set to 5 both for processing loci within individuals (M) and between individuals (n) when building the catalog. Appropriate values for M and n were determined in preliminary test runs. In these runs, M and n had the same value set between 2 and 7, and the total number of loci as well as the number of polymorphic loci was recorded. Following the recommendations of Viricel et al. [populations program in stacks to extract loci that were present in all three groups  in at least 70% of the individuals in each group (r). Data analysis was restricted to the first SNP at each locus in order to obtain unlinked SNPs required for population genetic analysis. Plink v 1.0.7 [p < 0.05 in one or both parental populations. Further, individuals with a genotyping rate < 90% were also excluded from the analysis leading to the exclusion of two individuals of the hybrid offspring. We also applied further filters to remove potentially paralogous loci resulting from the recent ‘salicoid’ duplication event [o) ≥ 0.6, FIS < 0, or a coverage depth that was greater than twice the standard deviation. Further, we also removed loci with a FST-value of 0 between S. purpurea and S. helvetica in order to restrict the analyses to loci with variation between the parental species. Ho, FIS, and FST-values for each locus were generated by the populations program. The filtered loci were aligned to the Populus trichocarpa genome [Salix suchowensis [The software  v. 1.44 , 74 was . 0.11.4 . Loci weing loci . The minl et al.  we chose v 1.0.7  was usedon event . Collapson event . Thus, wa genome  to detechowensis   of each hybrid individual with the program NewHybrids [2 hybrids, while the progeny as a whole was called second generation hybrids. Due to the young age of the hybrid zone on the forefield (approximately 20–30 years) it can be assumed that all individuals still belong to the early hybrid generations so that an assignment to exact hybrid classes is possible. NewHybrids cannot handle large datasets and thus we restricted the dataset to the first 300 loci of the whole dataset. We also ran NewHybrids with 300 loci that were selected randomly to ensure that the patterns were consistent across different subsets of the genome. The results were the same but we chose to use the first 300 loci to ensure the reproducibility of the results. Structure and NewHybrids were run using a burn-in period of 10,000 followed by 50,000 MCMC iterations. Longer run times were tested using reduced data sets, but did not change the results substantially.Pairwise genetic distances between individuals were calculated using the genetic distance measure of Smouse and Peakall  implemenenReport . Based ort [ade4 . We usedv. 2.3.4  to confio et al. . FinallywHybrids . We desiS. purpurea and S. helvetica for the analysis of segregation distortion so that the origin of an allele in a hybrid individual could be unequivocally determined. We checked whether all 45 F1 hybrids were heterozygous at these loci. Overall, 396 loci met both criteria. The R package introgress [2 hybrids, we tested for the deviation from the expected 1:2:1 distribution of homozygous and heterozygous genotypes found at each locus. In the backcrosses, we tested for the deviation from the expected 1:1 distribution of homozygous and heterozygous genotypes. χ2 goodness-of-fit tests were performed in R [2 hybrids, p-values were computed by Monte Carlo simulation due to the low number of individuals. We corrected for multiple testing using the false discovery rate (FDR) method of [We selected loci showing fixed differences between trogress  was usedmed in R . In the ethod of  with α =last search of all RAD-loci containing species specific SNPs against the S. purpurea genome using Phytozome 12 [We performed a Bozome 12  to deter1 hybrids at the forefield of the Rhône Glacier [− 2 s− 1) under equal soil and watering conditions. At this age, plants had leaves typical for adults , followed by the Games–Howell test as post hoc test. The type I error rate was α = 0.05. The ANOVA was performed using SPSS version 24 .The seedlings grown from five naturally pollinated F Glacier  were raiAdditional file 1:Figure S1. plot of the ∆K-values for the range of K-values tested in structure, Figure S2. examples of the leaf shape of S. purpurea, S. helvetica and their hybrids, Table S1. admixture proportions of 40 F1 hybrids, Table S2. assignment of hybrid class of 40 F1 hybrids. (PDF 288 kb)Supporting information for the Methods and Results section. Contains Additional file 2:Table S3. Distribution of homozygous and heterozygous genotypes found at each locus in the F2 hybrids and distribution of parental alleles in the backcrosses. (XLSX 29 kb)


Daphne rodriguezii (Thymelaeaceae) is endemic to the Balearic island of Menorca, where fragmentation and severe population decline are ongoing threats to this taxon. We developed a set of microsatellite markers to analyze the fine‐scale genetics of its few extant populations.The endangered shrub Daphne species.Fifteen microsatellite markers were obtained through Illumina high‐throughput sequencing and tested in two populations. Twelve of these loci showed no evidence of null alleles and were highly polymorphic, with a mean number of 8.3 alleles per locus. Levels of observed and expected heterozygosity ranged from 0.100 to 0.952 and from 0.095 to 0.854, respectively. Seven to nine of these loci were successfully amplified in five other Daphne species.This set of markers provides a useful tool for investigating the factors driving fine‐scale population structure in this threatened species, and it represents a novel genetic resource for other European  Daphne L. (Thymelaeaceae) comprises ca. 95 species with Eurasian and North African distributions  has recently reassessed its conservation status from “Vulnerable” to “Endangered” , all of them restricted to the eastern area of Menorca and the off‐shore islet of Colom  was prepared following the manufacturer's recommendations and analyzed on an Illumina MiSeq sequencing platform using the Nano 2 × 250 v2 format. A total of 1,507,789 raw reads were processed. The paired‐end reads that passed the Illumina's chastity filter were subject to de‐multiplexing and trimming of Illumina adapter residuals, and subsequently checked with the FastQC v0.11.7 software , the size of the amplified fragment (for optimization of the subsequent design of multiplexed reactions), and their successful amplification in all of the samples. Considering the allele size ranges and the apparent lack of null alleles across the seven individuals tested, we finally used 15 out of the 36 loci to evaluate their usefulness in revealing polymorphism with larger  sample sizes.Microsatellite development was conducted by Ecogenics GmbH  from genomic DNA extracted from silica‐dried Andrews, . FollowiAndrews, , which rAndrews, . MicrosaAndrews, . From thIn examining the levels of variability revealed by each SSR locus, we were constrained by the conservation status (EN) of the study species. However, we were able to obtain permissions to sample leaf material from two populations representing size extremes  linkage disequilibrium was found between loci Dro046 and Dro124, but only for population B.At the population level, the number of alleles per locus ranged from one to 11 Table . The levD. laureola, D. cneorum, D. oleoides), even relatively low sample sizes revealed that at least half of the amplified loci exhibited more than one allele (Table In addition, this panel of microsatellites rendered positive amplifications in a minimum of seven loci per species Table . The limle Table .D. rodriguezii is a powerful, cost‐effective tool for detecting substantial levels of genetic variation using a relatively low number of multiplexed reactions, even in small populations. Such a genetic resolution will allow us to assess parentage relationships in forthcoming studies on fine‐scale genetic structure. Additionally, the successful rates of cross‐amplification of these loci suggest that population genetic studies with these markers could be easily extended to other closely related Daphne species.The set of microsatellites characterized for C.G.‐V. and A.T. planned the study and collected plant tissue, J.C.I. and C.G.‐V. conducted laboratory work and allele scoring, and C.G.‐V. performed the analyses and wrote the manuscript, with input from J.C.I. and A.T.


Visual problem solving is essential to highly visual and knowledge-intensive professional domains such as clinical pathology, which trainees learn by participating in relevant tasks at the workplace (apprenticeship). Proper guidance of the visual problem solving of apprentices by the master is necessary. Interaction and adaptation to the expertise level of the learner are identified as key ingredients of this guidance. This study focuses on the effect of increased participation of the learner in the task on the interaction and adaptation of the guidance by masters. Thirteen unique dyads consisting of a clinical pathologist (master) and a resident (apprentice) discussed and diagnosed six microscope images. Their dialogues were analysed on their content. The dyads were divided in two groups according to the experience of the apprentice. For each dyad, master and apprentice both operated the microscope for half of the cases. Interaction was operationalised as the equal contribution of both master and apprentice to the dialogue. Adaptation was operationalised as the extent to which the content of the dialogues was adapted to the apprentice’s level. The main hypothesis stated that the interaction and adaptation increase when apprentices operate the microscope. Most results confirmed this hypothesis: apprentices contributed more content when participating more and the content of these dialogues better reflected expertise differences of apprentices. Based on these results, it is argued that, for learning visual problem solving in a visual and knowledge-intensive domain, it is not only important to externalise master performance, but also that of the apprentice. Visual problem solving is an essential skill in professional domains such as medicine and aviation. Professionals in these domains solve problems through the interpretation of domain-specific and complex visualizations. Radiologists, for example, make use of CT-scans to diagnose their patients. Several studies have revealed differences in the knowledge structures and problem solving strategies used by experts and non-experts during these tasks  and may also cause artefacts in the image. Moreover, the image ought to be ‘read’ at different levels of magnification, requiring the skill of microscope navigation. To summarize, in order to provide diagnoses, aspiring clinical pathologists have to obtain domain knowledge to understand the domain-specific visualizations of tissue and to identify abnormalities in them through the appropriate manipulation of the presented image.The knowledge described in the previous section is obtained by trainees in an apprenticeship-like training programme at the workplace. Typically, observation of expert performance (‘modelling’) is a crucial component of the learning process of apprentices  when collaboratively diagnosing tissue with expert pathologists (from now on: masters). The increase in participation consists of apprentices operating the microscope instead of the master (as in daily practice). The following two hypotheses are central to this study: (1) The interaction of learning dialogues in clinical pathology increases when the apprentice operates the microscope, compared to when it is operated by the master; and (2) The adaptation of learning dialogues in clinical pathology increases when the apprentice operates the microscope, compared to when it is operated by the master.Interaction is thereby understood as the equal contribution to the dialogue by apprentice and master, both qualitatively and quantitatively. This does not mean that the actual input of master and apprentice is equal—there obviously is a knowledge gap—but that both contributors discuss meaningful content, each to their own capabilities. Adaptation is understood as to whether the content of the dialogue matches the expected expertise level of the apprentice, based on expertise studies among clinical pathologists  and 13 residents , who formed unique dyads consisting of a master  and an apprentice (resident). Two groups of dyads were created based on the experience of the apprentice. Low-expertise (LE) apprentices (7 dyads) were in their first year of training and/or had no experience with the specific organ yet (colon). High-expertise (HE) apprentices (6 dyads) were in their second year of training or higher, and had experience with diagnosing colon tissue. The participants were recruited from three academic hospitals in the Netherlands on a voluntary basis and were rewarded with a small gift voucher after the experiment. The study was approved of by the department’s ethics committee and all participants gave written informed consent.The participants in this study were 13 clinical pathologists  on the two constructs, interaction and adaptation. These two constructs each had their own independent variable: conversational role (master or apprentice) for interaction, and the apprentice’s expertise level  for adaptation. Both constructs, interaction and adaptation, were operationalised using coding categories of features and elements of the conversations as dependent variables . This set-up led to two three-way interactions: microscope operation*conversational role*coding categories/descriptive variables (for the construct of interaction) and microscope operation*apprentice’s expertise level*coding categories (for the construct of interaction). A more detailed description of the variables, predictions, and hypotheses per construct is provided in the “Each dyad discussed and diagnosed six cases, which were digitally scanned microscope images of colon tissue. The images were obtained from the Atrium Medical Centre in Heerlen, the Netherlands. To ensure an actual learning dialogue, difficult cases were selected . Diagnostic accuracy confirmed the complexity, as none of the cases was correctly diagnosed by all dyads (ranging from 0 to 82%). Cases 3 and 5 were particularly difficult as they contained rare abnormalities . The six cases were presented according to a balanced Latin square. The microscopic images were viewed with the Aperio ImageScope digital microscope (version 11.2.0.780). To simulate the normal training situation—in practice a double-headed light microscope is used—two synchronised monitors were used .A demographic questionnaire was used to collect background information on the participants, including sex, age, vision and experience with diagnosing colon tissue and with the digital microscope.The dialogues between the master and apprentice were recorded by cameras with an in-built microphone that were mounted on both monitors.To simulate practice, the apprentices diagnosed the six cases individually (taking 30–45 min), prior to discussing them with the master. The actual experiment began with the participants completing the demographic questionnaire and being instructed on the experiment’s procedure. As they were not all used to a digital microscope, participants were given time to acquaint themselves with its operation. Every case started with reading out aloud the patient background information by the participant that operated the microscope. The same participant then opened the microscopic image and joint examination was started. Viewing time was unrestricted and each case ended with a diagnosis or—in cases where the dyad could not come to a conclusion—a request for further testing. Participants were instructed to behave as they would do in practice. After three cases microscope operation was alternated.The experiment resulted in 73 transcribed dialogues; five recordings (divided over 2 dyads) failed due to time restrictions or technical problems. All acknowledgments (including ‘hmhm’s’) were included as separate turns, as these were of interest to the study. QSR International’s NVivo 10 software was used for analysis, which automatically attributed dialogue turns to master or apprentice, based on the structure of the transcripts.The coding scheme was designed both in an inductive and in a deductive manner, following methods known as ‘descriptive coding’ and ‘hypothesis coding’, respectively Saldana . The hypThe 15 content categories are the measures for the two constructs, interaction and adaptation. Based on previous studies , questions asked by apprentice, and number of short turns by apprentice (1–3 words). The independent variable in this analysis was microscope operation. Because of the small sample sizes, non-parametric tests were used (Mann–Whitney).In addition, three quantitative measures of interaction were derived from the verbal data: To measure the effect of microscope operation on the adaptation of the dialogue to the level of the apprentice, the content of dyads with low-experienced apprentices (LE dyads) was compared with that of dyads with high-experienced apprentices (HE dyads). Based on literature , nor the number of words .The learning dialogues had a median time-on-task of 223 s and a median of 575 words. Microscope operation affected neither the time-on-task (This section discusses the results per construct.2(0) = 0, p = 1. This implies that its highest-order interaction (content categories*operator*contributor) was significant, χ2(14) = 98.68, p < .01. Masters and apprentices thus contributed different content to the dialogue, depending on the microscope operation. To break down this effect, separate Chi squares were conducted for microscope operation by masters and apprentices. The left pane of Table The three-way loglinear analysis produced a model with a likelihood ratio of χ2(14) = 209.36, p < .01. This means that masters and apprentices contributed different content to these dialogues. More in particular, when masters operated the microscope, they contributed more comments on the diagnosis, difficulty of the case, pathology knowledge and the knowledge of the apprentice. Apprentices contributed many turns that fell in the category of ‘acknowledgments’.When masters operated the microscope, there was a significant association between the content categories and the contributor, χ2(14) = 198.76, p < .01. When operating the microscope, apprentices contributed most comments on the givens of the case, simple and comparative findings, and their own knowledge and skills. Masters contributed most comments on further testing, pathology knowledge, and the experimental procedure. A visualisation of these results is given in Fig. When apprentices operated the microscope, there also was a significant association between content categories and the contributor, χ24 = 198.7U = 414.00, z = − 2.78, p < .01, r = − .33) and the number of questions asked by the apprentice . When they operated the microscope, apprentices had a larger share in the total words (Mdn = 47.52 vs. Mdn = 41.13) and asked more questions (Mdn = 2.50 vs. Mdn = 1.00) compared to when the master operated the microscope. Microscope operation did not affect the number of short turns by the apprentice .Microscope operation affected the coverage of the apprentice (2(0) = 0, p = 1. This means that the three-way interaction (content categories*operator* apprentice experience) was significant (χ2 (14) = 51.94, p < .01), indicating that the adaptation of masters to the apprentice was affected by who operated the microscope. To break down this effect, separate Chi square analyses were performed for those sessions operated by the master and by the apprentice. The right pane of Table The three-way log linear analysis resulted in a model with a likelihood ratio of χ2(14) = 70.92, p < .01. This means that there was different content being discussed by LE and HE dyads. More specifically, HE dyads discussed the givens of the case and the workflow more than LE dyads. The latter discussed more simple findings.When masters operated the microscope, there was a significant association between the content categories and the apprentice experience, χ2(14) = 86.80, p < .01. This implies that different content was discussed by LE and HE dyads in these dialogues: HE dyads discussed diagnoses, case difficulty, further testing, and the workflow more than LE dyads. LE dyads discussed more heuristics. Figure When apprentices operated the microscope there also was a significant association between the content categories and the apprentice experience, χTable This study tested two hypotheses on the effect of increased apprentice participation in a relevant workplace task on the interaction and adaptation in learning dialogues. Hypothesis 1 stated that the interaction between master and apprentice increases with increased apprentice participation . Apprentices were predicted to have a greater share in the dialogue, both in quantity and quality. Most of these predictions were confirmed. When operating the microscope, apprentices contributed more to the dialogue, asked more questions, and were less passive (expressed by number of acknowledgments) than when masters operated the microscope. These results suggest that there was more interaction between master and apprentice in the dialogues with increased apprentice participation.Hypothesis 2 stated that increased apprentice participation stimulates adaptation of the dialogue to the apprentice’s level, expressed in more basic findings for low-experienced apprentices and more ‘advanced’ content  for high-experienced apprentices. These predictions were mostly confirmed. When apprentices operated the microscope, dialogues with high-experienced apprentices contained more diagnoses, difficulty, further testing, and workflow, while low-experienced apprentices discussed more heuristics. These differences in content correspond with expertise differences between novices and intermediates in clinical pathology  and also much more than when they operated the microscope themselves. ‘Further testing’ entails additional tests to confirm or exclude the presence of abnormalities. These tests are thus only requested when no certain diagnosis could be reached based on the available material. This relatively high frequency could mean that masters do not feel certain about their conclusion when they are not acting hands-on themselves. They tried to overcome this by steering the apprentice to relevant areas, based on the high frequency of ‘process’ comments. However, it could be that they did not succeed in verbalising everything they wanted to check, and thus resorted to additional tests.What do these results teach us for the training of apprentices in clinical pathology and other visual and knowledge-intensive professions? Most importantly, the covertness of visual problem solving expertise does not only count for experts—as was argued in the Introduction—but also for apprentices: Just like it is difficult for apprentices to know what masters actually do, it is difficult for masters to detect what apprentices do. When apprentices’ participation increased, allowing apprecntices to show their ability level, the content of the dialogues changed for the better . The conversation focused more on their expertise, instead of that of the master. An interesting example is the increase in the heuristics that were discussed. Heuristics are rules of thumb on how to make sense of an image, for example to compare certain parts of the image with others. It is very likely that masters are prompted to share such rules of thumb when they actually see how an apprentice goes about diagnosing an image. When the master is controlling the image, this would either require a constant verbalisation by the master of the actions carried out (and why!) or questions from the apprentice to go beyond learning by mere observation. Similarly, the master could also choose to ‘interrupt’ an apprentice’s diagnostic reasoning when the followed routine would not lead to the correct diagnosis. As Moulton et al.  argue, tIt could thus be valuable for training purposes to externalise the knowledge of apprentices by increasing their participation in meaningful tasks. However, the training of clinical pathologists—as any form of workplace learning—is nested within an operational medical department. Clinical pathologists are diagnosticians just as much as they are educators. To determine whether microscope operation slowed the working process down, a quick analysis of the effect of microscope operation on time-on-task was carried out. When all cases were analysed together, no effect of microscope operation on time-on-task was found. However, when dyads were inspected separately, it turned out that for most dyads (8 out of 13) the cases in which the apprentice drove the microscope had a higher average time-on-task. In addition with more requests for further testing being made when apprentices operate the microscope, it is probably more beneficial for the productivity of the pathology department to have apprentices observe masters, rather than vice versa. However, investing in extra time to increase the participation of pathology residents could lead to more effective training of the future pathologists. As pathology might not be the most time-pressed and—in terms of patient well-being—most precarious medical specialty  it might form an appropriate context to increase learners’ participation. However, when considering the implementation of such a teaching innovation, it is important not to look only at its costs, but also at its benefits. If total training time could be diminished with a slightly decreased workflow, this still might be an efficient intervention.To determine whether the expected gain in effectivity outweighs the investment of an impaired work process, future studies should focus on the performance gain of increased participation in working tasks. Not measuring performance gain is the most important limitation of this study. Another missing aspect and therefore interesting direction for future studies would be to include the strategies that experts use in their guidance of apprentices. This could reveal which strategies are most effective, and how these differ with the expertise level of apprentices.To conclude, this study has explored new ground by studying in detail the learning dialogues as they take place at the workplace, in a highly visual and knowledge-intensive domain. Previous studies focused on parts of this process, such as visual but content-free puzzle tasks Gergle , or stud


Urban scaling research finds that agglomeration effects—the higher-than-expected outputs of larger cities—follow robust “superlinear” scaling relations in cross-sectional data. But the paradigm has predictive ambitions involving the dynamic scaling of individual cities over many time points and expects parallel superlinear growth trajectories as cities’ populations grow. This prediction has not yet been rigorously tested. I use geocoded microdata to approximate the city-size effect on per capita wage in 73 Swedish labor market areas for 1990–2012. The data support a superlinear scaling regime for all Swedish agglomerations. Echoing the rich-get-richer process on the system level, however, trajectories of superlinear growth are highly robust only for cities assuming dominant positions in the urban hierarchy. Urban scaling has evolved into an important paradigm for the study of socioeconomic agglomeration effects –3. It fintinents  based onanother” .A dynamic approach to urban scaling has been recently pioneered based on traffic data capturing time delays in 101 US metropolitan areas over time . While tHere, I use geocoded microdata on wage income from Swedish population registers for 1990–2012 to monitor the scaling trajectories of cities as their populations grow. My report provides compelling data to resolve this controversy and, taking a microlevel approach, provides a conceptual advance in the study of cities’ growth trajectories.A longitudinal perspective conflates variations in city sizes with economic development and social change and, to isolate the effect of city-size variations, we must partial out concomitant socioeconomic trends. Most importantly for wages as the observed urban output, these trends include gains in gross domestic product (GDP), educational expansion, increases in female labor force participation, and changing migration patterns. To exclude a large portion of socioeconomic change, I restrict my analysis to the Swedish-born working-age male population, scrutinizing a total of 1.12 million fully employed men nested in 73 labor market areas (LMAs), Sweden’s functional demarcation of metropolitan areas .age R2=0.05. SuperInset plots the variation of estimated r cities C. SuperlSo far, the trajectories include wealth creation due to economic development and social change. Inter alia, Sweden experienced 2 economic downturns in 1990–1993 and 2008–2012, leaving visible imprints—slight S curves—on cities’ growth trajectories. mployees 3, reprodBy design, the full microdata model approximates most closely the established cross-sectional interpretation of In favor of the paradigm’s predictive ambitions I find that superlinear scaling governs the trajectories of all agglomerations in Sweden’s urban system. At the same time, I also find superlinear growth to be nonrobust in LMAs with fewer than 75,000 inhabitants and the power law’s empirical fit much better for larger cities than for smaller ones. This qualitative difference signifies cities’ various positions in an urban hierarchy , 7, 8 anI use geocoded microdata assembled by Statistics Sweden covering the country’s entire urban system represented by 75 LMAs that, from the smallest  to the largest (2.51 million inhabitants), span 4 orders of magnitude. Government agencies, including tax authorities and educational institutions, collected and directly reported the data.I exclude all workers from the mining areas Gällivare and Kiruna in the far north of Sweden, whose wages depend primarily on the presence of natural resources. I further restrict my analysis to the 1.12 million Swedish-born males aged 18–60 y fully employed for at least 2 y during 1990–2012. I exclude women because of both marked fluctuations in female labor force participation  and a shrinking gender wage gap . I also exclude foreign-born men because of a strong increase in the migrant population in Sweden (from 9.2% of the full population in 1990 to 15.4% in 2012) and the concomitant changes in immigrants’ labor force participation and the varying disadvantages they face along different career paths. These system-wide trends must not affect the longitudinal estimation of I use individuals’ gross annual wage income  as an indicator of local wealth creation. Average annual wage increased 1.66-fold, from 195,000 kronor in 1990 to 324,000 kronor in 2012. In the city-level analysis 1 and 2 ITo estimate cross-sectional β A, I lineTo trace the scaling trajectories of l cities B, I substy level , capturiThe microlevel version of These models permit the adding of control variables to partial out socioeconomic change and to approximate the net size effect on per capita wage. To the aggregate-level model 2, I add el model 3, I add ficients : On the ficients —work exp