Breast cancer is the commonest malignancy in women worldwide. It is estimated to affect approximately 1.5 million women annually and responsible for the greatest number of cancer-related mortalities among women. In 2018, breast cancer mortalities stood at 627,000 women representing approximately 15% of all cancer deaths among women. In Ghana, breast cancer is the second leading cause of cancer deaths, with an incidence of 2,900 cases annually; one of eight women with the disease die. This gives impetus to the fight for improved early detection, treatment, and/management. In this light, we investigated the potential of death-associated protein kinase 1 (DAPK1) as a biomarker for breast cancer. As a tumour suppressor, its expression is activated by several carcinogens to influence cellular pathways that result in apoptosis, autophagy, immune response, and proliferation.  To investigate DAPK1 as a blood biomarker for breast cancer. p ≤ 0.05. Results and Discussion. DAPK1 levels were higher in sera and breast tissues of breast cancer patients than controls. The augmented DAPK1 expression can be interpreted as a stress response survival mechanism to remediate ongoing deleterious events in the cells orchestrated by carcinogenesis. In the presence of abundant DAPK1, the proliferative power of cells (both cancerous and noncancerous) is increased. This may explain why high DAPK1 expression strongly associates with aggressive breast cancer phenotypes like the ER-negative breast cancers, especially the triple-negative breast cancers (TNBC) which are the most aggressive, fast-growing, and highly metastatic.  Blood samples of participants diagnosed with breast cancer and healthy controls were collected and processed to obtain serum. Information on age, treatment, diagnosis, and pathology numbers was retrieved from folders. Pathology numbers were used to retrieve breast tissue blocks of patients at the Department of Pathology of the KBTH. Tissue blocks were sectioned and immunohistochemically stained with anti-DAPK1 and counterstained with hematoxylin to determine the DAPK1 expression levels. DAKP1 levels in blood sera were quantified using a commercial anti-DAPK1 ELISA kit. Case and control group means were compared using one-way ANOVA and Chi-square test. Statistical significance was set at  DAPK1 is highly expressed in sera and breast tissues of breast cancer patients than nonbreast cancer participants. The elevated expression of DAKP1 in circulation rather than in breast tissues makes it a candidate for use as a blood biomarker and potential use as therapeutic target in drug development. The World Health Organisation (WHO) report for 2018 indicates that cancer is the second leading cause of death globally accounting for 9.6 million deaths and further state that nearly 1 in 6 deaths globally is due to cancer with about 70% of these deaths occurring in low- and middle-income countries . CurrentThe incidence of breast cancer in developing countries in Africa particularly has increased, deviating from earlier reports . The disThe high mortality rates of the disease in developing countries are mostly attributed to late diagnosis and poor access to treatment. To improve breast cancer outcomes and survival, early detection is critical. Biomarkers have proven their usefulness in predicting disease incidence, disease progression, and disease outcomes. Death-associated protein kinase 1 (DAPK1) is part of a group of kinases like DAPK2, DAPK3, DAP kinase-related apoptosis-inducing protein kinase 1 (DRAK1), and DRAK2 . LocatedPhysical, chemical, and biological carcinogens which stimulate DAPK1 increase the expression of the protein via the activation of the dephosphorylation of ser 308 and p53 through the p14/p19ARF pathway, which results in programmed cell death (apoptosis), autophagy, immune response, and proliferation , 22. TheParticipants were women clinically diagnosed with breast cancer at Korle-Bu Teaching Hospital (KBTH), Accra, Ghana. Controls were apparently healthy individuals in the same facility and its immediate environs.Blood (5 ml) was drawn from consented participants at the chemotherapy suite of the Department of Surgery, Korle-Bu Teaching Hospital during regular treatment sessions of patients when patients' veins were punctured using a cannula for drug administration. This was done prior to drug administration to prevent double puncture of the veins of the participants who were prepared by assigned nursing officers. Blood samples were collected into serum separator tubes and allowed to clot at room temperature and centrifuged at 2500 rpm for 20 minutes. The sera were collected into fresh tubes and stored at -20°C until use. In total, 32 breast cancer patients and 32 apparently health women participated in the study as cases and controls, respectively.μm thick tissue sections, which were placed on poly-lysine-coated glass slides.Pathology identification numbers were noted from pathology reports of patients in their hospital folders , and their archived breast biopsy tissue blocks and haematoxylin and eosine (H&E) stained tissue sections were tracked and retrieved from the Department of Pathology of the Hospital. Retreived H&E stained tissue slides were screened, and the tumour grades confirmed by a pathologist. The corresponding paraffin-embedded tissue blocks of the H&E stained tissue slides were prepared for sectioning to obtain 4 μl of 1XPBS was gently dispersed in the wells and shaken gently for 30 seconds, then decanted. Further drying was done using filter paper or pad. Following this, 50 μl of each sample and standards was pipetted into designated ELISA plate wells precoated with anti-DAPK1. The samples were added to horseradish peroxidase-labeled recognition antigens specific to DAPK1 in the wells and incubated at 37°C for 1 hour. TMB substrate solution was added to each well and incubated for 10 minutes; after which, a stop solution was added to each well. The optical density (OD) was measured using a spectrophotometer at 450 nm. DAPK1 concentrations were determined from standard curves.Stored sera were retrieved and allowed to thaw at room temperature. Following manufacturer's instructions, the wells of the ELISA plate precoated with anti-DAPK1  were washed to condition them for the antigen-antibody interactions. 100 Tissue sections were dewaxed at 50°C (5 min) and in xylene (5 min). They were subsequently put in decreasing grades of ethanol; absolute, 90%, 70%, 50%, 40%, 25%, and finally into water. Citrate buffer (pH = 6.0) was subsequently used for antigen retrieval. Coplin jars containing 1x citrate buffer were heated to boiling point in a pressure cooker on a hot plate. At boiling point, the pressure cooker was opened, and the hydrated tissue sections were transferred into the coplin jars in the pressure cooker and the lid secured. As soon as the cooker had reached full pressure, the coplin jars and their contents were allowed to boil for 5 minutes after which the heat was turned off and placed in an empty sink. The pressure valve was subsequently released, and cold water was run over the cooker. Once depressurized, the lid was opened and allowed to sit and cool before slides were removed and transferred into distilled water.2O2) for 10 minutes at room temperature. Sections were thoroughly washed thereafter with 1XPBS. Subsequently, bovine serum albumin (BSA) was used for blocking. The sections were covered with BSA for 10 minutes at room temperature and then washed with 1XPBS. Tissue sections were then incubated with the primary anti-DAPK1 solution  for 5 hours at room temperature. The dilution factor for the anti-DAPK1 solution was 1 : 50 with 1XPBS as diluent. Sections were washed with 1XPBS after the incubation period after which they were incubated with a biotinylated secondary antibody for 20 minutes at room temperature. Sections were rinsed again in 1XPBS and treated with horseradish peroxidase-conjugated streptavidin (HRP)  solution for 10 minutes at room temperature. DAB substrate solution was then dispensed onto the sections for colour development. Tissue sections were rinsed thoroughly with tap water and counterstained with Mayer's hematoxylin  and mounted with DPX .To quench endogenous peroxidase activities, the cooled tissue sections were flooded with 3% hydrogen peroxidase solution . The scoring was based on the chromogenic intensity of the DAB reaction on the tissue sections under the microscope. Sections that showed no DAB reaction were scored zero (0). Sections with mild reaction were scored +1, those with moderate staining reaction were scored +2, and those that showed strong reaction scored +3. Thirty-two tissue biopsies corresponding to the 32 recruited breast cancer patients were successfully retrieved, sectioned, and stained. Also, thirty-two (32) breast biopsies that had been confirmed as noncancerous were randomly selected to serve as control (nonbreast cancer) and for comparison. Pictures of the sections were captured using an Olympus digital camera (model DP20) mounted on an Olympus microscope (model BX51 TF).t-test and one-way ANOVA were used to compare means of grouped quantitative data. Comparison between categorical data was done using Chi-square test. In all analyses, p value ≤ 0.05 was considered statistically significant. Values are presented as mean ± SD to two decimal places.Statistical analysis was performed using SPSS 23 software. Descriptive statistics were used to summarize the data. Student's The Institutional Review Board (IRB) of the Korle-Bu Teaching Hospital and the Ethical and Protocol Review Committee of the School of Biomedical and Allied Health Sciences, College of Health Sciences, University of Ghana both gave approval for the conduct of the study with clearance numbers STC/IRB/000100/2016 and MD/10550649/AA/5A/2016-2017, respectively.Sixty-four (64) participants were recruited; 32 breast cancer patients (cases) and 32 healthy individuals (controls). All participants were females living within the Greater Accra region of Ghana and its environs. Of the breast cancer cases, 10 were petty traders, 15 were unemployed and 7 were government workers . Their mp value of 0.039 (treatment) and those yet to be placed on treatment (pretreatment). Of the 32 cases, 17 were classified “treatment” and 15 “pretreatment”. From treatment group had slightly elevated serum DAPK1 expression than the pretreatment group, the difference was not statistically significant.DAPK1 expression in sera of breast cancer patients was significantly higher than in the controls with the values 4.95 (SD = 1.53) ng/ml and 3.49 (SD = 1.72) ng/ml, respectively, and of 0.039 . A furthp < 0.001).Immunohistochemically stained breast tissue sections were scored by two qualified experts. Regarding the staining intensities, there were no inter-observer differences. We have previously reported that DAPK1 was elevated in archived serum samples of breast cancer patients compared to nonbreast cancer individuals and thus the protein was associated with aggressive breast tumour phenotypes in Ghanaians . In furtPhysiologically, the expression of DAPK1 is activated by several carcinogens to influence cellular pathways that result in apoptosis, autophagy, immune response, and proliferation , 22. DAPFrom the immunohistochemistry results , tissue Of the 32 breast cancer cases, 1, 25, and 4 were of grade I, grade II, and grade III, respectively  and 2 untreatment” group was established which had participants who were already receiving chemotherapy for breast cancer prior to their enrollment into the study while a “pretreatment” group had participants who were not on chemotherapy though diagnosed of breast cancer. Participants in the “treatment” group were already on medications such as Doxorubicin, Cyclophosphamide, Paclitaxel, Trastuzumab, and 5-Fluorouracil; drugs which aim at destroying tumour cells in tissues and metastasized cancerous cells in the blood through apoptosis [treatment” group compared to the “pretreatment” group, the difference was not statistically significant. From the results, it appears that the chemotherapy drugs do not affect DAPK1 expression. This could be due to the quick degradation of the drugs by the body after their administration. Our observation corroborates the report of Lui and Chu [For the reliability of DAPK1 as a noninvasive biomarker, the effect of chemotherapy on serum DAPK1 expression was evaluated in breast cancer patients. A “poptosis , 29. Fro and Chu  who compIn summary, augmented DAPK1 expression in cancers can be interpreted as a stress response survival mechanism to remediate ongoing deleterious events in the cells orchestrated by carcinogenesis. DAKP1 was highly expressed in samples of breast cancer patients than in controls. Comparing levels of expression, the protein was highly abundant in sera compared to breast tissue biopsies. Also, chemotherapy did not interfere with DAKP1 expression levels and therefore makes the protein quite stable and reliable. DAPK1 may have potential application as a blood biomarker for breast cancer as well as the prospect for developing new therapeutic strategies. If increased expression is associated with aggressive tumours, then, targeting DAPK1 could be a treatment method as well for curtailing tumour aggression and progression.DAPK1 is highly expressed in sera and breast tissues of breast cancer patients than nonbreast cancer participants. The expression of the protein seems to have an association with tumour grades and thus increases with increasing tumour grade. Furthermore, DAPK1 levels are relatively substantial in sera than in breast tissue biopsies. The elevated expression of DAKP1 in circulation rather than in breast tissues makes it a candidate for use as a blood biomarker and holds prospect for developing new therapeutic strategies. With the observed increased expression of DAPK1 in association with breast cancer, the protein can well be a target for therapeutics and may potentially serve as a biomarker for tumour aggression and prognosis of the disease. Further studies in this direction will be insightful.


Isoetes, a group of mostly aquatic and semi-aquatic isoetalean lycopsids, which closely resemble fossil forms dating back to the Triassic. Rate variation observed in chloroplast genomes hampers accurate dating, but genome-wide nuclear markers place the origin of extant diversity within this group in the mid-Paleogene, 45–60 million years ago. Our genomic analyses coupled with a careful evaluation of the fossil record indicate that despite resembling forms from the Triassic, extant Isoetes species do not represent the remnants of an ancient and widespread group, but instead have spread around the globe in the relatively recent past.The fossil record provides an invaluable insight into the temporal origins of extant lineages of organisms. However, establishing the relationships between fossils and extant lineages can be difficult in groups with low rates of morphological change over time. Molecular dating can potentially circumvent this issue by allowing distant fossils to act as calibration points, but rate variation across large evolutionary scales can bias such analyses. In this study, we apply multiple dating methods to genome-wide datasets to infer the origin of extant species of  Determining the evolutionary relationships and divergence times between lineages is crucial for understanding the processes that generate diversity and evolutionary novelty –3. FossiGinkgo [Analyses of DNA sequences over the last few decades have resolved the phylogenetic relationships between many extant lineages, and large numbers of selectively neutral changes in the genomes allowed inferring accurate phylogenies even for the most morphologically uniform organisms , 22. MolGinkgo , howeverGinkgo , 32–37. Ginkgo , 38–41. Isoetes exemplifies many of the problems of “living fossil” taxa. The genus has long been of interest due to its status as the last lineage of the isoetalean lycopods. This group, known from at least the late Devonian, dominated terrestrial floras in the Carboniferous [Isoetes genus is a small herbaceous aquatic or semi-aquatic plant, generally lacking a stem and consisting of a number of stiff leaves atop a woody corm [Isoetes growth form are found in the Triassic onwards, although their exact affinities and relationships to Isoetes are unclear [Isoetes appear at this time, although no single fossil displays all of these features [Isoetites rolandii in the Jurassic represents the earliest clear example of a isoetalean lycopsid containing all the major features uniting modern Isoetes, including the loss of both vegetative leaves and an elongating stem [Nathorstiana persisted until the Early Cretaceous [Isoetes growth form (e.g. I. horridus) are subsequently found from the Early Cretaceous and into the Tertiary [Isoetes lineages, a number of reductions from three to two lobed corms have occurred [Isoetes appears to have persisted virtually unchanged since at least the Jurassic, and the general growth habit in the lineage is potentially as old as the Triassic.The lycopod genus niferous . The extody corm . It demoody corm  and aquaody corm . Fossils unclear –48. A vafeatures . The apping stem , 50, altetaceous . FossilsTertiary , 48. Witoccurred , along woccurred . In summIsoetes suggests the extant species could be the remnants of a very ancient genus. However, establishing the relationship between these fossils and modern Isoetes has proven difficult due to the highly conserved morphology of the genus [Isoetes species have a global distribution, yet display very little morphological variation–features such as spore morphology, corm lobation and habitat are currently used to distinguish extant species, but many of these features are homoplastic or variable within species [Isoetes, restricting our ability to understand the temporal origins of extant Isoetes species diversity.The close resemblance of fossil taxa to modern he genus , 48, 50. species –54. MorpIsoetes have been inferred using molecular phylogenetics [Isoetes represented the earliest split within the genus, but molecular phylogenetics falsified this hypothesis for extant species [Isoetes species diversity in the Triassic to Jurassic, with species distributions consistent with the breakup of the Gondwana supercontinent [Isoetes and its sister group Selaginella, resulting in a large genetic distance between the nearest calibration point and our node of interest [Isoetes using a combination of phylogenomic methods capturing markers spread across the genomes of numerous land plants.The relationships between extant species of genetics , 55, 56,genetics  hypothes species , 55. Recontinent , 57, 58.ontinent . This isinterest , 59. We Isoetes species and apply multiple molecular dating approaches to estimate the time to the most recent common ancestor of extant Isoetes based on nuclear and plastid genomes. Our results shed new light on the age and evolutionary dynamics of this “living fossil” lineage, and show how careful integration of large genomic datasets can help analyses of groups with a poorly informative fossil record.In this study, we generate transcriptomes and genomic datasets for multiple Live plants were collected from Cwm Idwal, UK, with permission from Natural Resources Wales, the Snowdonia National Park Authority, and the landowners . No permit was required for this collection and no protected species were sampled. DNA was acquired from preserved modern specimens permanently deposited at Kew Gardens, UK–specimen numbers are available in Isoetes for generating genome-wide DNA datasets–I. coromandelina . This resulted in an estimated crown date of Isoetes of 153.4 Ma (53.9–277.3 95% CI), only a 7% increase compared to the full species sampling. This indicates limited taxon sampling should not substantially alter the estimation of the Isoetes crown node date.In this study, we selected six d Rajput ), I. hum studies . Analysehe genus . Sparse  sequenced in other studies. The phylogenetic breadth of the datasets allowed the incorporation of fossil evidence providing calibration points spread across the tree.Previous studies of the chloroplast marker  Isoetes , 70, 71. Isoetes  as sugge Isoetes , 73. Genaracters , 75. A r species , 75. We Isoetes lacustris were sampled from Cwm Idwal, Wales and maintained at the University of Sheffield in 40 x 30 x 25 cm transparent plastic containers, with a substrate of sand to a depth of 5 cm, and the containers filled to the top with deionised water. These were placed in a Conviron growth chamber with a 12-h day/night cycle, 495 μmol m2s-1 light, temperature at 20°C during the day and 18°C at night, and CO2 at 400 ppm for six days. To maximise the number of transcripts retrieved, leaves from three individuals were sampled 3 hours after dark and 3 hours after light and stored immediately in liquid nitrogen. We also generated a transcriptome for Littorella uniflora (Plantaginaceae), another species of aquatic plant that shares aquatic CAM photosynthesis [I. lacustris–DW1, L. uniflora–DW2).Live ynthesis . IndividTM RNase inhibitor (Invitrogen) to 50 μl of extracted RNA to stabilise it. RNA was quantified using a gel electrophoresis, RNA 6000 Nano chips (Aligent) in an Aligent 2100 Bioanalyser, and a Nanodrop 8000. Samples were then prepared for Illumina sequencing using the TruSeq® RNA Sample Prep Kit v2 (Illumina). Paired-end sequencing was performed on an Illumina HiSeq 2500 platform available at Sheffield Diagnostic Genetics Service in rapid mode for 100 cycles, with 24 libraries pooled per lane of flow cell (other samples were from the same or different projects).RNA was extracted from the sampled leaves using the RNeasy® Plant Mini Kit (Qiagen), following the manufacturer protocol, with the addition of on-column DNase I digestion (Qiagen RNase-Free DNase Set). We then added 2.5 μl SUPERase-InIsoetes species were acquired from the DNA Bank from the Royal Botanical Gardens, Kew  were discarded either due to poor homology or alignment difficulties, or because sequences were obtained from less than 10 of the 33 chloroplast genomes analysed. The remaining 52 chloroplast genes were concatenated, producing a 55,542 bp matrix, with 33,582 polymorphic and 25,501 parsimony informative sites. A maximum likelihood phylogeny was generated using RAxML, with a GTR + G + I model of sequence evolution, determined to be the best-fit model using hierarchical likelihood ratio tests. The same matrix was later used for molecular dating.Chloroplast protein-coding genes were identified from all chloroplast genomes using DOGMA  and codiI. lacustris were assembled using Trinity v2.3.2 [L. uniflora, with an average length of 769bp. For each species, the longest open reading frames (ORFs) were extracted using TransDecoder, and for each unigene the contig with the longest ORF was used to build a reference dataset. Cleaned reads from the whole-genome sequencing for each of the Isoetes species were then separately mapped to this reference dataset using bowtie2 v2.3.2 [I. lacustris transcript.Cleaned RNAseq reads of y v2.3.2 , resulti2 v2.3.2  in local2 v2.3.2 . The SAMIsoetes, including in I. lacustris which has previously been identified as a decaploid [I. lacustris and the other land plants. Families of homologous ORFs generated by the method of Vilella et al. [Physcomitrella patens, Selaginella moellendorffii, Amborella trichopoda, Oryza sativa, Arabidopsis thaliana and Theobroma cacao) were used for subsequent ortholog identification.Gene duplication and losses are common in nuclear genomes–polyploidy is common in ecaploid . Therefoa et al.  were dowI. lacustris, L. uniflora and the additional embryophyte species to homolog families, with a minimum match length of 50 amino acids and e-value of 10−7. The expanded homolog families were then aligned according to their protein sequences using MAFFT, and phylogenies were constructed using RAxML and the GTR + G + I model, which fits most genes and is therefore appropriate for constructing large numbers of gene trees [I. lacustris, S. moellendorffii and either P. patens or Ceratodon purpurea, which were needed to use some of the fossil calibration points. By restricting our analysis to these 1:1 orthologs, we eliminate the possibility of non-orthologous genes resulting from gene or genome duplications being considered as orthologs. These 2,165 orthogroups were realigned, and consensus sequences of the genome skimming data were added to the alignments. Only the 782 orthogroups containing sequences for I. coromandelina sensu lato, which is necessary to capture the earliest split among extant species of Isoetes [Isoetes plus Selaginella, [Isoetes were considered phylogenetically uninformative and excluded. The remaining 292 datasets were deemed suitable for the phylogenetic problem addressed here, and were used for molecular dating. A phylogenetic tree was inferred separately for each of these markers, and a maximum likelihood phylogeny was also inferred using the 694,437 bp concatenated alignment, which was 41.14% complete with 443,864 polymorphic and 316,350 parsimony informative sites.Transcriptome and coding sequence data from seven additional species representing different embryophyte groups were retrieved from the literature –115 S3  and ORFsne trees –118. Theginella, ) or IsoeIsoetes, a fossil constraining the crown node of extant Isoetes would be ideal–such a constraint would require a fossil containing a synapomorphy from one of the two descendant branches of this node. Previous studies have identified a geographically diverse group of Isoetes including I. coromandelina sensu lato as the outgroup to the rest of the Isoetes [I. coromandelina complex itself contains a number of features initially thought to identify this as diverging earliest from other extant members of the group, its presence within “Clade A” identifies these features as derived [Isoetes appear to exist [Isoetes crown group, no morphological features clearly divide different clades [Isoetes.Time-calibrated trees were inferred from the different markers using the same set of calibration points. To date the crown node of extant  Isoetes , 56, 65. derived , 55. No to exist , 55. Thet clades , 42, 58.t clades , 120. ThIsoetes crown node for which reliable synapomorphies are available is the crown node of the Isoetopsida (Isoetes plus Selaginella). The Isoeptopsida are a well supported clade appearing in the Devonian, containing synapomorphies such as a heterospory and a ligule [Isoetes than Selaginella. This assessment is based on shared synapomorphies including bipolar growth from a shoot like “rhizomorph” structure and secondary woody tissue [The nearest node to the a ligule , 121. Isy tissue . Arboresy tissue , 123  were assigned by inspection of the traces using Tracer v1.6 . For indIsoetes species for which molecular data is available  is obtained via the implicit or explicit inference of a decrease in the rate of evolution along the stem branch, with rates in the crown branches being more similar to those in the rest of the tree. This assumption results from the model, and is not necessarily correct, urging for independent evidence.Isoetes clade was consistent with that of the chloroplast phylogeny, with I. coromandelina again being sister to all other species. Despite overall longer branch lengths in the concatenated nuclear phylogeny, variation among groups was reduced. Particularly, the total branch lengths from the common ancestor of Isoetes and Selaginella were much more similar than in the chloroplast phylogeny, with Selaginella having accumulated approximately 1.25 times more mutations than Isoetes since their last common ancestor . As with the chloroplast markers, the disparity between effective and assigned rates in crown branches was low across the range of smoothing values  and 45–60 Ma , respectively  [I. beestonii to provide a narrow lognormal prior with an offset of 245.5, a mean of 1.5 and standard deviation of 0.5Ma for the age of crown Isoetes, resulting in an estimate of the crown age at 251Ma. Pereira et al. [I. rolandii to provide a minimum age for the crown node of 145 Ma, resulting in an estimate of the crown age at 147Ma (145–154 95% CI). In both these studies, fossils are used to a priori strongly constrain the crown node of Isoetes to old ages. However, these fossils do not provide evidence that the split between “clade A” and the rest of Isoetes had occurred, as no synapomorphies are known from the extant members of these clades that could be preserved in fossils. The differences between these previous studies and our own emphasise the impact of fossil calibrations on date estimates. The study of Lars00E9n and Rydin (2015) [rbcL aligning with sequences outside of the genus. The markers that do not align outside Isoetes should not affect the crown node age estimate as they do not inform the ratio of crown to stem substitutions. However, including noncoding markers such as nrITS with much higher levels of substitution than rbcL may result in a young Isoetes crown node giving a poor model fit for a single partition. Indeed, maximum likelihood phylogenies of the full dataset have Isoetes crown branch lengths that are five times longer when compared to the same taxa only with rbcL  , 57, 58.n (2015)  used fosith rbcL . To inveIsoetes Click here for additional data file.S2 FigIsoetes crown group dates for individual nuclear genes in r8s that pass gradient checks for a range of assigned smoothing values, and the histogram of estimates where each gene is assigned its optimum smoothing value based on cross validation .Histograms showing estimated (PDF)Click here for additional data file.S3 FigIsoetes for individual nuclear genes in r8s that passed gradient checks for a range of assigned smoothing values, and the histogram of estimates where each gene is assigned its optimum smoothing value based on cross validation . Median values are displayed in the top righthand corner of each panel.Histograms of the ratio of effective vs. assigned branch rates for the stem (red) and average value for crown (blue) branches of (PDF)Click here for additional data file.S1 TablePublished with the permission of the Board of Trustees of the Royal Botanic Gardens, Kew.(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 TableSee main text for references.(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 TableLinear models in R (using the lm function) are used to identify the relationship between a number of alignment properties for individual nuclear genes and the resultant predicted dates in r8s and BEAST. Significant p-values (<0.05) are highlighted in bold.(DOCX)Click here for additional data file.S6 TableatpB-rbcL intergenic spacer removed, (iii) nrITS removed (iii) or iv) both the atpB-rbcL spacer and nrITS removed.Branch lengths from maximum likelihood phylogenies generated using RaxML (GTR+G+I model) for (i) all three markers used in the study, ii) the (DOCX)Click here for additional data file.S7 TablerbcL sequences for Isoetes species and other Embryophyte groups, and additional highly variable sequences for nrITS and the atpB-rbcL intergenic spacer for Isoetes only. Isoetes species lacking an rbcL sequence were excluded from the analysis. The entire dataset (i) gave similar estimates of the Isoetes crown age to Larsén and Rydin, 2015, but removal of the atpB-rbcL intergenic spacer (ii) reduced ages for the Isoetes crown, and removal of either nrITS (iii) or both Isoetes-specific markers (iv) resulted in ages consistent with the present study.The alignment from Larsén and Rydin (2015) was re-analysed using the same constraints and BEAST settings as the previous paper, with at least 3 independent runs reaching ESS > 100. The dataset contains (DOCX)Click here for additional data file.S1 FileFolder containing fasta files for each of the individual nuclear alignments.(GZ)Click here for additional data file.S2 FileFolder containing fasta file for the combined nuclear alignment.(GZ)Click here for additional data file.S3 FileFolder containing fasta file for the chloroplast alignment.(GZ)Click here for additional data file.S4 FileFolder containing nexus file for chloroplast phylogram.(GZ)Click here for additional data file.S5 FileFolder containing nexus file for concatenated nuclear phylogram.(GZ)Click here for additional data file.S6 FileFolder containing BEAST files used to reanalyse the Larsén and Rydin (2015) dataset.(GZ)Click here for additional data file.S1 Data(TXT)Click here for additional data file.S2 Data(FA)Click here for additional data file.


An increment pattern in the worldwide egg production, as well as in the farm’s capacity in laying hen housing systems, have been observed for the last fifty years. Also, animal welfare has become a subject of interest due to consumer awareness. These issues have introduced new challenges to respond to international markets’ demands while ensuring animal welfare and environmental footprint. Cage-free systems have been alternative systems to ensure the well-being of laying hens. Likewise, environmental control strategies have been implemented to improve air quality since airborne contaminants’ concentration can be high inside these systems. Thus, the analysis of the effect of these strategies on natural behavior and flock distribution is essential to assess a comprehensive analysis. Four strategies and a control were tested in an experimental cage-free system. Spatial occupancy and animal behaviors were tracked using video recordings. Neither the four environmental strategies nor the control applied in this experiment affected the natural behaviors of hens. However, changes in flock distribution and stress patterns were identified in the treatment with a reduction in litter allowance. This study provides evidence that it is possible to implement strategies to improve air quality without disrupting natural animal behaviors in cage-free systems.p < 0.05). Neither the four ECSs nor the control implemented in this experiment affected the natural behaviors of the hens. However, stress patterns and high activity were reported in the T17 treatment. This study shows that it is possible to use these ECSs without disrupting laying hens’ natural behaviors.Animal welfare concerns have been a challenging issue for producers and international marketing. In laying hen production, cage-free systems (CFS) have been identified as an alternative to ensure the laying hens’ well-being. Nevertheless, in CFS, important environmental issues have been reported, decreasing indoor air quality. Environmental control strategies (ECS) have been designed to enhance indoor air quality in CFSs. However, little information exists about the effect of these ECSs on natural animal behaviors. Four strategies and one control were tested in an experimental CFS, previously designed to track behavioral variables using video recordings over seven time-lapses of 1 hour per day. Spatial occupancy (SO) and laying hen behaviors (LHB) were registered. One statistical analysis was applied to evaluate the effect of ECS on SO and LHB using a multinomial response model. Results show lower chances to use litter area within the reduction of litter allowance treatment (T17) ( In the last fifty years, different laying hen production systems have been designed to improve animal welfare and respond to international market demands. Directives focusing on housing design and alternative methods to rearing laying hens were provided by the European Commission  to promo3) emissions, airborne dust production, and undesirable indoor air quality for hens and workers [CFS have been considered the best housing alternative for laying hens regarding animal welfare; allowing them to display a broader range of natural behaviors . These s workers ,8.3 concentrations (>25 ppm) in CFS can induce respiratory disease to bird subjects to aerial viruses [3 concentrations where animals can reduce preening and foraging activities while in all likelihood preferring areas with low NH3 concentrations [High NH viruses , and dec viruses ,11, as w viruses , and inc viruses . Furthertrations .10). These particles can be inhaled, reaching the lower respiratory tract of humans  [Airborne dust or particulate matter (PM) from litter disturbance can be transported as bio-aerosols carrying biological and organic substances , affecting the health of workers, hens, and people living close to confined laying hens housing systems ,16,17. Hiseases) ,19. Likeiseases) . Hypotheiseases) .3 and PM emissions include litter properties; environmental conditions such as air humidity, temperature, and ventilation rate, free animal movements; as well as manure management systems [3, directly or indirectly, several environmental control strategies (ECS) have been suggested, namely: (i) incorporation of inert materials in aviary diet, e.g., biochar, zeolite or bentonite, to improve N retention in poultry manure [3 volatilization, namely: N retention in manure storage, poultry manure amendment, and particulate matter abatement. Nevertheless, to our knowledge, animal behavior assessment, more specifically on laying hen behaviors, has been the subject of few studies, and the focus has been on animal physiognomy alterations. Thus, this study aims to evaluate the effect of four strategies on spatial occupancy and laying hen behavior. The outcomes of this study are expected to contribute to the implementation of animal-friendly ECSs without negatively impacting their natural behavior and thereby the welfare in laying hen CFS.Factors favoring NH systems ,22,23. Ty manure ,25; (ii)y manure ,27,28 ory manure ; (iii) sy manure ; (iv) ney manure ,32,33. TThe study took place in twelve independent bench-scale rooms . Each room was equipped with a variable-speed exhaust fan. The incoming air, drawn from outside, was preconditioned with an air conditioning unit and a heated electrical resistance unit to maintain an optimal temperature between 22 °C and 23 °C inside the rooms throughout the experimental period. The pre-conditioned air was distributed to all the rooms by using a series of ducts.2. Two nipple drinkers and a linear feeder were installed in the wire floor space. The bird droppings collected beneath the wire floor were dried with forced air and removed once a week. A perforated 7.5 cm diameter duct blew air from a 10 cm blower  fixed beneath the air inlet. Several holes , were positioned at a 45° angle. Then, based on airflow of 0.7 m3 h−1 hen−1, the blower was adjusted to obtain an air velocity of 2 m s−1. Two PVC-pipes (round profile) were installed over the wire floor area of each room (5 cm and 45 cm high), resulting in a linear space of 20 cm per hen.The rooms were laid out to meet the CFS housing requirements stipulated in the Code of practice for the care and handling of pullets and laying hens of the National Farm Animal Care Council of Canada (NFACC) . Hens haLohmann LSL-Lite laying-hens arrived at 19 weeks of age and were housed in the experimental rooms from February to June 2019 . Trimming of the beak (at one-day-old) and vaccination requirements were done before their arrival at the experimental facility. Hens were individually weighted, placing the animals in a plastic recipient (8 L) with non-adjustable cover (to reduce stress) put on a digital platform scale previously tared. Then, the 144 hens were randomly selected to distribute them into the twelve experimental rooms. Four hens were randomly selected from each room and identified by painting a blue spot on their back. Further information about blue spot identification would be mentioned in An adaptation period of two weeks was provided at the beginning of the experiment. The two-week period allowed (i) the hens recognize their environment and (ii) the caretakers to carry out flash hen behavior visual observations, ensuring well-being conditions and animal adaptability.Hens were fed with a commercial diet . Feeders were filled once in the morning. Then, they were verified three times per day (by the caretakers) and refilled by the end of the day. Water was provided by a solenoid-activated valve connected to a data logger. Access to feed and water was ad libitum, and the amounts consumed were daily quantified for each room. The lighting intensity was 10 to 15 lux with equal lighting periods for all the rooms . An immediate on/off light cycle was set for each room.Fédération de Producteurs d’Oeufs du Québec  to evaluate the possibility to decrease litter surface. Then, optimal parameters of the combined ECSs were selected following an experimental pre-test procedure focusing on economic and technical aspects (data not shown).Treatments included two combined ECSs previously selected by a panel of expert based on available literature. One treatment was proposed by the The four ECSs studied included: Decrease of litter surface area (T17); use of heated floor (HFOS), and litter amendment with biochar material (AOS) both combined with established oil sprinkling periods; as well as one single oil sprinkling treatment (OS). The experiment was divided into two consecutive experimental batches of eight weeks each (batch 1 and 2). A one-day time interval was used between batch 1 and 2 where the rooms were completely cleaned, ensuring any interference from the applied treatments in batch 1. The same laying-hens from batch 1 were housed for batch 2.−2 of litter area. Sprinkling was carried out only on the litter surface. In this treatment, the underlying floor temperature was kept at 27 °C using an electrically heated floor system (EHF). The AOS treatment included the addition of an activated spruce-fir based biochar , equivalent to 10% of the litter mass. The biochar particle size was ~1 mm, which was activated by acid protonation, after drying at 100 °C. The activation was made by immersion in a concentrated solution of sulfuric and nitric acid [−2. In this case, the litter area was kept at room temperature. The mass of litter placed in each room was 900 g for T17 and 1800 g for the other treatments.The HFOS treatment included the sprinkling of an acid emulsion made with vegetal oil and an organic acid solution. The application was carried out twice a week at a dosage of 585 mL mric acid . The OS Spatial occupancy and laying hen behavior were monitored for both 8-week experiments (batches 1 and 2). A video recording system  was set up inside the experimental rooms to record hens throughout the day. Four cameras were used for this study (POE Cameras) connected to a Network Video Recorder (NRV), saving 4TB HDD (Hard disk drivers) of data; that is, recording simultaneously four experimental rooms.To observe spatial occupancy (SO), each bench-scale room was divided into six main areas: Nest (N), Litter (L), Perches (P), Feeder (F), Drinker (D), and Nest-feeder (NF). The N-region was obtained by subtracting the number of observed hens in the other areas from the total number of hens since it was not possible to count the number of birds inside the nest at each observation. The NF-region was established as the free space located between the feeders and the nest according to the room design .Ten laying hen behaviors (LHB) were observed according to Blokhuis , KristenTwo and three random days were selected for the video recording of each treatment for batch 1 and 2, respectively. One day of observations included seven 1-hour recordings covering most of the day, that is: 6 h–7 h/8 h–9 h/11 h–12 h/14 h–15 h/17 h–18 h/20 h–21 h/21 h–22 h. Each observation was made over a 1-min video at the half-way time of each 1-h recording by two trained observers. A total of 154 and 252 videos were analyzed for batches 1 and 2, respectively. The quantity of videos was not equal for both batches due to a lag time between the beginning of the batch 1 and the video recording system installation. Both SO and LHB were analyzed using the video recordings . For SO NoHi is the number of hens observed in the i-th section over one day, THN is the total number of hens observed on the same day, and SO│j is the relative frequency of spatial occupancy in the j-th day in the i-th section. Furthermore, LHB analyses were carried out by summarizing the number of hens doing a specific behavior divided by the number of experimental rooms undergoing the same treatment.The SO were assessed using the relative frequency of an event as proposed by Kozak et al.  where thp < 0.05). The response category having the highest relative frequency, “Perches”, was used as the baseline in the model. The odds that an animal under treatment A occupied i-space rather than reference space, i.e., perches, is the probability ratio Odds (Treat A).i,treat A and Πp,treat A are the relative frequencies of SO in the i-space and perches areas under treatment A. Treatment comparisons over spatial occupancy were performed using estimations of odds ratios (Equation (3))The response variable of the SO analysis for each treatment was assumed to have a multinomial distribution. Therefore, a multinomial response model was applied using the PROC GLIMMIX procedure of SAS 9.4  and the general logic link function. Treatment effect was tested with the F-test (p < 0.05). Mean counts per treatment for each behavior were estimated and its standard deviations (±) are presented, respectively.For the LHB, observed counts of each behavior were analyzed using a generalized linear model. The procedure PROC GLIMMIX of SAS version 9.4  with logic link function was used to accommodate the assumption that counts have a Poisson distribution. The F-test for fixed effect was applied to assess the significance of the treatment effect on mean counts (p = 0.0001). It was observed that occupancy in feeders and nest-feeders area for the T17 treatment showed higher relative frequencies, with percentages of 27.8% ± 9.0% and 20.8% ± 2.0%, compare to 16%–22% and 9%–11% observed in the other treatments and the control, respectively. In fact, hens preferred the feeder area 1.69 times more in T17 when compared to the HFOS treatment (p = 0.04). Furthermore, the odds of occupancy in nest-feeders were 1.74 and 2.24 times higher in T17 treatment than that in the HFOS treatment (p = 0.05) and control (p = 0.0078).The relative frequencies (%) of SO for the first and second batches are shown in p = 0.0051) and OS (p = 0.0002) treatments than that of the T17 treatment. There was not any observed significant treatment effect in SO regarding random odds ratios between the HFOS and OS treatments, and the control.For the second batch, similar relative frequencies were observed for the perches, feeders, and drinkers area for the three treatments and the control with average frequencies of 24.8% ± 2.2%, 18.8% ± 1.0%, and 5.8% ± 1.0%, respectively b. MoreovThe average counts of laying hens performing a natural or species-specific behavior per day for batches 1 and 2 are shown in Species-specific behaviors such as dust bathing (DB) were reported for all three treatments and the control in both batches 1 and 2. These values were similar over the three treatments with average values and standard deviation of 3 ± 1 and 2 ± 1 hens observed for batches 1 and 2. Other animal behaviors, i.e., scratching, kneeling, ruffling feathers, body shaking, and drinking, did not show a particular trend. Furthermore, no significant effect of treatment was detected on any other behaviors following statistical analysis .Our results demonstrate that litter, perch, and feeder areas are the main locations where hens spend most of their time a,b. LittThe use of litter area was the lowest for the T17 treatment when compared to all other treatments and the control. There were 2 to 3 times more odds that hens would prefer litter in the other treatments. This means that reducing the litter surface area could lead to an unbalanced distribution of the flock within CFS. Furthermore, abnormal repetitive behaviors were identified while analyzing the video recordings for the T17 treatment, stress patterns as defined by Garner  were recDisplacement of animal behaviors could be observed if there were a motivational conflict. Taylor  defined Nesting activity in private places seems to be one of the preferred activities for laying hens, several studies have shown the preference for hens towards private spaces to lay eggs as a behavioral need ,36,38. UPreening, perching, and feeding were the most frequent behaviors observed in this experiment . These rIn this study, natural and species-specific behaviors were observed for all treatments. There was not any significant effect linked to the four ECSs in any of the nine laying hen behaviors considered . This waNowadays, animal behavior remains an important factor when assessing the quality of laying hen housing systems in light of animal welfare demands from markets and consumer’s needs. According to the literature, the application of ECSs inside these systems have proven the potential to improve air quality conditions for animals and workers in terms of airborne dust and gas concentrations. However, studies of the effect on laying hen behaviors are deemed necessary to better understand animal welfare.This study provided an animal welfare analysis based on spatial occupancy and natural animal behaviors after the application of different ECSs. This article indicates that the natural behavior of laying hens exposed in a cage-free experimental system using different ECSs was not affected. However, the reduction of litter surface produced significant differences in the spatial distribution of the flock. Abnormal behaviors, as well as high activity in the flock have also indicated a possibly stressful environment for hens where litter surface was limited. Moreover, it is noteworthy that reduced litter allowance could be the limiting factor to trigger some behaviors performed in the litter area. Though there was not any significant effect observed over the laying hen behavior analysis, reduction of litter area is not recommended as animals could undergo stress patterns and unbalance spatial distribution. Besides, video recordings covering private places, i.e., nest boxes, could be useful to improve the analysis of spatial occupancy and animal behaviors. Also, gradual on/off lighting programs must be applied to reduce anomalies within spatial distribution in the flock while resting during the night. Additional  research work should be conducted at an on-farm production scale with an online and integrated monitoring system to keep up with Precision Livestock Farming trends .


Gossypium hirsutum. We report that the expression of both the cry and cp4-epsps genes in wild cotton under natural conditions altered extrafloral nectar inducibility and thus, its association with different ant species: the dominance of the defensive species Camponotus planatus in Bt plants, the presence of cp4-epsps without defence role of Monomorium ebeninum ants, and of the invasive species Paratrechina longicornis in wild plants without transgenes. Moreover, we found an increase in herbivore damage to cp4-epsps plants. Our results reveal the influence of transgene expression on native ecological interactions. These findings can be useful in the design of risk assessment methodologies for genetically modified organisms and the in situ conservation of G. hirsutum metapopulations.After 25 years of genetically modified cotton cultivation in Mexico, gene flow between transgenic individuals and their wild relatives represents an opportunity for analysing the impacts of the presence of novel genes in ecological and evolutionary processes in natural conditions. We show comprehensive empirical evidence on the physiological, metabolic, and ecological effects of transgene introgression in wild cotton,  However, the consequences of transgene expression in natural ecosystems remain unexplored, leading to uncertainty about its impact on local evolutionary and ecological processes. Research on this topic has been challenging largely because, while agro-biotechnological innovations move rapidly, the information required for adequate risk assessment is still limited7. However, knowledge on transgene introgression effects in natural conditions and in particularly in the species centre of origin can offer valuable information for in situ conservation of crop wild relatives (CWR).The introgression of transgenes from economically important GM crops to their wild relatives10. However, there is great uncertainty and limited evidence regarding transgene behaviour in wild genotypes that survive in natural conditions under a set of complex ecological challenges7. Consequently, studying transgenes in the wild represents an excellent opportunity to integrate a new stage of scientific knowledge, combining genetic, ecological, evolutionary, and environmental information.The different disciplines interested in assessing the expected functions of genetically engineered genes have produced a fair amount of research in controlled environments, with the only purpose of applying the generated information to improving technological packages14. From these studies, we can infer that differences found in seed production and attraction of herbivores’ natural enemies are linked to the interaction of transgenes (i.e. cry and cp4-epsps). Such differences have been associated with defence mechanisms and/or main metabolic pathways involved in plant growth, development, and reproduction, such as the shikimate or the octadecanoid pathways16. As a consequence, the quantity and quality of metabolic products are modified, compromising not only plant performance but also their interactions with insects, bacteria, and soil fungi17.Recently, several studies have looked for differences in genetic expression, fitness, and diversity in non-target organisms, between transgenic organisms and their isogenic lines, as well as between experimental parameters and settings Gossypium hirsutum L.) and its associated ant communities in Mexico where the wild to domesticated complex exists. Upland cotton is a Mesoamerican native species in which introgression of transgenes conferring resistance to specific lepidopteran larvae  (Bt or cry genes) and tolerance to glyphosate (cp4-epsps) have been detected, at its center of origin, in seven out of eight metapopulations 3. We examined the potential effects of introgressed transgenes on G. hirsutum production of extrafloral nectar, which constitutes a reward for defensive ants who patrol the plant to repel herbivores19. Assessing the ant-cotton interaction is relevant because ants are not-target insects of transgenic technology and their role is protecting cotton plants against herbivores20. Furthermore, in the coastal sand dunes where the studied populations are established, ants play important ecological functions acting as soil engineers and seed dispersers, influencing plant germination, reproductive success, and distribution patterns. To this end, we analyzed three wild cotton genotypes, two of which are introgressed plants with transgenes. All of them are growing in the species centre of origin: (1) wild genotypes without transgenes; (2) wild genotypes with cry; and (3) wild genotypes with cp4-epsps . We evaluated the inducibility of extrafloral nectar (EFN) after exogenous application of methyl jasmonate (MeJA), which has been widely used as a tool when studying plant reactions to stress and understanding the interactions between the elicitation of plant responses and their consequences for herbivores and beneficial insects21. We also measure the response of the ant community and herbivore damage associated with the three cotton genotypes.To assess the consequences of transgenic expression in the absence of target pests and glyphosate as selection agents, we studied wild upland cotton .This study was conducted in the costal dunes of the Ría Lagartos Biosphere Reserve (hereafter RL), Yucatan, Mexico. We located it in the stub zone, where wild cotton, 22 Table . We chos2. DNA concentrations were analyzed with a Quibit 3.0 Fluorometer . DNA sequences were amplified through a PCR endpoint, DNA polymerase GoTaq Flexi , and the primers: Cry1Ab/Ac, Cry2Ab, and CP4EPSPS from Eurofins Scientific . The thermocycler was programmed for a denaturation period of 8 min at 95 °C, followed by 30 cycles of 20 s at 95 °C, 1 min at 60 °C, 1 min 72 °C, and then 8 min at 72 °C for the final cycle. Reactions were carried out in a volume of 25 µL containing 5 µL × buffer, 25 mM of magnesium chloride, 100 mM of dTTP, 10 µM primer, 70 nM of genomic DNA, and one unit of Taq polymerase (GoTaq Flexi by Promega). PCR products were separated in 2% denaturing agarose gel. We recorded the presence of three transgenes, cry1ab/ac, cry2ab, and cp4epsps, in individual and stacked form : (1) wild without transgenes, W (control); (2) wild with cry, Wcry; and (3) wild with cp4-epsps, Wcp4-epsps. For the purpose of this investigation we decided to exclude the plants expressing both transgenes, to avoid non additive effects. As described below, even with this limited amount of plants we were able to detect interesting results due to the genetic expression of these genes in a natural population, where plants coexist with their antagonists and mutualists. These results, of course, must be taken as a first line of evidence that requires further investigation with larger sample sizes.To determine the presence of the herbicide-tolerant transgenes and those with insecticide effects within the studied population, in March 2018 we collected the foliar tissue of 61 cotton plants (six leaves per plant) of comparable age, georeferencing each plant to recognize them for further experiments into a natural patch of cotton plants of 2 ha into a the RL to 6.1 km from the nearest Town, the most distance between plants was 280 m. Genomic DNA was isolated from foliar tissue using the Miniprep CTAB method23. Previously, we had evaluated whether induction through methyl jasmonate (MeJA) was active in cotton plants and found that indeed, cotton plants can respond to this elicitor by inducing extrafloral nectar in the branch where it is applied, hence the response was local and not systemic21. Considering this, control and induction treatments were applied in the same individual on different branches, using ten leaves that were sprayed with 2 mL of distilled water or MeJA solution (450 mM), respectively. To exclude insects from extrafloral nectaries, we coated plant branches with Tanglefoot in its original concentration  24. Induction was carried out at 6:00 a.m. during three days of the summer of 2018 and concentration measurements of soluble solids were taken six hours later, at noon, based on previous observations on the peak of extrafloral nectar production. Nectar secretions from 10 leaves in each treatment(one nectary per leaf) per plant, in total 84 branches and 420 leaves were quantified; the amount of soluble solids was measured with 3 µL graduated micropipettes, and nectar concentration was obtained with a temperature-compensated portable refractometer . To remove the nectar, we applied 3 µL of distilled water to each nectary and repeated the procedure until we reached concentrations lower than 1%. Then, we added values from all collections in one leaf, to quantify leaf overall production of solid EFN compounds25.Each experimental plant of the three genotypes was divided into two zones, in which we applied the control and induction treatments in the most distal branchesAfter nectar measurements concluded, MeJA induction was carried out for two more days following the same experimental design as described above but allowing ant foraging. We conducted two censuses per day during a two-day period, at 10:00 a.m. and 4:00 p.m., when ant activity was at its highest (according to previous observations by our lab group). We registered the number of ants feeding and patrolling extrafloral nectaries and collected a sample of each ant species for taxonomic identification.Laboratorio de Invertebrados del Suelo . Voucher specimens for all collected species were deposited in the latter collection  for each branch of the genotypes described above. We used a Canon EOS Rebel T6i 18 MP camera without flash, positioned at 15 cm from the leaves placed above a scaled portable background. Pictures were taken in the field, preventing the leaves from being removed from the branches. Images were saved as JPG format and automatic quantification of leaf damage was performed with BioLeaf Analyze Foliar (version 1.0)27. Consequently, we individually evaluated the effect of the induction treatment on EFN for each genotype using the Generalized Lineal Model with Quasipoisson distribution with the package GLM Ime4 in R version 3.5.0. Likewise, ant abundance and the percentage of herbivore damage were evaluated with Generalized Linear Models with Quasipoisson distribution  and the genotype of the wild plants  were considered as a fixed factor. We compared the effect of the induction treatment, genotype, date, and their interactions. Finally, ant richness and composition were expressed as the total of morphospecies per genotype. The scripts utilized for the analyses are available online at https://github.com/conservationgenetics/Ongoing-ecological-and-evolutionary-consequences-by-the-presence-of-transgenes-in-a-wild-cotton-popu.Given that our data did not fit normal assumptions, our simples were analyzed using Generalized Linear Models with Quasipoisson distribution (GLM Ime4 package in R version 3.5.0), based on the dispersion of the data.The Poisson distribution and zero inflated models were also consideredP(χ2) = 0.048, df = 1). In contrast, transgenic genotypes (Wcry and Wcp4-epsps) did not respond to this stimulus (Wcry: P(χ2) = 0.375, df = 1; Wcp4-epsps: P(χ2) = 0.388, df = 1), because of different reasons regarding the genotypes with introgressed genes. Whereas Wcry control plants had a high EFN production equivalent to the induced state of W plants, EFN production in Wcp4-epsps plants was inhibited, yielding less than 8 µg/mL in both treatments  < 0.001, df = 2)  = 0.637, df = 1).Exogenous application of MeJA significantly increased EFN secretion in wild plants without transgenes (W) , meanwhile in Wcry and Wcp4-epsps we registered 33 and 34 ants, belonging to four and six species, respectively .We collected 109 ants associated with the EFN of cp4-epsps had an exclusive ant species, while Wcry didn't have exclusives ants, and four of the ants collected were presented in all genotypes  < 0.001, df = 1), with C. planatus being the most abundant ant species. In contrast, in the W and Wcp4-epsps genotypes the induction treatment did not have any effect on ant abundance (W: P(χ2) = 0.436, df = 1; Wcp4-epsps: (P(χ2) = 0.210, df = 1)  < 0.001, df = 2) when compared to W and Wcry genotypes. The lowest percentage of herbivore damage value was registered for Wcry plants  and invasive ant species (P. longicornis) in the same proportion. Furthermore, herbivore damage and its associated ant community were different according to the introgressed transgene.In this study, we showed that the expression of bt rice and glyphosate-tolerant sunflowers living in natural conditions where introgressed plants are different from their wild relatives5.In general, it has been assumed that introgressed and wild genotypes should display similar phenotypes in the absence of the selection agents targeted by transgenes. However, when we compared the control group and the three genotypes, we registered different nectar secretion patterns among them Fig. . Similar29. However, unlike wild plants without transgenes, individuals with transgenes were not sensitive to the induction treatment with MeJA for increasing their EFN production , in which direct defences such as gossypol terpenoids (160%), hemigossypolone (160%), helicoids 1|4 (213%) and indirect defenses, such as volatile compounds (VOCs) (171.2%) and extrafloral nectar (EFN) (133%), were reported to increase in plants sprinkled with JA and MeJA30.Most plants are able to induce responses after herbivore damage and/or phytohormone exogenous application ion Fig. . These rcry control plants had a high EFN production equivalent to the induced state of W plants, EFN production in Wcp4-epsps plants was inhibited. Contrasting these findings with results obtained under controlled conditions (i.e. greenhouse and crop conditions)21, we suggest that EFN production is linked to genotypes with transgenes and abiotic stress in the coastal dunes, because transgenes are connected to main metabolic pathways that respond to stressful conditions21.The inability of plants with transgenes to have the production of extrafloral nectar induced in them was related to different processes dependent on the identity of the transgenes in question. Whereas Wcp4-epsps exhibited large differences compared to wild plants without them. Their low nectar production (> 8 µg/mL) 33: therefore, we suggest an interference between the SA and JA pathways given previous reports that an over-expression of the cp4-epsps gene modifies the second part of the shikimate pathway (post-chorismate), which leads to the synthesis of essential amino acids as phenylalanine, tryptophan, or tyrosine, the latter being a precursor of benzoic acid BE, and SA35  Fig.  could becry genes continuously produced EFN as a constitutive defence 11. This is consistent with observations of a synergy between maize direct defences and Bt genes, after exogenous applications of JA , in addition to Ghppo1, which confers natural resistance to lepidopteran pest, such as H. armigera38. The interaction of cry with other genes could modify the production of EFN in Wcry plants.Wild cotton plants with nce Fig. , in equiG. hirsutum41.We identified eight species of ants harvesting EFN Table , but wit42. In our study, the availability of EFN was modified. Although species richness was the same as in W plants . Interestingly, although the amount of nectar did not vary between treatments, the abundance of ants was significantly different. The dominance of a single ant species could have benefited the plants with increased indirect defence, reducing herbivore damage and promoting a greater seed production per plant, as described in Turnera ulmifolia44, Schomburgkia tibicinis45, and Opuntia stricta42. However, considering the aggressive and dominant behaviour of C. planatus, there may be ecological costs through antagonistic relationships with pollinators. Ants can interrupt pollination and affect plant fitness47. The outcome of these mutualistic and antagonistic interactions requires further study.In W50. However, a study carried out on wild upland cotton reported that plants tolerate intermediate levels of leaf damage inflicted by leaf-chewing insects (< 50%)51. Thus, the increased herbivore damage observed in Wcp4-epsps plants  does not necessarily jeopardize plant growth or reproductive success. For example, in the absence of glyphosate, transgenic hybrids of rice and soybean show changes in their phenology (i.e. early flowering and shorter germination times) and an increase in their fitness (i.e. larger fruit and seed sets) compared to their wild relatives5. It has been suggested that the over-expression of the epsps gene is responsible for these compensation effects on plant fitness, in view of the biotic and abiotic stressors that activate the shikimate pathway15. Further investigation to find out if this is the case for Wcp4-epsps cotton is currently being developed under natural conditions.Considering that the type of mutualism that cotton sustains with ants is defensive, we suggest that the change we observed in the composition of ants is likely to have influenced herbivore damage in the different genotypes, which in turn has the potential to reduce fitness as shown by other studies of cottoncry plants exhibited the lowest herbivore damage, although not significantly different from wild plants without transgenes , increasing their seed production by 55%4. Nonetheless, in G. hirsutum we did not find differences in herbivore damage when compared to the W and Wcry genotypes, probably because lepidopteran species targeted by Bt cotton were not present in our study site, hence, cry expression might not have a role in increasing fitness. However, indirect effects could be present through its interaction with native herbivores. For example, experiments under laboratory and experimental conditions conducted on maize with cry genes, have shown non-lethal effects in the physical condition of non-target caterpillars 52. Likewise, if the nutritious quality of herbivore tissues changes after consuming Bt toxins, these effects could cascade to higher trophic levels54, increasing mortality and decreasing longevity or development of predators. This has been shown for chewing predators  because they ingest the gut of the prey, where most of the toxins are concentrated54. Parasitoids have also been found to be affected after the consumption of Bt toxins contained in their preys55. When the wasp Microplitis mediator (parasitoid of H. armigera) was fed with larvae containing cry toxins, they extended its egg and larval development time by 1–2 days, significantly decreasing its pupal weight by 35%, and its overall longevity when the toxin concentrations were high (4–8 μg g−1). Hence, a question that warrants further investigation is how Wcry cotton genotypes in the wild affect herbivore and predator communities and their interactions.In contrast, Wnes Fig. . The expcry and cp4-epsps gene expression in wild cotton plants. As a result, we obtained the following noteworthy results: (1) differential response in the induced defence mechanism  concordat to plant genotype; and (2) modification of biotic interactions between introgressed cotton and relevant organisms, under natural conditions.By means of an integrative methodology, we evaluated the effect of the 2, and recently, introgression in the Baja California Sur, BCSM, Central Pacific, CPM, and Yucatan Peninsula YPM metapopulations has been reported3. In this study, we reaffirm the presence of transgenes in the YPM, but it's important to consider that the establishment of these has been fast. Whereas during the first monitoring in 2008, YPM metapopulation didn't register the presence of transgenes, in 2018, 60.64% of 61 plants had them , these have only been tested under controlled conditions. We found that some theory-based concerns can be confirmed when performing functional experimental designs in the wild. First, it is possible to investigate ecological and evolutionary impacts of new genes under natural conditions by studying community processes, such as changes in tri-trophic interactions. Second, we detected physiological and possible metabolic alterations generated by the expression of transgenes in wild cotton plants without the pressure of selection agents (pests and herbicides) targeted by those genes. Third, until 2008, 4 wild metapopulations of upland cotton showed evidence of introgression with GM cottonIn natural ecosystems, changes at different scales , following the introduction of novel genes lead to endless research possibilities. Through them we can integrate broad information regarding biological control, agro-biotechnologies, and conservation biology, with promising further applications. Although we do not know the routes of transgene dispersion, we provide evidence of some of the mechanisms that could favour the establishment and persistence of these new genes, mainly due to their interaction with key defence metabolic pathways. However, transgene frequency in wild populations and the associated ecological consequences must continue to be monitored and evaluated so as to contribute information that allows us to make decisions for the conservation of the primary genetic pool and the ecological and evolutionary processes that have shaped its diversity.in-situ conservation of the primary gene pool without transgenes. All these efforts would contribute to the integration of the available disperse information so we may understand the consequences of transgenic plants coexisting with their relatives. As we demonstrated in the current study, the presence of these genes can cause intrinsic changes in wild populations of cotton , and changes in their ecological interactions. If we want to conserve in-situ the primary gene pool of wild relatives, we must work to identify the ecological and evolutionary processes affected by the existence and permanence of these transgenes within their populations. Upon the detection of these genes, mitigation strategies to reduce the magnitude of the damage can be promptly designed.Alterations in the defence mechanisms of wild relatives in one of the most important crops for humanity represents critical evidence on the threats of introgressed genes to biological and cultural heritage for the following generations. Such negative consequences would be enough to envision GMO liberation into the environment, short and long distance from its origin centre, from a different perspective. At this point, we are in a watershed moment to: (1) develop the necessary research to mitigate the ecological, evolutionary caused by the introgression of GMOs into the wild-to-domesticated complex of the species used, as well as reformulating risk assessments for protection goals; (2) fill gaps in our knowledge of the complex dispersion routes of transgenes from crop to wild relatives and native varieties; and finally, (3) prioritize the Supplementary Table S1.Supplementary Table S2.


ObjectivesPremenstrual syndrome (PMS) is a common disturbance among women of childbearing age. Aromatherapy is a commonly used form of complementary and alternative medicine (CAM) to treat PMS. The purpose of this study is to quantify and summarize the effects of aromatherapy on premenstrual syndrome symptoms. Methods. PubMed, Scopus, and Cochrane Library databases were searched through relevant search terms until October 2020. The effect sizes were pooled as weighted mean difference (WMD) and 95% confidence interval (CI) using the random effect model. Egger tests and visual inspection of the funnel plot were performed to identify the existence of publication bias. The I-squared (I2) test was applied to measure heterogeneity. Results. Eight studies (n = 8) were included in this analysis. The quantitative synthesis of evidence found that aromatherapy decreases PMS scores , I2 = 94.5%), total psychological symptoms of PMS , I2 = 82.6%), anxiety of PMS , I2 = 94.2%), depression of PMS , I2 = 93.7%), and fatigue of PMS , I2 = 89.7%) compared to the control group. Conclusion. Aromatherapy is an effective tool for the relief of PMS symptoms. Additional randomized controlled clinical trials with different durations and essential oils should be conducted to confirm our findings. Premenstrual syndrome (PMS) refers to unpleasant changes in psychological, physical, and behavioral health that occur in the last week of the menstrual cycle and resolves at the beginning of the new menstrual cycle . This syPMS affects 20% to 40% of women of childbearing age all over the world . BecauseAromatherapy is a component of CAM which involves the inhalation of plant extracts as essential oils . EssentiOne previous review evaluated the effectiveness of aromatherapy in conjunction with Iranian herbal medicines on PMS and primary dysmenorrhea . HoweverThis study was designed according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) .Databases such as PubMed/Medline, Cochrane Library, and Scopus were searched to recognize relevant published articles until October 2020. The advanced search was performed using a prepared syntax based on Medical Subject Headings (MeSH) and related keywords including  AND . To identify all possible studies, the search was conducted without the use of filters. In addition, the reference lists from eligible papers and pertaining review articles were manually inspected to ensure no other articles were missed.Based on the predefined eligibility criteria, two independent investigators (SE and MQ) reviewed the title and abstract of every article for possible inclusion in this study. The inclusion criteria were (1) randomized clinical trials (RCTs) with either parallel or crossover design; (2) studies conducted on females with premenstrual syndrome; (3) studies assessing the effect of aromatherapy on premenstrual symptoms; and (4) studies reporting mean and standard deviation (SD) of symptoms of PMS. Studies which used other methods in addition to aromatherapy were omitted unless the control group also received the additional treatment. This was done to ensure that the aromatherapy treatment was the only difference between the groups.Data extraction was conducted by two separate authors who thoroughly searched each eligible article to extract the following data: author's name, publication year, country, essential oil selection, control group intervention, duration of intervention, duration of the treatment session, the total number of sessions, participants mean age or age range, sample size, study design, outcome assessment tool, and PMS symptom values (mean and standard deviation) before and after the intervention.The risk of bias for each study was assessed by two independent examiners using the Cochrane Collaboration tool . This sc2 + 2 – ) [R value in the abovementioned formula [n), where n is the number of participants.Data were reported as weighted mean differences (WMDs) with 95% confidence intervals (CIs). The random effects model was used to assess the weighted mean difference between values of PMS symptoms. For studies which did not provide mean change with standard deviations, we calculated these data using the following formula: mean change = final values − baseline values; SD = square root ((SD baseline) final)) . A corre formula , 20. To I2 index, and I2 values >50% were considered to be evidence of heterogeneity. Subgroup analyses were conducted based on predefined factors, including sample size, duration of intervention, outcome assessment tool, and study design. Sensitivity analyses were performed to examine the influence of each study on the overall effect size. Potential publication bias was identified by Egger's test and a visual inspection of funnel plots. In the presence of publication bias, Duval and Tweedie's trim and fill method was used to control the analysis for its effects [P value below 0.05.Heterogeneity was evaluated using the  effects . All staA total of 253 articles were identified via online databases, and no additional articles were found through the additional manual search. A total of 234 articles remained after the elimination of duplicates. The abstracts of these 234 papers were screened, and the full text of 22 studies was evaluated. Of those 22 studies, 14 articles were omitted for the following reasons: 12 studies reported irrelevant outcomes, and 2 studies were review articles. This left a total of 8 studies in this analysis. The flow diagram of the study selection process is shown in Citrus aurantium blossom [n = 2) [Citrus junos Sieb. ex Tanaka) [Study participants were women who had moderate to severe PMS. Four of the studies measured PMS symptoms with PSST questionnaires , 23, 26, blossom , 23 and  [n = 2) , 23. Yuz Tanaka) , geraniu Tanaka) , and cla Tanaka) –26, thre Tanaka) , 22, 23, Tanaka) , and one Tanaka)  as the mFive studies were categorized as high quality, and the remaining articles were classified as fair, based on six domains of the Cochrane Collaboration tool. Only three studies described the exact method used for randomization, and only two studies reported blinding. The details of quality assessment for articles included in the present systematic review are illustrated in I2 = 82.6%), anxiety of PMS , I2 = 94.2%), and depression of PMS , I2 = 93.7%) in the intervention group compared to the control group. However, aromatherapy did not produce a significant effect on confusion as a symptom of PMS , I2 = 66.5%)  .I2 = 94.6%) as well as fatigue from PMS , I2 = 89.7%) based on the random effects model   .P < 0.001 for all)  .By omitting each study and reanalyzing the data, we found that none of the studies affect the significance of the summary effect size of aromatherapy on psychological symptoms of PMS (Supplementary P=0.039) identified publication bias regarding the effect of aromatherapy on psychological symptoms of PMS (Supplementary P=0.22) and the overall score of PMS (P=0.38).The funnel plot and Egger test  + bergamot (Citrus bergamia)) compared to the control group [Psychological symptoms of PMS may occur following the decrease in quantity or function of serotonin, tryptophan, and estrogen level , 28. A pol group . In a stol group .The present study found that aromatherapy could decrease the severity of psychological symptoms of PMS such as anxiety and depression, as well as fatigue as a physical symptom of PMS and the total score of PMS. Systematic review sand meta-analyses have found that aromatherapy can decrease depressive symptoms  and preoβ-caryophyllene) has been found to improve psychological symptoms such as depression and anxiety [α-pinene, also has alleviating effects on autonomic stress response to novel environments [Citrus sinensis), made up of limonene (88.1%), myrcene (3.77%), and α-pinene (1.19%), female patients had lower anxiety, higher peace, and a more positive mood in a waiting room of dental office [One important factor to consider when assessing the effectiveness of aromatherapy is that essential oils are plant extracts. Therefore, each batch differs with regard to the chemical volatile compounds contained within the oils used in these studies. The volatile compound of essential oils contains flavonoid and trepan chemicals such as limonene, gamma-terpinene, linalool, and linalyl acetate that have shown to act as anxiolytic antidepressants and contain sedative properties , 35. Lim anxiety . A majorronments . Lehrnerl office .One reason for the variation in previous clinical studies on aromatherapy might be the variation in distance between the aroma and the nostrils, which affects the overall dose of inhalation. Most studies in our analysis used a diffuser or eye pads, both of which are effective methods for producing favorable effects .One explanation for aromatherapy's effects on PMS is the way in which these volatile compounds affect the limbic system. The volatile odorant molecules are inhaled through the roof of the nose where cilia enable them to reach the receptor cells in the nose. When these volatile odorant molecules reach these cells, the olfactory bulb and olfactory tract transmit an electrochemical impulse to the primary olfactory areas in the brain that are contained in the hypothalamus, hippocampus, and the limbic system. These systems are responsible for controlling autonomic homeostasis, managing conscious thought processes, and creating emotional feelings, respectively .The small number of studies that examined symptoms of PMS is a primary limitation of this study. Publication bias in the studies that examined the impact of aromatherapy on PMS scores produces another limitation although publication bias should be interpreted with caution given the small sample size. Furthermore, the heterogeneity among studies is high. The high amount of heterogeneity among studies may be due to the differences in duration of the treatment session, the total number of sessions, frequency of the treatment, forms of essential oils, different volatile compounds, and outcome assessment tool. In addition, three out of the 8 studies included in the systematic review were only found to be of fair quality.The meta-analysis provides evidence that aromatherapy reduces overall symptom scores and both physical and psychological symptoms of PMS. To reproduce these results, a pretest is recommended before using aromatherapy, ensuring that participants have healthy olfactory function and do not experience negative responses to the oils selected. In addition, an increase in inhalation time and a higher number of sessions should be considered for future aromatherapy treatments.


Melospiza melodia). We captured 208 Song Sparrows at seven sites along a gradient of urbanization in and around Blacksburg, VA, USA, then genotyped them using a panel of fifteen polymorphic microsatellite loci. We found that genetic diversity was comparable among the seven study sites, and there was no evidence of genetic structuring among sites. These findings suggest that over a gradient of urbanization characterized by low density urban development, Song Sparrows likely exist in a single panmictic population.Urbanization fragments landscapes and can impede the movement of organisms through their environment, which can decrease population connectivity. Reduction in connectivity influences gene flow and allele frequencies, and can lead to a reduction in genetic diversity and the fixation of certain alleles, with potential negative effects for populations. Previous studies have detected effects of urbanization on genetic diversity and structure in terrestrial animals living in landscapes that vary in their degree of urbanization, even over very short distances. We investigated the effects of low-intensity urbanization on genetic diversity and genetic structure in Song Sparrows ( Urbanization and habitat fragmentation are major concerns in a changing world, and can have large effects on population genetic diversity and structure. Habitat loss and fragmentation are characterized by the disruption of a previously continuous landscape and the separation of natural areas into smaller patches with reduced area and increased isolation . Human nParus major) surveyed across an urbanization gradient displayed a weak but detectable effect of urbanization on genetic structure and a small reduction in genome-wide diversity in more urban areas [Passer domesticus) across a gradient of urbanization exhibited greater genetic structure in the urban habitat center, yet there was no significant effect on genetic diversity [Habitat changes due to urbanization have been shown to affect genetic diversity and structure in a variety of animal species. For example, an evaluation of genetic structure and diversity in several species of small terrestrial vertebrates in Los Angeles, CA suggested that high-density urbanization may have a severe effect on animal populations, resulting in reduced genetic diversity and increased genetic structure in more isolated patches . Other ran areas . Similariversity ,14. The iversity .Fully understanding the consequences of urbanization for wildlife genetic diversity requires examining population genetic structure in species living along gradients that vary in urbanization intensity , as well2 area around each site into 100 m × 100 m cells and scores the abundance of vegetation, buildings, and paved surfaces, such as roads and parking lots, in each cell. These scores are used to calculate an ‘urbanization index’ for each site using principal component analysis . Birds were captured primarily through mist netting accompanied by song playback; this method, which relies on territoriality, primarily attracts males. Mist nets were set up at the center of each study site; we did not locate the nests of individual birds. After capture, approximately 150 μL of blood was collected from the brachial vein into a heparinized capillary and 2–5 μL of blood was blotted onto filter paper  for subsequent genotyping. The birds were banded with United States Fish and Wildlife Service bands for individual identification before release.This study was conducted under permits from the U. S. Fish and Wildlife Service (MB08005B-0), the U. S. Department of the Interior (23818), the Virginia Department of Game and Inland Fisheries (053668), and an approved protocol from Virginia Tech’s Institutional Animal Care and Use Committee (protocol 13–074).We extracted total genomic DNA from the blood blots using the Qiagen DNeasy Blood and Tissue kit animal blood protocol . We modified this protocol for the removal of blood from the filter paper by changing the volumes of Proteinase K and phosphate buffered saline  used per reaction from the volumes of 20 μL kit-supplied Proteinase K and 200 μL PBS recommended in the manufacturer’s protocol to 30 μL and 190 μL, respectively.2, 0.15 mM deoxynucleotide triphosphates, 0.5 μM each of fluorescently labeled forward primers and non-labeled reverse primers, 0.5 units of AmpliTaq Gold DNA polymerase , and 1 μL DNA template (10–60 ng/μL concentration). These reactions were run with an initial denaturization step of 95°C for 9 min, 40 cycles of 95°C for 30 sec, a locus-specific annealing temperature for 30 sec . Fragment analysis was performed on an ABI 3130xL Genetic Analyzer at either the University of Texas at El Paso or New Mexico State University; a subset of samples were analyzed on both machines and this analysis confirmed that fragment lengths were comparable across sequencers. Alleles were scored in iBin v.2 .GenePop 4.2 [GenAlEx 6.5 [FIS.We used ePop 4.2 ,34 to teAlEx 6.5 ,36 to caFST values between each pair of study sites using GenAlEx 6.5 [P-values for FST between sites. We applied α = 0.05 and a false discovery rate (FDR) [FST values. We also implemented a Mantel test in GenAlEx to test for the presence of isolation by distance [We calculated pairwise AlEx 6.5 ,36, usinte (FDR)  of 0.1 wdistance .We conducted a hierarchical AMOVA  using the R package “poppr” v. 2.8.4 . This anStructure 2.3.4 [K from one to 10, then used both the L(K) and Δ(K) methods implemented in Structure [Structure Harvester [Structure run to refine our estimates of cluster membership. We increased the number of replications to 5,000,000, with all other model parameters remaining the same as in earlier analyses.We also applied two Bayesian clustering methods to estimate the number of genetic clusters represented by our samples. First, we estimated the number of genetic clusters using re 2.3.4 , using ttructure  and Struarvester  to deterarvester . After tGeneland 4.0.8 [Geneland runs to determine the most likely number of genetic clusters, we ran an additional analysis with K set at the most likely value to refine estimates of the proportional membership to each cluster for each individual bird. Parameters for this analysis were the same as the initial Geneland runs, except a single run was conducted with the number of MCMC repetitions increased to 5,000,000, and K was set at the most likely value obtained from the initial analysis.Analysis in nd 4.0.8 , which iSosp014 and Sosp141 were each out of HWE at two sites, and Sosp012 was out of HWE at four sites. We detected evidence of linkage disequilibrium between Sosp003 and Sosp012. Consulting the Song Sparrow linkage map [Sosp003 and Sosp012 were in the same linkage group, and that all other loci used in our study were in different linkage groups. Sosp012 was removed from further analysis because in addition to the LD, it was also out of HWE at four sites. After the removal of Mme012 and Sosp012, 13/15 loci were retained for subsequent analyses.A total of 208 individuals, 94% of which were males, were successfully genotyped . We did kage map  confirmeFST ranged from 0.010–0.023 and showed no significant genetic differentiation between any pairs of sites .Observed heterozygosity was generally similar across the seven study sites and did not appear to be related to urbanization index . Pairwisof sites . AlthougStructure suggested that the most likely value of K was one  method [K) and the barplots suggested that that true number of clusters was one.Using the L(K) method,  was one . We obseof three . Howeverividuals . If K we) method , and botGeneland analysis identified a most likely K of eight, with the two runs with the highest likelihood settling on K of eight. Four of ten runs overall indicated that the most likely value of K was seven. We ran a follow-up analysis in Geneland with K set to eight, in case Geneland analysis had detected cryptic subdivision in one of our seven sampling sites. However, the parameterization run instead found that birds from 6/7 sites were predominantly assigned to the same cluster, and that birds from the seventh site (Kentland Farm) were largely assigned to that cluster as well  ,24? DespPasser domesticus) [Tachycineta bicolor), significant genetic structure was not detected, but researchers found higher levels of genetic diversity in less rural areas [Setophaga chrysoparia, formerly Dendroica chrysoparia) living across a broad expanse of urban area  did not differ between sites, but low, yet significant, levels of genetic differentiation were detected [Athene cunicularia) did not show differences in genome-wide heterozygosity, but the urban populations did significantly display weak genetic structure [It is also possible that there simply is no genetic differentiation among Song Sparrows living at the sites that we studied. This possibility is consistent with some other studies of population genetic structure in songbirds, which have documented results ranging from little to no genetic differentiation between urban and rural populations, to cases where urban populations unexpectedly had higher genetic diversity than did rural populations. Notably, a previous study of Song Sparrows in the Bay Area of California did not detect significant genetic structuring, even among morphologically recognized subspecies . Howeveresticus) . In Treeal areas , a resulal areas . Geneticdetected . Similartructure . Our fintructure ,46. SuchFinally, although we have focused here on studies of genetic diversity and structure in avian systems, an extensive recent review of the effects of urbanization on both animals and plants suggested that inconsistent and even contradictory results among studies may not be unusual at all . Miles eS1 Data(XLSX)Click here for additional data file.