Skin biopsies (5 mm) taken from behind the dorsal fin on Atlantic salmon post-smolts were followed over a 2 month period. The healing process was dominated by hemostasis, acute inflammation, and epidermal repair the first 14 days post wounding (dpw), as shown through imaging, histological evaluation, and transcriptomics. Most of the immune genes showed decreased expression after two weeks, approaching the levels of intact skin, as also reflected in sections where reduced inflammation in the wound bed was observed. Transcriptional events suggest recruitment of lymphocytes to the wound site during the acute phase, with activation of humoral responses from 14 dpw and onward. From the histology, a more adherent mucus was observed that correlated with altered transcription of glycosyltransferases. This may indicate different properties and functions of the mucus during the wound healing process. Wound contraction started between 14 and 36 dpw. The occurrence of these events was concurrent with granulation tissue formation, melanocyte migration and up-regulation of genes involved in extracellular matrix formation. The presented description of the wound healing processes in Atlantic salmon gives insight into comparative ulcerative biology in mammals and fish and provides both novel and updated knowledge that can be applied for improved best operational practices for fish welfare in aquaculture. Salmo salar), is a coherent and dense barrier that protects the interior of the fish against the outer environment. The skin covers the entire outer surface, including the head, fins and eyes. Lesions in the skin are a major economic problem for the farmer and a welfare issue for the fish1. There are two types of wounds, mechanically induced wounds and lesions caused by pathogens. Mechanical wound development is often associated with events that may cause skin damage such as handling, de-lousing, acute panic episodes, storms, and predators1. Pathogens that may cause wounds includes sea lice that feed on skin and mucus, and the winter ulcer bacteria Moritella viscosa and Tenacibaculum spp1. Most studies on skin problems in Atlantic salmon focus on the interactions between the skin and wound causing pathogens5. However, there is little knowledge about the actual wound healing process in Atlantic salmon skin without the presence of pathogens.The skin of Atlantic salmon  that migrate from the intact surrounding tissue to cover the wound surface10. In Atlantic salmon, small superficial wounds may close within a few hours, but this re-epithelialization process is dependent on factors such as temperature, wound dimensions, stress, and nutrition13. Following re-epithelialization, a preliminary epidermis with mucus production is formed and this initial barrier protects the wound from the external environment15. Reconstitution of the epidermis is an important part of the healing process, but for deeper wounds, the dermis also needs to be regenerated. Since the dermis consists of several structures and cell types such as scales, bone cells, dense and loose connective tissue, fibroblasts, pigment cells, and capillaries16, complete regeneration depends on an orchestra of responses involving many cell types and tissues. This process is well studied in zebrafish, in which granulation tissue is already present at 2 days post wounding (dpw) and at 28 dpw the damaged skin is almost indistinguishable from intact tissue6. In contrast, the process of granulation tissue formation in salmonids is poorly understood. In salmonids, the formation of granulation tissue is reported to start between 10 and 42 dpw18, variations depending most likely on the methods used to assess the wounds, life stage, and temperature.All the classical stages of wound healing: re-epithelialization, inflammation, cell proliferation with granulation tissue formation, and tissue remodeling, are conserved in fish19, creating a full thickness wound on the dorsal flank of the fish. The advantage with full thickness wounds compared to other wound healing models such as scale loss and skin abrasion is that both dermal and epidermal repair may be studied, as well as wound contraction. By combining several methods, including photography, histology, immunohistochemistry, scanning electron microscopy and transcriptomics, important phases in the wound healing process in the skin of post-smolt Atlantic salmon was elucidated.The aim of this study was to thoroughly investigate and describe the molecular processes involved in the different phases of dermal wound healing in post-smolt Atlantic salmon. The fish were wounded with a 5 mm punch biopsy tool, as previously described by several authorsEach fish was wounded with a 5 mm punch biopsy needle Fig. , and theAt 1 dpw, the wound surface was fully or partly covered by an amorphous exudate Fig. , with miAt 3 dpw, a neo-epidermis covered the wound surface Figs  and 4a1.At 7 dpw, the epidermal layer was no longer clearly separated from the wound bed and several keratocytes were found interspersed among the damaged muscle fibers Fig. . These kAt 14 dpw, both inflammation and tissue repair were prominent in the wound bed. The epidermal layer was more organized compared to 7 dpw, with a clear separation to the wound bed Fig. , howeverAt 36 dpw, the wounds were partly contracted Fig. , and filCollagen deposits filled the wound bed, but the fibrils did not have a uniform direction Fig. . In addiAt 43 dpw very few PCNA+ cells were found in the wound bed and in the epidermal layer Fig. . The colAt 57 dpw the wounds were fully contracted Fig. . In the Wounding had a major impact on the transcriptomic response with >2000 differentially expressed genes (DEG) at three or more of the analyzed time points. Thus, clustering of DEG with known functions (N = 1444) was performed to reveal general trends in the transcriptomic profiles Fig. . The majmatrix metalloproteinase 9 and 13) were the most up-regulated genes in this study, with a log2 fold change of 7 (128-fold change) at 3 dpw  also peaked in transcription between 1–3 dpw. Further, many genes with diverse immune functions and innate effectors, such as components of oxidative burst complex (cytochrome b-245 and neutrophil cytosolic factor1), lipid signalling (eicosanoids) and multiple responders to cellular stress (heat shock proteins) and acute phase protein (serum amyloids) had a log2 fold change ≥2 (4-fold change) at 1–14 dpw. Genes known as activators of B and T cells  and genes involved in B and T cell differentiation  stayed activated for the entire duration of the study (1–57 dpw).To understand the ongoing inflammatory and regenerative processes, we examined transcription of selected genetic markers activated in the early (1–14 dpw) or late (36–47 dpw) healing phases Fig. . The matvascular endothelial growth factor), endothelial migration (angiopoietin), proliferation of hematopoietic stem cells (interleukin 11), and vasoconstriction (angiotensinogen). At 1 dpw several genes involved in coagulation/anticoagulation  were also activated . Further, several genes associated with epithelial migration , cell to cell adherence , and components of the basement membrane  were active at 1 dpw. Transcription of the glycosyltransferases alpha 2,3-sialyltransferase and Alpha-N-acetylgalactosaminide-alpha-2,6-sialyltransferase6 increased during the early healing phase.Several genes involved in haemostasis and tissue repair also showed early activation Fig. . These ited Fig. , togethemajor histocompatibility complex −1 and −2a and cluster of differentiation 4, a key marker of T helper cells  and proteins involved in maturation of fibrils. Late activation was also shown for several growth factors such as transforming growth factor-beta, connective tissue growth factor and epidermal growth factor.Late activation was shown for many genes involved in the synthesis of immunoglobulins together with antigen-presenting proteins such as the lls Fig. . Other gWe observed a clear separation between the early (1–14 dpw) and late (36–57 dpw) healing phases. The separation was manifested in the wound morphology (expanded vs. contracted), histology (inflammation vs. fibrous tissue) and gene expression data (inflammation vs. fibrous tissue repair). The early healing phase could be further separated into re-epithelization and hemostasis which was activated at 1 dpw and acute inflammation at 3 to 7 dpw, while 14 dpw represented a transition stage between acute inflammation and the onset of fibrous repair. In the late healing phase, proliferation and collagen deposition was prominent at 36 dpw, whereas tissue remodeling was established at 43–57 dpw. The timeline of the most important wound healing events in the skin of Atlantic salmon are summarized in Fig. integrins and fibronectin, which are used by platelets to mediate aggregation and fibrin clot formation21. In mammals, platelet activation also results in the release of a number of signaling molecules such as vascular endothelial growth factor22, and connective tissue growth factor that can induce sustained fibrosis23, these were also up-regulated immediately by wounding. In the gills of rainbow trout (Oncorhynchus mykiss), eicosanoids play a significant role in vasoconstriction24. As several genes involved in eicosanoid metabolism were up-regulated immediately by wounding, a similar role might be present in fish skin. Genes involved in blood coagulation such as antithrombin, urokinase, and serpine1 were also up-regulated immediately by wounding. Since the absence of blood clot formation is considered a hallmark in the healing of fish skin6, these genes may have other functions in fish. Nevertheless, an amorphous exudate of unknown origin covered the wound surface at 1 dpw. A similar amorphous exudate has been reported in wounds of juvenile Atlantic salmon12 and in rainbow trout18. Thus, we assume the formation of fibrin-like structures would initially protect the wound surface and provide a smooth surface aiding epithelial migration. A novel finding in this study was the presence of mucous cells together with the migrating keratocytes at the wound borders at 1 dpw. This finding suggests that differentiation of mucous cells takes place as the keratocytes migrate on the wound surface. This contradicts findings in a previous study in which it was suggested that differentiation and proliferation of the keratocytes only occur when the migrating keratocytes cover the entire wound area25. The re-epithelization process was finished somewhere between 1 and 3 dpw, which is in concordance with previous research on 5 mm punch biopsy wounds in Atlantic salmon post-smolts11.At 1 dpw, re-epithelialization and hemostasis were activated. At the wound margins, both migrating keratocytes and mucous cells were found. Several genes involved in epidermal repair and hemostasis were up-regulated, such as 27. Given that melanin pigment has antioxidant activity and protects against environmental stressors30 and pathogens31, this pigmentation may be an initial mechanism protecting the wound surface.Wounding also led to immediate hyperpigmentation at the wound edges and spreading of pigmented bodies at the wound surface. These pigmented bodies were found on the wound surface at all investigated time points, but not on intact skin. Such a phenomenon is reported in several previous wound healing studies35.Between 3 and 7 dpw, the histological analysis showed infiltration of inflammatory cells into the wound bed accompanied by tissue degradation. The transcriptomic results supported these findings and indicated the activation of an acute immune response and proteases from 1 dpw. This included transcription of immune effectors such as chemokines and interleukins that are involved in the recruitment of macrophages and other immune cells to the wound site. In general, the first two weeks post wounding showed a characteristic early innate immune response, similar to that observed in previous studiesmatrix metalloproteinases. These proteases are secreted by both keratocytes and macrophages, and they are essential components of several parts of the wound healing process36. Further, they degrade extracellular matrixes such as laminins37, and fibrillar collagens, and they control inflammation by regulating the activity of cytokines and chemokines38. Thus, most of the observed tissue deterioration in the wound bed is likely linked to the high activity of these proteases and part of the natural wound healing process.At 7 dpw, histological analysis showed that there was no clear separation between the epidermal layer and the damaged muscle fibers. This might be a side effect of the inflammatory response and protease activity in the wound bed. The genes with the greatest overall change in expression ratio in this study were the 39, and alter the glycosylation pattern40, and the proportion of acidic and neutral mucins41. The observed alteration in the mucus response happened simultaneously with the high inflammatory response in the wound bed, and could possibly represent an additional epidermal protection mechanism.At 3 and 7 dpw, pink-purple stained mucous cells were present in the epidermis, whereas very few pink-purple mucous cells were found at the other time points. At 7 dpw, the percentage of acidic and neutral mucous cells was at the greatest abundance. At this time point, mucus was also sticking to the surface of SEM samples and to the AB/PAS samples. Indeed, for the SEM samples, the mucus gel completely covered the wounded surface, covering the epithelial cells. Being the only time point with these characteristics, increased adherence properties of the mucus gel is suggested. It has been shown that a large number of inflammatory mediators can directly enhance mucin transcription in mammals42. At 14 dpw, myofibroblast-like cells were also present in the granulation tissue. In humans, these cells express α-smooth muscle actin, which plays a major role in wound contraction43.At 14 dpw, both inflammation, granulation tissue, scale formation, and collagen fibrils were present in the wound. In this regard, 14 dpw represented the beginning of the transition between the early and late healing phase. The collagen structures were distinct, with three to four rope-like structures protruding from the intact muscle tissue. Given the distance between the rope-like structures, we suggest that fibroblasts may migrate from the myocommata, which are the major connective tissue compartment in teleost muscle17 suggesting that fish skin lacks a primary contraction mechanism. Such rapid contraction is a common feature of animals with loose skin including, mice, cats, dogs, and horses46. In humans and porcine, the dermis is firmly attached to the muscle and therefore primary wound contraction is limited47. Firm attachment of the muscle to the skin dermis is also the case in fish42. Therefore, wound contraction in salmonids is most likely driven by granulation tissue formation, similar to what is driving wound contraction in humans and porcine47.The wounds started to contract between 14 and 36 dpw, thus the appearance of these cells may indicate the onset of wound contraction. Before 14 dpw, the wounds were expanding rather than contracting. Similar results are reported in rainbow trout48. At 36 dpw there were few PCNA+ cells around the new scales, and the scales had features similar to late differentiated scales with epidermal folding in zebrafish49. In a parallel wound healing study, we recently showed that post-smolts reared at high fish densities had delayed scale mineralization50. In adult zebrafish both scales and pigmentation pattern completely recover after deep cutaneous wounding6, however a similar study in rainbow trout did not report scale regeneration during the healing of a 6 mm deep cutaneous wound17.At 14 dpw early scale differentiation with proliferating bone cells and deposition of mineralized matrix were present in the wounds. Osteoblasts proliferate as they differentiate from a mesenchymal stem cell to pre-osteoblast, thus the PCNA+ cells at 14 dpw indicate scale formation12. Here, we also report onset of fibrous repair at 14 dpw for 5 mm punch biopsy wounds. Thus, suggesting the possibility that onset of fibrotic repair may be independent to wound size and life-stage. In zebrafish, inflammation is important for both fibroblast recruitment and granulation tissue formation6, indicating fibroblast-stimulating signals from inflammatory cells in fish. This is also the case in mammals where macrophages promote granulation tissue formation and neo-vascularization51. Thus, inflammation is most likely needed to activate dermal repair in teleosts.Further, a previous study following the healing of narrow incisional wounds in Atlantic salmon juveniles (reared at 10 °C) found that fibrous tissue repair starts at 14 dpwtransforming growth factor-β and epidermal growth factor were activated. Remodeling is the final phase of wound healing, and in this phase, the cell density and metabolic activity in the granulation tissue decreases. Changes also occur in the type, amount and organization of collagen, leading to an enhancement of the tensile strength of the tissues36. Given our findings, we suggest that  the wounds have entered the remodeling phase at 43 and 57 dpw.The late healing phase (36–57 dpw) was characterized by wound contraction and the formation of granulation tissue and scarring. At 36 dpw, an abundance of dividing cells was located in the wound bed, together with randomly distributed collagen fibrils. At 43–57 dpw, there were very few proliferating cells in the wound bed. The collagen fibrils were also more mature, being assembled into thicker structures aligned parallel to the epidermis. From 36–57 dpw, several genes involved in collagen synthesis, fibril maturation and growth factors such as 53. Here we show that similar processes are likely occurring in fish. In mammals, the role of T-cells in wound healing is best described, with CD4+ cells suggested to enhance the wound healing process54, while other T-cell populations either have positive or negative effects on collagen deposits in the wounds57. B-cells also play a critical role in wound healing, as they may modify the immune cell populations and accelerate the healing process59. In this study, there was no indication of secondary infections in the wounds. Nevertheless, mechanical wounding will expose the tissue to an unsterile environment which is likely to also impact the immune response. In earlier trials with experimental virus infections of salmon smolts, we observed activation of B- and T-cell marker genes at 6 to 8 weeks post-infection60; thus, in the present study, the adaptive response was activated one or two weeks earlier compared to previous observations. Therefore, the results suggest that the activation of the adaptive immune response is connected to the wound healing and we suggest a role of activated lymphocytes in the fibrotic repair process in Atlantic salmon.The adaptive immune response was strongly activated from 36 dpw. In mammals, both the innate and adaptive immunity is required for wound healingLabeo rohita)61. Thus, it appears that the epidermal layer at the edges of the wounds has a different function to the cells located at the wound center.In the late healing phase, the epidermal layer was also under reconstruction. Proliferation of epidermal cells at the wound margins, together with few proliferative cells in the wound center, suggests a growth zone at the margins and a remodeling zone in the wound center. The proliferative activity in the epidermis decreased alongside with the proliferating activity in the wound bed. Analysis of the flat keratocytes at the wound surface also showed a different cell morphology of the cells located at the wound margins and those at the wound center. A similar appearance of flat keratocytes at the margins and elevated epithelial cells at the wound center has been reported in healing wounds of the teleost fish rohu carp  were reared in a single 500 L tank). During the experimental period, the mean fish density was 14 kg/m3. From the 4th to the 6th of November the fresh water in each tank was gradually replaced with seawater. The specific water flow was adjusted to 0.5 L/kg/min at the start of the experiment. The dissolved oxygen level in the outlet water was kept higher than 80% saturation by automatic oxygenation of the water in the header tanks (Oxyguard Commander). Both temperature (ranging from 9.4–10 °C) and oxygen saturation were measured daily . Following transfer to seawater, the fish were exposed to a regime of 12 hours light and 12 hours dark. The fish were fed a commercial dry diet .This study was carried out at the Industrial Laboratory  between November 4rd of December, skin biopsies were taken on 90 individuals with a 5 mm biopsy punch (IntegraTMMiltexTM) as described by17. Prior to wounding, the fish were fully anesthetized with MS-222 (Sigma-Aldrich). Directly after wounding, the three layers of skin, epidermis dermis and hypodermis were removed, leaving a deep cutaneous wound with damaged muscle fibers. Samples (N = 12 fish per sampling point and group) were taken intensively during the first week , and thereafter 14, 36, 43 and 57 dpw. Mortalities during the experiment period were low (<1%). Upon completion of the experiment, fish were euthanized with an overdose of anesthetic (MS-222). Samples for gene expression analyses were frozen immediately in liquid nitrogen and transferred to −80 °C for storage. The skin samples were fixed in a 4% paraformaldehyde solution (Electron microscopy science) overnight and then washed in 1 x PBS (Sigma Aldrich), before stepwise dehydration to 70% ethanol and finally transferred to −20 °C for storage. Skin samples for SEM were stored in PBS and not dehydrated.On the 3Photographs of the fish and wounds were taken with a Sony Cyber-shot DSC-RX100 Zeiss camera . Unstained tissue samples were magnified under a Leica stereoscope (Leica wilde M3B), N = 6 for each time point, photos were taken through the optical lens with a Sony Cyber-shot DSC-RX100 camera.2O pH 4.3 for 2 min. The Picro Sirius Red Stain Kit (Polysciences inc.) was used according to the manufacturer’s instructions. Staining of Proliferating Cell Nuclear Antigen (PCNA), was done with mouse anti-PCNA IgG2a (Millipore) and VECTASTAIN® Abc-HRP kit, anti-mouse IgG (Vector Laboratories) according to the manufacturer’s instructions (N = 3). Microscopy was carried out on a Zeiss Axio Observer Z1 (Carl Zeiss). Measurements were done in Aperio ImageScope (v12.3.2.8013). For evaluation of mucous cells (AB/PAS), a 10X objective was used. Mucous cells within the 10x magnification frame were counted. One frame approximately covered one-third of the wounded area at 1–14 dpw, and the half of the wound area at 36–57 dpw. The number of mucous cells was counted manually in two standardized areas on each sample, further differentiated between acidic (blue cells), neutral mucins (pink cells) and a mix of neutral and acidic mucous cells (purple cells).Skin samples for histological evaluation were embedded in paraffin using a Leica TP1020 (N = 4 at 1–14 dpw and N = 3 at 36–57 dpw). Staining with Alcian blue and periodic acid-Schiff (AB/PAS) was done by staining the slides for 15 min in Alcian blue (Sigma-Aldrich), pH 2.5 followed by oxidizing the sections in 0.5% periodic acid solution (Sigma-Aldrich) for 5 min, followed by Schiff reagent (Merck) for 15 min and counterstaining in Mayer’s hematoxylin (Sigma-Aldrich) for 30 s Hematoxylin (Sigma-Aldrich) and eosin (Sigma-Aldrich) staining was done according to manufacturer’s instructions. Staining with alizarin red (Sigma-Aldrich) were done in a solution of 2 g Alizarin red in 100 mL dHSkin samples for SEM (N = 3) were dehydrated from PBS to absolute ethanol and dried using a Critical Point Dryer  with liquid carbon dioxide as the transitional fluid. The samples were then mounted on stubs with carbon tape and coated with gold-palladium  and examined with SEM . Sample preparation and analysis were performed at the Imaging Center at the Norwegian University of Life Sciences.Tissue samples for quantitative real-time PCR (RT-qPCR) were stored at −80 °C prior to RNA extraction. Frozen skin sections (N = 6 per time point) were transferred directly to 1 mL chilled TRIzol  and homogenized in a Precellys®24 homogenizer. RNA was extracted from the homogenized tissues using a PureLink™ Pro 96 well purification kit (Thermo Fisher Scientific) with on-column-DNase  digestion according to the protocol for TRIzol-homogenised samples. The concentration of extracted total RNA was measured with a NanoDrop 1000 Spectrometer (Thermo Fisher Scientific) and RNA integrity was determined with an Agilent 2100 Bioanalyzer with RNA Nano kits . Samples with an RNA integrity number (RIN) of 8 or higher were accepted.62. Microarrays were fabricated by Agilent Technologies. All reagents and equipment were purchased from the same source . All kits were used according to the manufacturer’s protocol. In brief, RNA amplification and labeling with Cy3 was performed with Low Input Quick Amp Labeling Kits  and Gene Expression Hybridization Kits were used for fragmentation of labeled RNA and preparation of the hybridization setup. Microarrays were hybridized for 17 h in a hybridization oven at 65 °C and rotation speed of 10 rounds per minute, washed for one minute with Gene Expression Wash Buffer I at room temperature, and one minute with Gene Expression Wash Buffer II at 37 °C. Washed slides were scanned with an Agilent SureScan Microarray scanner. Eight biological replicates from intact skin (control samples) and ten replicates per time-point were included in analyses, in total 38 arrays were used. Nofima’s bioinformatics package STARS32 was used for data processing and mining.Microarray analyses were performed using Nofima’s Atlantic salmon DNA oligonucleotide microarray SIQ-6  containing approximately 15,000 probes of genes selected by annotations and expression profiles2-Expression Ratio >|0.8| (1.75-fold) and p < 0.05 relative to the un-wounded controls. A complete gene list of DEG, gene identifier and their respective STARS category can be found in Supplementary File 2 fold change relative to controls were calculated for each gene and time point. Only genes significantly different from the controls at 3 or more time points were included in the analysis (1444 genes). The Euclidean distances were calculated, and the complete linkage clustering was drawn as a heat map. The dendrogram was pruned in order to identify 8 clearly-defined sub-clusters. One cluster contained only two genes and was thus excluded from the plot. Enrichment analyses of functional categories were calculated for each cluster with one-tailed Fisher tests to test for significant over-representation in the given cluster. Filtering, statistical analyses, and plotting of results were performed in R .For the microarray results, the differentially expressed genes were selected by the following criteria: logo >|0.8| .75-fold This study was approved by the local responsible laboratory animal science specialist under the surveillance of the Norwegian Animal Research Authority (NARA) and registered by the national ethics committee (the Norwegian Food Safety Authority) (ID7058). The methods were carried out in accordance with the relevant guidelines and regulations.Supplementary Dataset 1


Additionally, we observe an asymmetrical distribution of mtDNA haplogroups in the ancient population, with certain haplogroups appearing more frequently in the islands closer to the continent. This reinforces results based on modern mtDNA and Y-chromosome data, and archaeological evidence suggesting the existence of two distinct migrations. Comparisons between insular populations show that some populations had high genetic diversity, while others were probably affected by genetic drift and/or bottlenecks. In spite of observing interinsular differences in the survival of indigenous lineages, modern populations, with the sole exception of La Gomera, are homogenous across the islands, supporting the theory of extensive human mobility after the European conquest.The Canary Islands’ indigenous people have been the subject of substantial archaeological, anthropological, linguistic and genetic research pointing to a most probable North African Berber source. However, neither agreement about the exact point of origin nor a model for the indigenous colonization of the islands has been established. To shed light on these questions, we analyzed 48 ancient mitogenomes from 25 archaeological sites from the seven main islands. Most lineages observed in the ancient samples have a Mediterranean distribution, and belong to lineages associated with the Neolithic expansion in the Near East and Europe . This phylogeographic analysis of Canarian ancient mitogenomes, the first of its kind, shows that some lineages are restricted to Central North Africa , while others have a wider distribution, including both West and Central North Africa, and, in some cases, Europe and the Near East . In addition, we identify four new Canarian-specific lineages  whose coalescence dates correlate with the estimated time for the colonization of the islands (1 Due to pulation . Europeath century, the Spanish kingdom of Castile gradually conquered all of the Canary Islands, after previous European attempts. In most of the islands, the indigenous people resisted the European conquest [Chroniclers were amazed to discover that the Canarian natives were unaware of navigational methods and had remained isolated from the African continent ,3. Durinconquest . The cruconquest . In spitconquest . Those tThe geographic origin of the Canarian indigenous people was initially inferred from both the interpretation of historical written sources and the analysis of archaeological evidence. Most archaeological and anthropological data support a North African origin for the Canarian indigenous people, relating to the Berber populations ,8. Key est century BCE were obtained from sediment, wood and charcoal samples that could be older than the archaeological site where they were excavated.In regards to the time of the arrival of the first population groups, some authors have proposed the first millennium BCE as the upper bound for human presence in the archipelago , based oMitochondrial DNA (mtDNA) is a powerful tool for inferring the geographic origin of populations . MtDNA ith century [Regarding the colonization model, linguistic research has pointed to at least two migration waves from North Africa ,34. Also century . This id century  and Y-ch century  in the mThe direct analysis of ancient remains from the Canary Islands, using mtDNA by means of PCR techniques, confirmed the presence of North African markers in the indigenous people, including the U6b1a haplogroup, as well as some of the Eurasian lineages observed in the modern population . AdmixtuAlthough previous ancient DNA (aDNA) studies have been fundamental to understanding the origin and evolution of the Canarian population, most of the ancient mtDNA data produced so far has been obtained using PCR amplification. This classical aDNA technique has provided valuable information, but results have always been hindered by the risk of sample contamination. This is due to the fact that aDNA from warm climates is often extremely degraded and the PCR technique is highly sensitive, thus minute amounts of modern contaminant DNA can be preferentially amplified . AdditioA recent NGS study of the Canarian indigenous people presented the first complete mtDNA genomes and low-coverage full genomes from this population, and, more specifically, from the central islands of Tenerife and Gran Canaria . HoweverIn order to obtain a comprehensive mtDNA perspective on the origin of the indigenous people of the Canary Islands, we have applied aDNA protocols and NGS to assemble ancient mtDNA genomes from all seven sub-populations. Since human remains from warm regions like the Canary Islands are expected to have low endogenous DNA content, we applied an enrichment technique  to improSamples were collected in collaboration with both Canarian universities, La Laguna (Tenerife) and Las Palmas de Gran Canaria (Gran Canaria), as well as the insular museums of Gran Canaria (El Museo Canario), La Palma (Museo Arqueológico Benahorita) and La Gomera (Museo Arqueológico de La Gomera). Information about specimens used in this study is available in Best-conserved samples were selected for DNA extraction. Although the petrous bone is considered the best source for aDNA , we usedRequired precautions were taken during the handling of samples, and all experiments that included aDNA were carried out in dedicated, clean lab facilities at the Paleogenomics Lab, University of California Santa Cruz, to avoid contamination. DNA extraction was performed following Dabney et al. . Bone saAfter the screening of shotgun libraries, those samples with an endogenous DNA content lower than 10% were enriched using whole-genome in solution capture . BrieflyIn order to perform population-based analyses, we included in our study previously published –39 and uWe included in this study several current Canary Islands mtDNA genomes, analyzed using both whole-genome and Sanger sequencing. Complete genomes were obtained in Instituto Tecnológico y de Energías Renovables (ITER) by whole-genome sequencing from a set of 18 unrelated Canarians. Briefly, DNA samples were processed with a Nextera DNA Prep kit, with dual indexes following the manufacturer’s recommendations . Library sizes were checked on a TapeStation 4200  and their concentration determined by the Qubit dsDNA HS Assay . Samples were sequenced to a depth of 30X on a HiSeq 4000 instrument (Illumina) with paired-end 150-base reads. Sanger sequencing mtDNA genomes were obtained at University of La Laguna following previously published methodologies , for samSubmission to the Stanford Institutional Review Board was waived because this research does not involved human subjects, we did not interacted with study subjects and because the specimens were collected for purposes other than the current research. Consent was not obtained for this study because data were analyzed anonymously.Shotgun sequencing reads were trimmed and adapters removed using AdapterRemoval version 1.5.4 . SpecifiDamage patterns were assessed using MapDamage v2.0 . Insert http://www.phylotree.org) [http://www.ncbi.nlm.nih.gov), phylogenetic trees were built using median-joining networks [MtDNA reads were directly mapped to the revised Cambridge Reference Sequence (rCRS)  and filtree.org) . MtDNA hree.org) . Modern networks . Indels networks . We usednetworks . Accompanetworks . For higNewly reported HVR sequences were analyzed using BioEdit software v.7.0.9.0 , and hapST [NGS data was combined with previous HVRI sequencing data to perform population-based analysis. Published samples used for comparisons are detailed in ST  as in ArST . MultidiST . AdmixtuST , which wST .The average endogenous DNA content for the Canarian ancient samples is 7.92%, a relatively high value considering the warm and humid environmental conditions of the archipelago . HoweverAfter capture, we obtained complete mtDNA genomes from 48 ancient human remains sampled in 25 different archaeological sites . Our samWe also obtained complete mtDNA genomes from a set of 18 modern Canarians . More thIn order to compare our samples to previously published data, we combined the newly generated mtDNA genomes with HVRI data from the Canarian indigenous population  33,36–3–336–39. As previously observed, the indigenous populations of the Canary Islands in the past were not homogenous ; Fig 3. p = 0.0117), L3b1a (p = 0.0038), U5 (p = 0.0117) and U6c (p = 0.0012). Though also present in the western islands, haplogroups T2c1 (p = 0.0168) and U6a (p = 0.0029) appeared more frequently in the eastern islands. However, these results can be artifacts caused by the high frequency of H1cf in El Hierro and U6b1a in La Gomera. After removing these two populations from the western group, only differences in the distribution of U6c remained significant (p = 0.0332).By directly comparing the mtDNA types found in the indigenous population of each island, we observe that H4a1e, L3b1a, U5 and U6c haplogroups are present only in the eastern islands . Differences between eastern and western islands were shown to be significant for the four haplogroups, when all the ancient samples were considered: H4a1 (p = 0.2364).In contrast with the heterogeneity we observe in pre-Hispanic times, mtDNA haplogroup frequencies in modern populations of the Canary Islands are homogenous , with thIn order to determine the admixture pattern at an insular level, we compared modern Canarian samples with their principal parental populations: indigenous people, Iberians, and sub-Saharan Africans . Global To determine if a more specific origin for the Canarian indigenous population could be ascertained, the ancient mtDNA sample was combined with a reference modern DNA database containing samples from the Canary Islands, Europe, North Africa, Sub-Saharan Africa and the Near East . In the The HVRI has been proven to be of limited value in providing a clear picture of the origin of the indigenous people of the Canary Islands. In order to conduct a better assignment of the geographic origin of the maternal Canarian indigenous lineages, we performed detailed phylogeographic analysis of all the lineages observed in the aDNA dataset , includiWe observe five different H sub-lineages in the indigenous people of the Canary Islands: H1cf, H1e1a9, H2, H3 and H4a1e. H1cf  seems toTwo sublineages of haplogroup J are observed in the indigenous population of the Canary Islands: J1c3 and the newly defined J2a2d1a1. J1c3 is present in Europe, North Africa and the Near East, and more interestingly, in ancient Neolithic samples from Spain and Sardinia . AlthougPhylogenetic analysis of the Canarian T2 sequences places them within T2b and T2c1d, two haplogroups thoroughly observed in Neolithic and Bronze Age sites from Europe. The inclusion of ancient and modern Canarian samples allows us to define four new T2c1d subhaplogroups . T2c1d3 We identify several ancient samples within macrohaplogroup L, belonging to L1b1a and the newly defined L3b1a12. Although current mtDNA analysis evidences the presence of Sub-Saharan lineages into North Africa in the Early Holocene , Later SCanarian ancient sequences belonging to X haplogroup are classified within the X3a clade . This liFinally, several U6 sublineages are observed in the indigenous population of the Canary Islands, including U6a1a1 , U6a7a1 Our mtDNA results on the indigenous people of the Canary Islands shed light on the prehistory of North Africa. Our data are in agreement with recent aDNA data from Morocco  and furtIn our phylogeographic analysis of complete mtDNA sequences from the Canarian indigenous population, we found lineages that are only observed in Central North Africa and the Canary Islands , while others have a wider distribution including both West and Central North Africa, and, in some cases, Europe and the Near East . These results point to a complex scenario, where different migration waves from a dynamic and evolving North African population reached the islands over time. Every island experienced their own evolutionary path, determined by the environmental conditions and limitations of insularity. Those islands with the capability of sustaining large populations retained variability, while others with more restricted means (La Gomera and probably El Hierro) had to develop cultural practices to avoid inbreeding, like mandatory exogamic practices ,94.th century BC [Although the North African Berber origin is the most widely accepted hypothesis, other lines of research have proposed that certain funerary practices and religious beliefs observed in the indigenous population of the Canary Islands could be linked to Punic-Phoenician influence , thus prntury BC . This Cantury BC  publisheth century, and all except one are from after the 10th century. The only site with an older date is Guayadeque; however, we have to take into account that this is a large site, with evidence of human occupation extending until the 14th centuries AD [Previous genetic analyses of the modern Canarian population detected an asymmetrical distribution of maternal and paternal lineages in the archipelago ,35. Our uries AD , and theth - 12th centuries [th and 13th centuries [Archaeological data has evidenced significant changes in the productive strategies of some islands around the 11enturies ,104–106.enturies ,108. Theenturies . Again, enturies ,110. FutS1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.S1 FigCombined calibrated radiocarbon per archaeological site (A) and per mtDNA lineage (B).(PDF)Click here for additional data file.S2 FigNumber along links refers to nucleotide changes, whereas “@”, “d” and “i” indicates back mutations, deletions and insertions, respectively. Recurrent mutations, such as 309iC, 315iC and 16519, have not been taken into account.(PDF)Click here for additional data file.S3 FigThe most probable geographic origin of the sequences is indicated using a color code.(PDF)Click here for additional data file.S4 FigThe island of origin and time period (ancient vs. modern) are indicated using a color code.(PDF)Click here for additional data file.S5 FigGenBank accessions and geographic origin are indicated for each complete sequence taken from the bibliography. The origin of the samples is indicated using a color code: the indigenous people of the Canary Islands (yellow); modern Canarians (orange); North Africans (green); Europeans (light blue); sub-Saharan Africans (red); the Near East (violet); the Americas (brown) and other ancient populations (blue).(PDF)Click here for additional data file.S6 FigAll the coalescence ages have been calculated in this study, except for H2a, H3 and T2b, whose ages have been obtained from previous results . The red dotted line correspond with the colonization time of the Canary Islands based on radiocarbon data. Haplogroups considered as autochthonous of the Canary Islands are indicated with an asterisk.(PDF)Click here for additional data file.S7 FigGenBank accessions and geographic origin are indicated for each complete sequence taken from the bibliography. Sub-haplogroups in dark grey and white fonts indicate newly defined branches. Color codes are as in (PDF)Click here for additional data file.S8 FigGenBank accessions and geographic origin are indicated for each complete sequence taken from the bibliography. Sub-haplogroups in dark grey and white fonts indicate newly defined branches. Color codes are as in (PDF)Click here for additional data file.S9 FigGenBank accessions and geographic origin are indicated for each complete sequence taken from the bibliography. Color codes are as in Figure S5.(PDF)Click here for additional data file.S10 FigGenBank accessions and geographic origin are indicated for each complete sequence taken from the bibliography. Sub-haplogroups in dark grey and white fonts indicate newly defined branches. Color codes are as in (PDF)Click here for additional data file.S11 FigGenBank accessions and geographic origin are indicated for each complete sequence taken from the bibliography. Sub-haplogroups in dark grey and white fonts indicate newly defined branches. Color codes are as in (PDF)Click here for additional data file.S12 FigGenBank accessions and geographic origin are indicated for each complete sequence taken from the bibliography. Sub-haplogroups in dark grey and white fonts indicate newly defined branches. Color codes are as in (PDF)Click here for additional data file.S13 FigGenBank accessions and geographic origin are indicated for each complete sequence taken from the bibliography. Color codes are as in Figure S5.(PDF)Click here for additional data file.S14 FigGenBank accessions and geographic origin are indicated for each complete sequence taken from the bibliography. Color codes are as in Figure S5.(PDF)Click here for additional data file.S15 FigGenBank accessions and geographic origin are indicated for each complete sequence taken from the bibliography. Color codes are as in Figure S5.(PDF)Click here for additional data file.S16 FigGenBank accessions and geographic origin are indicated for each complete sequence taken from the bibliography. Color codes are as in Figure S5.(PDF)Click here for additional data file.S17 FigGenBank accessions and geographic origin are indicated for each complete sequence taken from the bibliography. Color codes are as in Figure S5.(PDF)Click here for additional data file.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.


With globalization, more and more people travel to countries where they are at risk of injuries and travel-related diseases. To protect travelers’ health, it is crucial to understand whether travelers accurately perceive medical assistance resources before and during their trips. This study investigated the need, awareness, and previous usage of overseas emergency medical assistance services (EMAS) among people traveling abroad.n = 500) at a travel clinic in Taipei, Taiwan.Anonymous questionnaires were distributed to patients , emergent medical repatriation (87.6%), and assistance with arranging hospital admission (87.4%). Patients were less aware of the following services: arrangement of appointments with doctors (70.7%) and monitoring of medical conditions during hospitalization (73.0%). Less than 5% of respondents had a previous experience with EMAS., and the percentage of travelers who have previously received medical assistance through these services is extremely low. The discrepancy between the need and usage of EMAS emphasizes the necessity to adapt EMAS materials in pre-travel consultations to meet the needs of international travelers.EMAS are considered important to people who are traveling abroad. However, approximately 20–30% of travelers lack an awareness of EMASThe online version of this article (10.1186/s12992-018-0382-5) contains supplementary material, which is available to authorized users. The number of international travelers is increasing each year. In 2016, international tourist arrivals in all countries reached a total of 1235 million, representing a 3.9% increase over the prior year [People who travel abroad have a greater risk of morbidities or death. Travelers to developing countries have twice the risk of injuries from traffic accidents than their counterparts in their home countries . Approxi, middle- or high-income countries. Furthermore, aeromedical evacuation (AME), which can cost between $25,000- $250,000, is not affordable for most travelers without travel insurance [In an era of extremely high medical expenses for emergency medical care, severe illness or injuries may result in financial catastrophe or impoverishment for international travelers. Considering the increased risk of injuries and illness during international trips, travel insurance is critical for travelers with medical needs during travel , 9. Travnsurance . OverseaDue to the expansion and heightening of international tourism, the frequencies of travel injuries and illnesses have been increasing every year. In an Australian study, 25.3% of insured travelers reported the use of emergency assistance during travel . HoweverThis study was a cross-sectional survey using a well-structured, three-part questionnaire that was distributed to all patients at the travel clinic in National Taiwan University Hospital (NTUH) from April 2014 to May 2014. Return and completion of the questionnaire represented the subject’s consent of participation. The questionnaire was completed anonymously.The targeted participants were all adult patients over 18 years old with prior experience traveling abroad. Patients who were unwilling or unable to complete the questionnaire were excluded. The design of the study and selection of subjects were approved by the Research Ethics Committee at National Taiwan University Hospital in Taiwan (201402078RINB) before the study was conducted.The three-part questionnaire included questions on socio-demographic characteristics, attitudes, and awareness towards the EMAS commonly provided to insured travelers. The questionnaire was pretested for face validity by a panel of ten physicians with experience in the clinical practice of travel medicine, including family medicine specialists, infectious disease specialists and pediatricians, from NTUH and Centers for Disease Control (CDC), Taiwan. A literature review and consensus opinion from five physicians at NTUH and CDC, Taiwan, were also conducted to test the content validity of the questionnaire. However, few studies have investigated the perceived importance and awareness of preparedness measures during travel, such as EMAS provided by insurance companies in this study. To the best of our knowledge, other measures are not available to correlate our results with. The five physicians were asked to verify whether the factors regarding travel risk assessments in part I of the questionnaire were appropriate and whether the EMAS content listed in parts II and III of the questionnaire were services that they considered important or that travelers mostly mentioned in clinical practice. Each item was evaluated on a scale of one (low) to five (high) for clarity and relevance to clinical practice, and items with a rating of at least four were included. After the process, we added the service of “medical consultation via instant messaging” and deleted “the transportation and transfer of medical supplies” in parts II and III of the questionnaire. We also used reliability analysis to test for internal consistency. Cronbach’s alpha was 0.907 for items of importance and 0.945 for items of awareness and usage experience  24-h telephone medical advice, (2) instant messaging of medical advice, (3) arrangement of appointments at nearby hospitals, (4) arrangement of appointments with doctors, (5) medical record translation and transfer, (6) arrangement of hospital admission, (7) monitoring of medical conditions during hospitalization, (8) emergency medical repatriation, and (9) arrangement of appointments with doctors after travel. The scoring system used a five-point Likert Scale, ranging from “very unimportant” (1 point) to “unimportant” (2 points), “no comment” (3 points), “important” (4 points) and “very important” (5 points). Higher scores indicated positive attitudes regarding the need of certain services. Services with a score of 1 to 3 points were considered “not important” and those with a score of 4 to 5 points were considered “important.”Awareness and previous experience of EMAS: This part sought information on whether people had heard about the abovementioned nine different features of travel insurance services and whether they had experience using these services in the past.The included socio-demographic characteristics were sex, age, education, medical history, trip destination, special activities during travel, and prior travel-related health problems. The other two parts of the questionnaire included the following components:P value less than 0.05 was considered statistically significant. Missing data were excluded from analysis.Data management and statistical analysis were performed using SPSS17.0 statistical software . Demographic data were represented by frequency distributions. Chi-square test was used to compare the proportions of the importance and awareness of EMAS between different socio-economic variables. A A total of 615 patients who visited the travel clinic were given the questionnaire and 501 responded (effective response rate = 81.3%). The unbiased selection and high response rate represented good interval validity. After eliminating one incomplete questionnaire, 500 respondents were included in the final analysis ., and 14.2% had previously experienced travel-associated illness while abroad. Furthermore, 45.8% of the respondents planned to travel to a developing region, such as China, Southeast and South Asia, Middle and South America, or Africa, at the time of their visit. A total of 16.6% the respondents planned to participate in activities that may increase their risk of health problems, such as mountain climbing or jungle trekking, during their travel.Table Figure Table Table Among the respondents in this study, 14.2% of subjects reported previous travel-associated illness while traveling. In other studies, approximately 20–60% of travelers experienced health problems during travel , 12, whiIn our study, the services that our respondents found to be important include 24-h telephone medical consultation (91.8%), emergent medical repatriation (87.6%), and providing a referral for hospital admission to tourists abroad (87.4%). In an Australian study, emergency telephone service provided by the travel insurance company was reported in 17.1% of all general claims . The perThis study demonstrated that emergent medical repatriation was considered one of the most important EMAS by travelers. Emergent medical repatriation is an interprofessional collaborative practice, which includes the field of emergency medicine and travel medicine . TraveleOur results showed that many travelers were not aware of the service that provides medical consultation through instant messaging because this service is not routinely offered by insurance companies in Taiwan. The pre-travel consultation rate is relatively low in Taiwan at 0.1% compared to the 40.5% rate for all ill GeoSentinel travelers . AnotherPrior travel experience was an inclusion criterion in this study. Regarding the perceived importance and awareness of EMAS, we believe that compared to first-time travelers, more experienced travelers would be more aware of EMAS and would consider EMAS to be more important because of possible previous travel-associated illness experiences. However, our results demonstrated that even experienced travelers may lack an awareness of EMAS during travel. Therefore, there is a need for primary care providers and public health officials to incorporate travel medical assistance materials in pre-travel consultation and refine travel clinic practices and health policies.. The respondents in this study could be representative of the majority of the travelers visiting travel clinics in Taiwan.There are several limitations in this study. First, 81.3% of the surveyed patients returned a completed questionnaire. A crowded clinic setting might have influenced the effective response rate and might have resulted in selection bias. Second, due to a lack of collected data regarding the previous travel experience of the respondents, we did not adjust the outcomes in this study. This work was a pilot study on EMAS, and we would like to further clarify the risk level of the travelers’ previous trips in a subsequent study. Third, since most of the respondents at the travel clinic were young, had high education levels, and had fewer pre-existing diseases and travel-associated illness experiences, some of the results of this study might not apply to all travelers. Lastly, because most of the travelers at the travel clinic in NTUH came from northern Taiwan, the respondents in this study may not be representative of travelers from all areas of Taiwan. However, the travel clinic in NTUH serves approximately 30–59% of all travelers visiting travel clinics. A total of 5,914 travelers visited the travel clinic in NTUH in 2017, whereas approximately 10,000- 20,000 travelers visited travel clinics in Taiwan over a year . The resDespite the above limitations, few studies have investigated the need, awareness, and experience of EMAS while traveling abroad. The questionnaire in the study was developed for this work and has not been used before. We believe that the perceived importance, awareness and usage experiences of EMAS are good preliminary indicators for exploring whether the availability of EMAS or other medical assistance services during travel is indeed an issue. This investigation revealed that approximately 20–30% of travelers lacked an awareness of EMAS during travel despite their overall positive attitude towards the importance of these services. In a European study, most travelers sought advice from their primary care physician for pre-travel consultation and about one-third of travelers visited a travel clinic specialist . PreviouThe cross-sectional survey in a travel clinic clearly demonstrates that EMAS is necessary for international travelers. EMAS are considered important for most international travelers. However, approximately 20–30% of the travelers lacked an awareness of EMAS, and the percentage of travelers who had previously received medical assistance through these services is extremely low. Primary care physicians and travel medicine specialists are encouraged to offer more information about emergency medical assistance resources during pre-travel consultation, empowering travelers to manage their health throughout the trip.Additional file 1:Questionnaire: Assessment of Travel Risks and Needs for Travel Medical Assistance of Patients at Travel Clinics. (DOCX 21 kb)


Eisenia fetida, inserts voltage-regulated channels into artificial lipid membranes containing sphingomyelin. The voltage-induced gating leads to a strong static hysteresis in conductance, which endows lysenin with molecular memory capabilities. To explain this history-dependent behavior, we hypothesized a gating mechanism that implies the movement of a voltage domain sensor from an aqueous environment into the hydrophobic core of the membrane under the influence of an external electric field. In this work, we employed electrophysiology approaches to investigate the effects of ionic screening elicited by metal cations on the voltage-induced gating and hysteresis in conductance of lysenin channels exposed to oscillatory voltage stimuli. Our experimental data show that screening of the voltage sensor domain strongly affects the voltage regulation only during inactivation (channel closing). In contrast, channel reactivation (reopening) presents a more stable, almost invariant voltage dependency. Additionally, in the presence of anionic Adenosine 5′-triphosphate (ATP), which binds at a different site in the channel’s structure and occludes the conducting pathway, both inactivation and reactivation pathways are significantly affected. Therefore, the movement of the voltage domain sensor into a physically different environment that precludes electrostatically bound ions may be an integral part of the gating mechanism.Lysenin, a pore forming toxin (PFT) extracted from  E. fetida, induces cytolysis and hemolysis of cells that contain sphingomyelin in their plasmalemma [Lysenin, a pore forming toxin (PFT) found in the coelomic fluid of the red earthworm smalemma ,2,3,4,5.smalemma ,7 and atsmalemma ,9,10 invsmalemma ,12. Remasmalemma ,12. The smalemma . This meThe most striking feature of lysenin channels is their unique voltage regulation, which has been extensively explored in multiple studies ,7,14,15.For a population of lysenin channels, the open-close transition is described by a Boltzmann distribution within a two-state model for which the transition is relatively slow, being characterized by a relaxation time of several seconds . The sloopen) is invariant. This stable reactivation pathway leads to a static hysteresis [This unusual feature may be related to the mechanism by which lysenin channels respond to voltage stimuli. In experiments investigating the effects of temperature on lysenin gating , it has steresis , which csteresis ,20,21, wIt has been suggested that lysenin channels alter their conformation by the movement of a gate coupled to a charged voltage domain sensor when under the influence of an external electric field , which i0.5, the voltage at which the Popen equals 0.5) and the number n of elementary gating charges in various experimental conditions comprising electrostatic screening induced by counterions. Our results show that electrostatic screening has a major influence on channel inactivation, while the reactivation pathway is basically invariant. However, investigations conducted by employing ATP, which modulate the macroscopic conductance by binding to the channel lumen and partially occluding the conducting pathway [The present work was undertaken to produce evidence for the hypothesis that the voltage domain sensor moves into a physically different environment during gating. Our investigations considered electrostatic screening elicited by monovalent and multivalent metal cations acting as counterions for the voltage domain sensor. A simple two-state gating model that assumes a Boltzmann distribution of the states ,30,31,32 pathway , show a The macroscopic ionic currents through a large population of lysenin channels, which were inserted into the bilayer membrane and exposed to increasing monovalent ion concentrations, underwent visible changes with regards to their magnitude and voltage required to initiate the open-close transitions during ascending voltage ramps , and these points were not represented in the plots. KCl addition yielded a significant rightward shift of the Popen for ascending voltage ramps ) to calculate the number of elementary gating charges n , from which both water and bound counterions are excluded by the large Born energy penalty [These intricate results show that the history of channel conformation  influences its further response to applied voltages, which is the essence of molecular memory . This ma penalty ,36,37,38 penalty ,41,42,433+ may elicit only minor changes of the macroscopic conductance, such small concentrations may not sufficiently screen a gating charge which is simultaneously exposed to substantially larger concentrations of monovalent ions in the support electrolyte. However, it appears that the binding of multivalent ions is more specific and much stronger than monovalent ions [3+ ions on the voltage gating of lysenin channels while using the same experimental approaches adopted for KCl. Addition of small amounts of La3+ (no more than 2 µL of various stock LaCl3 solutions with concentration in the range 0.1 mM–1 mM for each addition) to the bulk electrolyte (1 mL of 100 mM KCl) to achieve target La3+ concentrations (up to 1.4 µM) elicited great changes in the I–V characteristics plotted for ascending voltage ramps  indicated that La3+ addition elicited negligible changes with respect to the solution conductivity or ligand-induced gating. Comparative analysis of the I–V curves  upon sub-µmolar La3+ addition, showing great screening effectiveness compared to KCl. Such a result was expected, based on the assumption that both monovalent and multivalent cations elicit ionic screening. Nonetheless, steadier values of V0.5 and n were obtained during descending voltage ramps, similar to KCl. This constitutes supplementary evidence for a gating mechanism that implies the movement of the voltage domain sensor into a more hydrophobic environment and supports the hypothesis that multivalent and monovalent cations act similarly, but with different affinities with respect to electrostatic binding to the voltage domain sensor.The experimental V0.5 a and n  was the smallest recorded from all the experiments described in this work. This may be explained by considering the consistently lower number of channels inserted in the membrane for the ATP experiments; previous studies reported that crowding in the membrane plane (achievable at large channel densities) may influence the voltage gating of lysenin channels [Differences between metal cations and ATP were also observed in the estimated Vtrations . Both V0 lysenin . Intereschannels . In the open plots may not represent true steady states. Nonetheless, in such situations, the channels that closed at lower voltages resided for longer times in the closed state, yet the reactivation pathway was invariant. Also, the Boltzmann distribution considered in this work , which is much larger than the characteristic relaxation time of lysenin channels (seconds). However, addition of cations or anions may substantially change the relaxation time, and therefore the I–V and Pproaches ) did notThe vertical bilayer membrane chamber consisted of two polytetrafluoroethylene (PTFE) reservoirs (~1 mL volume each) separated by a thin PTFE film in which a small hole (~ 70 µm diameter) was pierced by an electric spark. The reservoirs were filled with electrolyte solutions  and connected to the Axopatch 200B electrophysiology amplifier  via Ag/AgCl electrodes. The analog signal of the amplifier was further digitized and recorded with the DigiData 1440A digitizer controlled by the pClamp 10.6.2.2 software package . The bilayer was produced using a mixture of 1 mg asolectin , 0.4 mg cholesterol , and 0.5 mg sphingomyelin  in 100 μL n-decane . The bilayer integrity was verified by both capacitance and conductance measurements. Channel insertion was performed by addition of small amounts of lysenin  to the ground reservoir under continuous stirring with a low noise magnetic stirrer . Individual channel insertion was monitored by observing the discrete, step-wise changes of the ionic current through the membrane biased by − 60 mV transmembrane potentials [3 , and ATP  were prepared by dissolving the powders in 20 mM HEPES (pH 7.2). To achieve the target concentrations in the bulk, corresponding amounts of stock solutions were added to both reservoirs under continuous stirring. This procedure was repeated for all target concentrations achieved for the experiments.Stock solutions of KCl , LaClFor each ionic concentration, the voltage-induced gating of lysenin channels was investigated by recording the ionic currents in response to linear, triangle-shaped voltage ramps. The ramps were created with the pClamp 10.6.2.2 software package and delivered to the electrophysiology amplifier via one of the digitizer’s outputs. Each voltage ramp stimulus had a period of 20 min  in the range 0 mV: + 60 mV, at a sampling rate of minimum 1 s, with a 1 kHz hardware filter. Each recording was saved as a separate file and further analyzed with Origin 8.5.1 , pClamp 10.6.2.2 and Mathematica 10.4 .For analysis and modelling, we considered that the voltage-induced gating of lysenin channels is described by a two-state model :(1)Open⇔open of the channels in response to applied voltages is described by the Boltzmann distribution [n is the total number of elementary gating charges e of the voltage domain sensor (e = 1.6 × 10−19 C), V0.5 is the midway voltage of activation (the voltage at which Popen = 0.5), V is the applied voltage, R is the universal gas constant (8.34 J/K), F is Faraday’s number (F = 96485.33 C/mol), and T is the absolute temperature (T = 295 K).Within this model, the open probability Pribution ,46:(2)Pomax is the ionic current that would be recorded at the same voltage if all the channels were open. For each particular voltage, Imax was estimated from a straight line constructed by fitting the linear portion of each I–V curve recorded at low transmembrane voltages .Within the two-state model, the experimental open probability for each applied voltage was derived from ,32,47:(3open, plotted for each of the ionic conditions, and further used in the theoretical model to determine n. Since even a closed lysenin channel may present a small leakage current [open only for the two highest KCl concentrations used in our experiment, which presented visible leakages. No other correction was performed for experimental data. All graphs have been prepared using the Origin 8.5.1 software. The midway voltage of activation was experimentally determined from the P current ,17, this


Poa badensis, a rare steppe plant of calcareous rock/sand vegetation. To explore potential differences in reproductive success and genetic composition of peripheral populations, we analysed the absolute (north)westernmost occurrences in Western Germany and populations at the western margin (Eastern Austria) and the centre  of the Pannonicum, representing a part of the continuous range. Specifically, we discuss the genetic and reproductive constitution of the (north)westernmost exclave and draw conclusions on the species’ biogeographical and conservation history in this region. Therefore, we used two independent molecular marker systems  and a set of performance parameters. Overall, lowest regional genetic diversity was found in Western Germany, which is mainly a result of the specific history of two populations. However, this low genetic diversity was not accompanied by reduced reproductive success. The Eastern Austrian populations showed reduced genetic diversity and predominantly reduced performance, interpreted as a consequence of small population sizes. Central Hungarian populations showed the overall highest genetic diversity and comparatively high performance values. We observed high admixture and haplotype sharing between Austrian and Hungarian populations, indicating gene flow among these regions. In contrast, we interpreted the increased population differentiation within, and the clear distinctiveness of the German exclave as a long-term isolation of these (north)westernmost occurrences. Our results, overall, prove the good constitution of these populations and, together with their particular biogeographical history, highlight their conservation value.Many steppe species reach their (north)westernmost distribution limit in western Central Europe. This also applies to The online version of this article (10.1007/s10531-019-01722-x) contains supplementary material, which is available to authorized users. Investigating the constitution of (rare) species at their absolute distributional periphery, we can test commonly and controversially discussed biogeographical hypotheses, e.g. the ‘abundant centre’ distribution . At its (north)westernmost distribution limit the species grows at isolated sites in Central and Western Germany  and concentrated mostly at a regional scale westernmost distribution of this thermophilous (rock) steppe species  and chloroplast (cpDNA) sequence data. We also tested the reproductive success of this rare species across the same populations by estimating different performance parameters in a germination experiment. Based on these datasets, we evaluated the predicted general characteristics of peripheral populations. Specifically, we aimed at answering the following questions and finally draw conclusions on the species’ history in the German exclave: (1) Can we detect different patterns of genetic composition and reproductive success along our study transect, following predictions on centrality-marginality? Or do the patterns rather reflect a particular history of the three study regions, indicating long-term  refugia at the edge of the species’ distribution? (2) Do genetic and performance parameters vary in parallel: are populations of the (north)westernmost exclave less viable, i.e. showing decreased genetic diversity, highly  isolated populations and reduced reproductive success? Finally (3) we conclude what can be deduced from our results with respect to the species’ biogeographical and conservation history in the (north)westernmost exclave in Central Europe.In this study on Poa badensis as a diagnostic species of the order Stipo-Festucetalia pallentis  and the software FlowMax  likely connected occurrences, i.e. a distinct exclave, outside its continuous Pannonian distribution range.Focusing on the Central European distribution of P. badensis were later collected from the same populations  by taking one, randomly chosen, mature panicle per fruiting plant.To obtain an equal sampling across our study transect, we chose four populations per study region, i.e. twelve populations in total. In addition to this main dataset, we sampled material from the fifth population known in the Western German study region, the Mainzer Sand (G5). This population was not included in our main dataset due to the low number of individuals (i.e. four) found there, in our sampling year 2010). For molecular analyses, we collected leaf material from randomly chosen individuals across the respective population. Additionally, the number of flowering individuals was estimated at the same time to obtain population-size categories for each sampled population  and the number of seeds per panicle were counted. The seeds were distinguished as mature and undeveloped seeds; total seed mass was then determined for all mature seeds per individual panicle. For germination, usually ten seeds per individual mother plant were used, whereby undeveloped, shrunken seeds were excluded. The seeds were placed in petri dishes on a double-layered filter paper following a definite scheme, which allowed tracking each seed, individually. Petri dishes were placed in a climate chamber for 41 days. The settings for light and temperature mimic a day/night cycle with 12 h and 25 °C (with light) and 12 h and 15 °C (dark). The germination process was documented by differentiating four states for each seedling: ‘not germinated’, ‘radicle’ (visible), ‘cotyledon’ (visible), ‘dead’. Individuals, which successfully germinated (i.e. showing breakthrough of the radicle), were transplanted in multi-pot-plates filled with a standardised mixture of plant soil and quartz sand at a ratio of 2 to 1. After 2 months, seedling survival was determined by recording the plants survived in the multi-pot-plates.To isolate total genomic DNA from silica dried leaf material, ca. 20 mg per sample were ground with sterilized glass pellets and processed using the DNeasy™ Plant Mini Extraction Kit  according to the manufacturer’s protocol.Amplified Fragment Length Polymorphisms (AFLPs) profiles were generated for 7–23 individuals per population according to Vos et al. . These combinations have been applied successfully in previous studies  and were selected due to their potential for higher intraspecific variation. The amplification was carried out under standard PCR conditions as described in Plenk et al. . The parameter number of seeds only includes the mature seeds counted in the respective panicle. However, as we must assume that shed spikelets contained mostly mature seeds, this parameter might be underestimated. To assess the magnitude of this possible influence on our results, we calculated the proportion of shed spikelets compared to the total number of spikelets, which was only 17.6%. The number of seeds was also used to calculate a mean weight per seed (i.e. seed mass) from the total seed mass of all mature seeds within a single panicle. Fruit set was calculated as the proportion of mature seeds from the number of flowers whereas the germination rate and seedling survival rate were determined as the proportion of germinated seeds/survived seedlings related to the number of seeds applied in the germination experiment. Since our data does not meet the assumptions of parametric analyses, we performed a non-parametric Kruskal–Wallis rank sum test , fruit set (%), germination rate (%), and seedling survival rate (%). The K) by means of stochastic optimisation. Clustering of individuals was performed with a maximum of K = 13 clusters and nine replicates for each K. We performed the analyses with and without geographical coordinates of populations as an informative prior. A Bayesian admixture analysis . To reduce potential unbalances caused by the unequal sample size of our AFLP data , which are expected at each locus, for this adjusted sample size. Further, the percentage of polymorphic loci (PLP) was also calculated with this standardised sample size, whereby a locus is considered as polymorphic when Br ranged from 1.05 to 1.95 (5% level) or 1.01 to 1.99 . The mean seed mass per seed showed the highest values in the Hungarian (mean 0.3490 mg ± 0.4337) and the lowest in the Eastern Austrian region (mean 0.2899 mg ± 0.1990), demonstrating highly significant differences between regions (p < 0.001). In detail, seed mass was significantly different between the German and the Austrian, as well as the German and the Hungarian study region.In total, 1874 seeds from 223 mother plants were included in the germination experiment. On a regional scale, the number of mature seeds per panicle was highest in Central Hungary  and lowest in Eastern Austria (mean 22.63 ± 31.05), differing significantly among regions  due to significant differences between the Austrian and Hungarian and the German and Hungarian region. Detailed results of all performance parameters are summarised in Table Overall, the level of germination rate after 41 days was high: i.e. 80.8% ± 24.1). The values varied only slightly between 87.9%  and 71.1% . On regional level, germination was highest in the German exclave (mean 82.8% ± 22.4) and lowest in Central Hungary (mean 77.3% ± 26.8); albeit without statistical significance. A similar pattern was obtained for seedling survival with the regionally highest survival rate in Western Germany (mean 64.3% ± 24.5). The lowest survival rate was found in the Hungarian region (mean 47.5% ± 25.9); here, differences between regions were highly significant , indicating gradually higher survival rates towards the (north)westernmost exclave. However, none of the other performance parameters was significantly correlated with this estimate of peripherality.None of the performance parameters was significantly correlated with population size, although there was a slight trend to interdependence between fruit set, number of seeds and number of flowers. A significant positive correlation was shown for the seedling survival rate and geographical longitude  showed a heterogeneous composition (cf. Online Resource 2).For each study region about 80 individuals i.e. 241 in total) were included in the  in totalAMOVA) without regional structure, we found variation of 21.8% among and 78.2% within all populations (Online Resource 3). However, using a hierarchical approach based on the geographical origin (i.e. the three study regions), 11.0% of variation originated from the differentiation among regions, whereby 13.1% represented population differentiation within regions. The partitioning of data into two groups, representing the (north)westernmost exclave versus the Pannonian region (i.e. Austrian and Hungarian populations), revealed 12.3% of the variation among these groups and 14.1% among populations within groups. To test patterns of within-region population differentiation, we calculated two additional AMOVA analyses, i.e. one for the (north)westernmost exclave and one for the Pannonian region, resulting in 23.5% and 12.3% population differentiation, respectively (cf. Online Resource 3). The linear regression of pairwise transformed FST-values and geographical distances between populations (i.e. km) revealed a significant isolation-by-distance pattern . Within subsets we found significant isolation by distance in the Pannonian region , but not in the (north)westernmost exclave .Testing the molecular variance . German individuals were differentiated into the two other groups, one containing the populations Eckelsheim (G1) and Weilersberg  and the other one Heidesheim (G3) and Eierfels .admixture analysis based on spatial clustering of individuals  were generated for 94 individuals, resulting in an alignment of 1507 base pairs length and seven haplotypes. We found two main haplotype groups, separated by one specific mutation at bp 883 (atpI-H): one German group and one Pannonian group . Within the German group individuals from the population Eckelsheim (G1) formed one specific haplotype, whereas the second haplotype in this group contained all other German populations . In contrast, the Pannonian group is characterised by further structuring (five haplotypes), but most of the individuals  formed one major haplotype . The four individuals analysed were allocated to both haplotypes detected within the German exclave  of our studied populations with values compiled by Nybom  and highest  diversity values were found in the populations Eckelsheim  and Pilisszentkereszt , respectively. Interestingly, the population Weilersberg (G2), showed the second lowest genetic diversity , although being a large population (PSC: 4). After standardisation of AFLP band richness to the minimum populational sample size . The estimates after rarefaction were slightly higher in some of the populations . However, the highest and lowest values were again found in the populations Pilisszentkereszt (H3) and Eckelsheim (G1), respectively.Different genetic diversity estimates based on our ts Table . Compariby Nybom  for longHE, I) were highest in Central Hungary  and lowest in Western Germany . Genetic diversity of Eastern Austrian populations was only slightly higher  than in the (north)westernmost exclave. However, the distribution of genetic diversity across regions varied slightly for PLPs: here, the lowest regional diversity was found in the Eastern Austrian study region and this pattern retained also after rarefaction.On regional average, genetic diversity values  was monomorphic , reproductive success and genetic composition along our study transect. Regarding the demographic characterisation of our studied populations, Stipa capillata westernmost populations  and an increasing genetic differentiation among the (north)westernmost populations, as expected for a central-marginal gradient . The populations Heidesheim  and Eierfels  did not show reduced levels of genetic diversity; the values observed there are comparable to those of the continuous distribution in Central Hungary , a possible explanation for the high performance parameters observed in the (north)westernmost exclave might be the occurrence of one (or more) polyploidization events, likely occurring during warm  stages. This may have led to a stronger reproductive potential, probably as an evolutionary strategy to overcome the more unfavorable conditions at the absolute range limit. Moreover, polyploid populations may also show generally higher levels of genetic diversity  may have aided to retain a certain level of genetic diversity, as observed in the populations Heidesheim (G3) and Eierfels (G4). On the contrary, the populations Eckelsheim (G1) and Weilersberg (G2) are likely less viable and show low genetic diversity, which is driven by their histories. Although population size did not correlate with the genetic diversity in general, at least one of these two populations is characterised by small population size . Nevertheless, low genetic diversity of populations does not necessarily imply that they have to be less viable. Habel and Schmitt , 2018 arPoa badensis among other steppe species as a relict, surviving on sandy/rocky grassland sites in Central Europe since the last glacial. However, they also remarked that most of the populations occurring today are likely younger westernmost exclave as genetically impoverished. However, there are two populations, Eierfels (G4) and Heidesheim (G3), showing comparatively high levels of genetic diversity. As their history is very different, we will go into more detail. The population Eierfels (G4) is situated on a flat ridge of basic conglomerate in the Lower Nahe region which is protected as natural monument and harbours a unique xerothermic vegetation Korneck . The popP. badensis had been introduced to this site by taking (seed) material from the closely located population Heidesheim (G3). However, in our AFLP data we found the population Weilersberg grouping together with Eckelsheim and not with the assumed source of the seed transfer  is again located on a rocky outcrop belonging to the Waderner Schichten as part of the Rhinehessian Plateau , the population Weilersberg G2) is characterised by a different and considerably younger history. This area was originally used as a sandpit and later designated as a nature reserve. To our knowledge,  is charaeau Fig. ; 2018 miOur AFLP data revealed admixture between the populations Heidesheim (G3) and Eierfels  in situ survival, probably since early postglacial times, which we especially hypothesise for the populations Eckelsheim (G1), Eierfels (G4), and Mainzer Sand (G5). We moreover conclude, that the populations of the Waderner Schichten  may originate from the probably slightly earlier populated occurrences along the northern edge of the Rhinehessian plateau (e.g. Mainzer Sand and other previously existing ones) and spread there along the river systems of Nahe and subsidiary streams to the west and south  event. In this case, we would expect low genetic diversity and less genetic differentiation of these founder populations from a potential source in more central/eastern areas  adapted to low levels of genetic diversity Supplementary material 2 (PDF 2609 kb)Supplementary material 3 (PDF 96 kb)Supplementary material 4 (PDF 778 kb)Below is the link to the electronic supplementary material.


Nepeta × faassenii was the highest conductor (13.65 mmol H2O m–2s–1), while Panicum virgatum was the lowest conductor (2.67 mmol H2O m–2s–1) (p < 0.001). There was significant variation in percent N of leaves and soil, which did not relate to the higher water conductance in bioswales. Significantly higher C, N, and water content on the high end of bioswale slopes suggest storm water run-off is mostly absorbed on the inlet side. Bacterial and fungal communities were significantly clustered by bioswale and by plant species within each bioswale implying there are micro-environmental controls on the soil microbial composition, and that plant composition matters for microbial assemblages within bioswales. Plants with higher transpiration rates were associated with greater fungal and bacterial diversity at the level of the bioswale and at scale of the individual plant, suggesting a possible link between plant physiological traits and soil microbial communities. These data suggest that the specific plant palette selected for planting bioswales can have deterministic effects on the surrounding microbial communities which may further influence functions such as transpiration and nutrient cycling. These results may have implications for bioswale management to improve urban water quality and reduce stress on sewage systems after storm events by revising plant species palette selection based on the functional consequences of plant-microbial associations in engineered green infrastructure.Bioswales and other forms of green infrastructure can be effective means to reduce environmental stresses in urban ecosystems; however, few studies have evaluated the ecology of these systems, or the role that plant selection and microbial assembly play in their function. For the current study, we examined the relationship between plant transpiration rates for five commonly planted herbaceous species in three bioswales in New York City, as well as bioswale soil microbial composition and soil chemistry. Soils were sampled near individual plants, with distinction made between upper  and lower slopes . We found high variation in transpiration rates across species, and that  Cities with older infrastructure that combine both sanitary, industrial, and storm sewers are at risk of overwhelming the capacity of these sewer systems during wet weather events, which trigger combined sewer overflows (CSOs) into nearby waterways . These CWhile plants are the prominent biological design feature of bioswales, microbes that reside within and around the plants are a relatively unexplored aspect of bioswales that have the potential to significantly increase green infrastructure functionality . Plants In this study, we evaluated plant-associated microbial communities in bioswales in New York City, and measured relative rates of transpiration in the plants they were associated with. We chose five herbaceous target plant species that are common in NYC bioswale plant palettes and hypothesized that (1) each plant species would host unique soil microbial assemblages; (2) water conductance of the five-plant species both within and across bioswales would reveal species-specific patterns; and (3) variation in plant physiology would correlate with variation in soil microbial composition. These results present potential improvements for the design of bioswales for optimal hydrology and other desired functions such as pollutant removal.Aronia melanocarpa (Rosaceae), Eupatorium dubium (Asteraceae), Nepeta × faassenii (Lamiacae), Panicum virgatum (Poaceae), and Spiraea nipponica (Rosaceae), were selected as our focal study species, as these are commonly included in bioswale plant palettes in this region, and all form relationships with arbuscular mycorrhizal (AM) fungi. Soils from the bioswales examined in this study were derived from the same homogenous mixture installed by the same contractor, and have not been modified since planting.Three bioswales in Rego Park Queens, New York, were chosen that were all established in 2013, 3 years prior to sampling. The bioswales were chosen based on their similar planting design to keep the distribution of species controlled. Five native plant species, –2 s–1) was measured for each of the five-plant species using a steady state leaf porometer (Decagon Model SC-I). Four measurements were taken from each plant species within each of the three bioswales between 10:00 and 14:00, on September 17, 2016. A portable weather station recorded photosynthetically active radiation, wind speed, temperature and the transpiration rates.Stomatal conductance (mmol mTo solve for the Vapor Pressure Deficit (VPD), we use the following equation:To relate transpiration (E) to the VPD, we use the following equation:2O m–2s–1, VPD is the vapor pressure deficit in kPa, BP is the barometric pressure in kPa (101.3 at sea level), and gsw is the stomatal conductance to water vapor , soil samples were collected at the base of each plant for evaluation of species-specific soil microbial assemblages. Each bioswale was constructed with a slope to facilitate entry storm water to inlet, or high end of the slope, and exit of water through the low end, or outlet. Two individuals of each plant species were identified that were closest to the high and low end of each bioswale, and these were selected as our focal individuals for sampling. Soil adjacent to four individuals of each target species were sampled within each bioswale. Individual soil cores were passed through sterile 2.0 mm sieves and placed into cryovials that were flash frozen in liquid N on-site for subsequent molecular analysis. The remaining soil from each core was placed in a sterile Whirl-Pak bag  for soil physicochemical analyses . The Whirl-Pak bags and cryovials were frozen at −80°C on the same day of collection. Soil pH was analyzed with a gas electrode using a 1:2 ratio with water. Total C and N content was quantified by combustion using an Elementar Vario-Macro CNS analyzer at the Lamont Doherty Earth Observatory . To calculate volumetric water content, 5 g of wet soil was weighed and recorded, dried at 105°C for 24 h, and reweighed for each sample.2O, 12.5 μL GoTAQ 2X MM mix , 0.5 μL of both the forward and reverse Illumina-barcoded primers, and 1 μL of genomic DNA. PCR cycles were performed at 94°C for 3 min, 35 cycles of 94°C for 45 s, 50°C for 60 s, 72°C for 90 s, then 10 min at 72°C. All the PCR reactions were run in duplicate, and visualized using gel electrophoresis. Successful reactions were quantified using a spectrofluorometer, pooled, and sent for Illumina MiSeq paired-end sequencing. Reads were processed using the UPARSE pipeline software pipeline . Genomic DNA was amplified using the ITS1-F and ITS2 primers for fungi  and the pipeline  and sequg UPARSE . Taxonomp ≤ 0.05) with log transpiration rate were extracted for each plant species and analyzed for correlations with other metadata variables. P values were derived using Anova and Tukey’s Honestly Significant Difference (HSD) tests  tests . All perD) tests . FunctioD) tests . Guilds Nepeta as the highest water conductor with values averaging to 13.65 mmol H2O m–2s–1. Panicum conducted the least water with 2.67 mmol H2O m–2s–1, and intermediate similar results for Aronia, Eupatorium, and Spiraea, which averaged ∼6.41 mmol H2O m–2s–1 (p < 0.001). These patterns among species were relatively consistent, despite some variation across bioswales.Transpiration readings show p < 0.05), but not significantly by species, and there was no significant difference in soil C variation. However, the slope (high or low) of the bioswale showed significant variation in percent soil N, C, and water content (p < 0.05). The inlet end of the slope had nearly 1.6 times more C, double the N, and nearly 1.5 times more water content. Percent water content did not vary significantly across bioswales or species. The C:N ratios and soil pH were not significantly different in slope or across species.Percent N varied significantly across bioswales . There were also significant interaction effects between plant species and bioswale location, plant species and slope position, as well as bioswale location and slope position (p < 0.05). Similarly, plant species and bioswale location had a significant effect on fungal community composition (p < 0.05) and an interaction effect between plant species, bioswale location and slope position was also found (p < 0.05).Multiplicative PERMANOVA tests found significant effects of plant species, bioswale location, and slope position on bacterial community composition  with plant transpiration rate across plant species  with at least 10 sampled plants of the same species for bacteria and at least four sampled plants of the same species for fungi. Following that correction, a total of 66 bacterial OTUs and 47 fungal OTUs were significantly correlated , a widely used herbaceous plant in NYC green infrastructure, maintained the highest rates of water conductance across our bioswale study sites and may be beneficial to include and maintain in bioswale plant palettes. By contrast, Panicum (Poaceae) consistently had the lowest transpiration rates, probably due to its C4 physiology traits adapted for water conservation. While C4 plants may be beneficial for climates that experience frequent drought, they may not be the best choice for plant palettes in cities such as New York, where precipitation occurs frequently. Our study focused on the herbaceous understory plants of the bioswales, but other life forms such as trees and shrubs are often planted in these types of green infrastructure installations, and would be beneficial to examine in future studies to get integrated estimates of plant water movement from soils. One study of bioswale trees near Chicago found similar results to ours in that tree species varied widely in their transpiration and growth rates, and had dramatic differences on estimated bioswale water budgets  transpiration rates and a subset of the soil microbial taxa are independently affected by the same abiotic factors or (2) microbes residing in and around the rhizosphere of plants influence plant physiology, or (3) the plant transpiration is influencing the microbial communities in the soil in some way. While we could not distinguish between these hypotheses in the current study, there is accumulating evidence on the associations of microbes with plastic plant phenotypes and physiological processes . Some stin situ experiments.Further research is necessary to examine the particular taxa that influence plant transpiration rates in controlled and Glomus and Rhizophagus, both of which are cosmopolitan in their distributions and are widely regarded as having generalist life history strategies due to their large genomes and frequent occurrences in disturbed environments . Our reference number is PRJNA549763.OB, KM, and MP conceived the ideas and designed the methodology. OB, JH, and SB collected the data. OB, DD, AG, KS, KM, SB, and MP interpreted the results. OB, KM, DD, and KS wrote the manuscript. All authors contributed to the drafts and gave final approval for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.