Diplodus bellottii, may be used as a model to understand the population genetics of these invasions. In the last decades, this species has undergone an outstanding range expansion from its African area of origin to the Atlantic coast of the Iberian Peninsula, where now occurs abundantly. Mitochondrial and nuclear markers revealed a striking high haplotypic nucleotide and genetic diversity values, along with significant population differentiation throughout the present-day geographical range of the Senegal seabream. These results are not consistent with the central-marginal hypothesis, nor with the expectations of a leptokurtic distribution of individuals, as D. bellottii seems to be able to retain exceptional levels of diversity in marginal and recently colonised areas. We discuss possible causes for hyperdiversity and lack of geographical structure and subsequent implications for fisheries.The displacement of species from equatorial latitudes to temperate locations following the increase in sea surface temperatures is among the significant reported consequences of climate change. Shifts in the distributional ranges of species result in fish communities tropicalisation, i.e., high latitude colonisations by typically low latitude distribution species. These movements create new interactions between species and new trophic assemblages. The Senegal seabream,  The increase of sea surface temperatures (SSTs) in the last decades has promoted shifts in the distributional ranges of species  with individuals moving into areas best corresponding to their physiological optimum. Additionally, the ability of a species to colonise new habitats is influenced by oceanographic currents, the existence of adequate resource availability  and life-history patterns . These movements lead to the colonization of more poleward habitats by low latitude species, and create new interactions between species and new trophic assemblages. In commercial species, these shifts due to climate change can be magnified by fishing pressures, as reported for the North Sea cod .Tropicalisation, the displacement of species from equatorial latitudes to temperate locations, is one of the major reported consequences of climate changes10). As a general rule, organisms follow a leptokurtic distribution type, in which the majority of individuals stay at or near the original area, and only a fraction disperse to longer distances. This range extension is usually done in a stepping-stone manner, implying that each settlement has fewer individuals compared with the previous one. Theoretically, this process corresponds to multiple successive genetic founder events associated with the corresponding genetic implications of the downsize in the effective population numbers: the erosion of the genetic diversity by genetic drift induces allele loss, the “southern richness and northern purity” paradigm11. Additionally, the “central-marginal hypothesis”12 posits that populations at the centre of the distribution have higher population sizes and gene flow, and peripheral (leading edge) populations, are smaller in size, have lower genetic diversity and will be more genetically differentiated12.Poleward colonization by organisms with a typically equatorial distribution was described almost three decades ago for terrestrial organisms in association with postglacial recolonisation routes  panmictic populations without latitudinal variation in genetic diversity (e.g. Lipophrys pholis15); (2) significant population structure with similar levels of genetic diversity throughout the entire species range ; and (3) sharp decline of genetic diversity from south (west European) to northern (Scandinavian) populations . It is less common the observation of geographical range expansion of marine species without loss of genetic diversity but, for examples, see21. Contemporary and increasingly visible tropicalization will contribute to more records of fast range expansion events.Phylogeographic studies using mitochondrial and nuclear genes revealed that species with similar environmental requirements and life-history traits often present distinct genetic and demographic historical patternsDiplodus bellottii Steindachner, 1882 (Sparidae), displayed an outstanding range expansion from its endemic origin  to the Atlantic coast of the Iberian Peninsula within a decade  estuaries28, and Ria de Aveiro in the north Atlantic coast of Portugal29  coherent with a predictable loss of overall genetic diversity. We hypothesise that similarly to several other marine fish species in this area of the Northeastern Atlantic such as Halobatrachus didactylus33, Symphodus melops34 or Labrus bergylta19, the recent observed range expansion will lead to a substantial loss of diversity. Also, the expanding low-density leading edge is by definition constituted by edge patches, which are most typically affected by founder events35, where demographic and allelic composition stochasticity36 is predictably stronger. If no significant reduction in singleton haplotypes is observed, and no differences among haplotype frequencies are found, there would be no evidence for a founder event or genetic bottleneck and the species may be panmictic. Alternatively, if no significant reduction in singleton haplotypes is observed but there are differences among haplotype frequencies, the species may be genetically structured. This study is the first appraisal on the genetic variability of this species across its range, a key information to understand its astonishing northward expansion. Additionally, we used species distribution modelling with present-day conditions to evaluate suitable areas for potential colonization, and explored whether the sampling coverage allowed an effective representation of the genetic diversity of the species.In this study, we used the documented expansion of CR = 0.997 to 1.000 and hS7 = 0.960 to 1.000) and nucleotide diversities were high, ranging from πCR = 0.4% (Cadiz) to 10.4% (Mauritania) and πS7 = 0.0% (Cadiz) to 2.7% (Mauritania) with πCR = 7.9 and πS7 = 0.1% for the entire data set  and for S7 . The same parameters behaved differently in pairwise comparisons for both markers  involving four pairs of sampling sites in CR and six in S7, including locations that are not the most distant ones, such as Algarve and Cadiz.In the entire data set the three estimates of fixation The haplotype accumulation curves failed to reach the asymptote for any of the markers Fig. , indicatThe main characteristic exhibited by the two markers’ haplotype networks is their bush-like complexity, with no central or otherwise extremely abundant haplotype. The networks displayed a large number of singleton haplotypes and very few shared haplotypes among sampling sites (coloured circles), a pattern characteristic of DNA hyperdiversity Fig. . The CR p = 0.036) and not for the S7 .The neighbour-net network Fig.  showed tOne of the main features of the BIOMOD2 software is the ability to combine predictions made in single models in an ensemble. The species distribution modelling produced six models  that can be considered good to excellent while CTA, Maxent and SRE have a poor fit with TSS and ROC values under 0.7 Fig. . Among tD. bellottii without apparent anomalies  ranged from 65 to 92% considering all models individually, with the highest value for the GBM model and the lowest for SRE . Nevertheless, the diversity levels recorded in D. bellottii were similar to values observed in widely distributed pelagic species such as sardines (e.g.41) or other fish species with larger population sizes (see Table 1 in42). Interestingly, in the congeneric D. vulgaris43 and D. puntazzo13, the overall CR haplotype (0.983 and 0.998 respectively), and nucleotide diversity (1.8% and 4.1%) are also very high. Contrariwise, in another species of Diplodus, D. sargus, diversity measures were much lower (h = 0.700 and π = 0.8%)13. The differences between D. puntazzo and D. sargus genetic diversity values were attributed to a suite of fortuitous occurrences such as extinction and colonisation rather than to ecological differences between the two species13.Benthopelagic fish such as GST, G’ST, θ, and D) were very different with GST; and θ, showing few pairwise significant differences, and G’ST, and D returning all pairwise comparisons significant. It is important to refer that both θ and GST suffer from the fact that this dataset is hypervariable, therefore restricting these estimators to values close to zero and overestimating gene flow (for a review see44). On the other hand, both G’ST and D, because they measure differentiation as the fraction of a deme's population that consists of private or near-private alleles45, will always be significantly different from zero. The hypervariable mtDNA in D. bellottii leads to a lack of shared haplotypes (with rare exceptions) among locations  and very few among-location shared haplotypes, 4% and 13%, for CR and S7, respectively. This fact makes all locations virtually significantly different from one another, with statistically significant values of D ranging between 0.847–1.000 and 0.849–1.000 for CR and S7, respectively. The expanding low-density leading edge is by definition constituted by edge patches, which are typically affected by founder events35, where demographic36 and allelic composition stochasticity are predictably stronger. However, alleles present in the leading edge patches, even if absent from other marginal locations are frequently shared with sites at the centre of the distribution47, which is not observed in our results.The high diversity recorded in D. bellottii and the paucity of shared haplotypes throughout all the sampled locations, including the native southern site, place our findings in apparent conflict with “southern richness and northern purity”11 and the peripheral populations  hypotheses. Non-expected high diversity was also found in the leading edge population of other species  suggesting that marine organisms with a long planktonic stage and high numbers of colonisers can travel long distances and export their diversity to the newly colonised areas16. In these cases, bottlenecks may not occur because populations are not close to the carrying capacity of the habitats. Diplodus bellottii is a prolific species at least in its southern distribution limit48 and it may have sufficiently large numbers of individuals to colonise in significantly large waves of individuals.The extremely high level of genetic diversity found across the entire distribution of 49. The haplotype network . The mitochondrial control region is a high-mutation region organized in hypervariable regions flanking a central conserved region and is considered the most variant non-coding gene in fishes (e.g. for groupers51). This mitochondrial region is a marker of choice in phylogeographic and connectivity studies (but on the use of hyperdiverse mtDNA for population differentiation see46). The gastropod Melarhaphe neritoides revealed hypervariable mitochondrial DNA markers  as a consequence of high mutation rates49. The second hypothesis used to explain high mutation rates in tropical areas , suggests that temperature and UV tend to generally increase mutation rates in the mtDNA of vertebrates52. The third hypothesis, the metabolic rate theory, posits that smaller animals accumulate mutations quicker than large animals54. However, it has been shown in the cold-water blue crab Callinectes sapidus, that in each recruitment event, the genetic diversity could be just as high as the one found in the adult population, although with different composition55. This would be exactly the opposite of a ‘sweepstakes effect,’ in which a small proportion of the gene pool contributes to the recruitment of the population56, therefore promoting reduced genetic diversity of populations. This way, in each seasonal recruitment, a new combination of alleles may constitute the new generation, which could help to explain the high genetic diversity in this blue crab species, even in populations near the limits of the species range55. Indeed, a similar pattern was found in two fish species, Lipophrys pholis and Atherina presbyter, with contrasting life-history patterns in a nursery rocky coastal area in Portugal57. In L. pholis only two out of 171 haplotypes were shared between three sampling periods, and in A. presbyter 25 haplotypes out of 155 were shared, which reveals an increase of private alleles in the gene pool over the years, even without a significant corresponding increase in diversity indices.High genetic diversity in CR may have several explanations. The most plausible hypotheses involve: (1) the mutation rate of the fragment, (2) the evolutionary rates hypothesis, or (3) the metabolic rate theory , while other species present contrasting high values (as the congeneric D. sargus61). While the dispersal distance is often used as a function of the pelagic larval duration (PLD)63, there is ample controversy on the subject, with studies finding evidence for uncorrelated individuals’ PLDs and net distance travelled65. Although the exact PLD for our species is unknown, it is expected to be around 17 days, value reported for other Diplodus species (e.g.66); this value may however vary with latitude (a proxy of sea temperature). Finally, there are no apparent physical barriers between sampled locations, which may promote considerable admixture among individuals from different origins. However, the species distribution modelling evidenced stretches of coast along northwestern Africa that seem to be an unsuitable habitat for the species, which could theoretically foster some level of geographical isolation. Apparently, D. bellottii overreaches those soft barriers keeping its genetic diversity undisturbed.Despite the results of this study in which all sampling sites are significantly different from each other, no geographical associated structure could be inferred from the networks. Lack of geographic genetic structure can be rooted in large effective population sizes, high potential for dispersal and weak physical barriers to gene flow67. Spatial selection would favour dispersal, and since both dispersal and reproductive rate limit invasion (colonisation) speed, high reproductive rates promote faster range expansions. Because D. bellottii is under thermal stress at 30 °C68, a scenario of spatial selection in the most southern range is rather probable. The reduced tolerance to higher temperatures, coupled with the species preference for estuaries with large areas and high volumes but low depth nursery grounds, means that an increase in sea surface temperatures may result in habitat loss for juveniles, as the estuaries of southern Europe may get too warm. During the summer of 2006, D. bellottii was first detected at 40° N  showing the quick continued northward expansion of this species. Although the results from the species distribution modelling do not show a particular affinity of this species for areas north to Lisbon, the continued raising of sea surface temperature will make these areas suitable for continual range expansion of D. bellottii. The impacts of this species in the recently colonised ecosystems, mainly in what concerns interactions with other species occupying the same ecological niche, has not been fully evaluated. The spatial overlap of D. bellottii with other sparid species is not consistent, across the literature, ranging from 47%69 to 94%30. These large differences may be explained by the dynamics of the estuaries, which suffer major changes in their hydrological features across the years. A noteworthy observation is that the spatial niche overlap between D. bellottii and D. vulgaris has been shown to increase with latitude69.The concept of “spatial selection” includes the idea that more available resources coupled with less competition may lead to spatial allele sorting and increased growth at the invasion front, with the corresponding increase in reproduction70. Sometimes it is hard to realize that any particular genealogy is the single outcome of an infinite number of equally possible genealogies resulting from stochastic outcome71.The origin of the two groups Fig.  is debat31, and the existence of more valuable commercial species, has not made the Senegal seabream a main fisheries target. In Lisbon, the species is mostly a bycatch of a traditional shallow artisanal fishery31 and for official statistics, D. bellottii is merely recorded as Diplodus sp. The fact of this species may constitute a future target fishery should prompt an adequate biological survey to assist its proper management. The unexpected pattern of very high diversity in recently-colonised areas within a climate change scenario, deserves further investigation and this species can serve as a useful model for studying the impact of a changing environment on the genetic diversity of a coastal species. Surveys need to be implemented, ecological implications evaluated, and a genomic temporal approach assessed to understand the causes for a successful colonisation without the loss of genetic diversity levels.This species is an important commercial species in Mauritania, but its smaller length in the Lisbon regionD. bellotti were collected from five locations along its distributional range in the northeastern Atlantic  and a final extension at 72 °C for 7′. The same primers were used for the sequencing reaction, and the PCR products were purified and sequenced in STABVIDA .Total genomic DNA was extracted with the REDExtract-N-Amp Kit (Sigma-Aldrich) following the manufacturer's instructions. The mitochondrial control region (CR) and the first intron of the nuclear S7 ribosomal protein gene (S7) were amplified, with the same kit, in a Bio-Rad Mycycler thermal cycler, using L-pro1 and H-DL1https://www.codoncode.com/index.htm) and sequences were aligned with Clustal X 2.174. For the S7 gene forward and reverse strands were obtained and some samples were sequenced twice for error checking. All sequences were deposited in GenBank .Chromatograms were edited with Codon Code Aligner 77 and nucleotide diversity (π)76 and respective standard deviations.The standard measures of genetic diversity were computed for the five location sites separately and after pooling . We used a variety of packages developed for R v.3.6.1FST‐like statistics78, such as: (1) Nei's GST,77, (2) Hedrick's G′ST79, (3) Weir and Cockerham θ80 and (4) Jost's D81 using diveRsity R-package82. While the first three estimators measure nearness to fixation, the last quantifies the relative degree of allelic differentiation45. We report these four estimates to provide complementary information on the genetic structure among the Senegal seabream sampled locations. The diveRsity R-package was used to estimate 95% confidence intervals for all estimators using 10,000 bootstrap iterations.To assess population structure, we estimated spider R-package83 was used to obtain haplotype accumulation curves by 1000 random permutation subsampling using the functions haploAccum and plot.haploAccum supplied in the spider R-package83 and the Chao 1 estimator84, with chaoHaplo function. Haplotype accumulation curves provide a graphical way to assess the extent of haplotype sampling by representing the extent of saturation as a function of the number of individuals sampled and the number of haplotypes accumulated. Curves displaying weak asymptotic behaviour suggest further sampling is necessary to capture the genetic variation of the species, while curves with rapid saturation indicate that much of the intraspecific haplotype diversity was sampled. The Chao1 estimates the appropriate minimum sample size to account for all haplotype diversity based on the sample size and number of detected haplotypes as well as the number of singleton and doubleton sequences (those occurring once and those appearing twice) in the dataset.The D. bellottii was investigated by reconstructing haplotype networks using a statistical parsimony algorithm85, implemented in TCS v.1.2186. The raw output was visualized in the web implementation of tcsBU37. Additionally, an unrooted neighbour-net network was constructed using SplitsTree 487 to investigate the genealogical relationships among haplotypes. The network was built using HKY + I + G nucleotide substitution model for both markers, as the best selected with the modelTest function of the phangorn88 R-package according to the Akaike Information Criterion89 and compatible with the distance algorithms available in SplitsTree 4, and equal-angle split transformation90 with 1000 bootstrap replicates.Population genetic connectivity in Diplodus bellottii was obtained from occurrence records from two databases: OBIS (http:// www.iobis.org/) and GBIF (https://data.gbif.org/). We used 94 records of Diplodus bellottii compiled from the Global Biodiversity Information Facility  and 72 points lifted from the Ocean Biogeographic Information System  and added five own records. Spurious geo-reference errors and duplications were deleted from the initial data. Presence points were limited to within 200 m depth to consider the recorded depth range for this species (between 10 and 90 m depth)91. Finally, the remaining 39 presence cells were georeferenced to a 0.083° × 0.083° (= 5 arc min ~ 9.2 km) grid resolution.The georeferenced locations of the observed distribution of 92 and MARSPEC93 with a spatial resolution of 5 arcmin using the R package sdmpredictors94. All predictors were delimited up to 200 m depth, given the species known bathymetric preference95, which enhances modelling performances by limiting the extent of extrapolation.The occurrence data was linked to 43 environmental variables available from Bio‐ORACLE96 implemented in R package uSDM98. We used a stringent VIF value (< 2) to retain predictors, as recommended for ecological studies see99. The final set of variables was reduced to four  complying with two criteria: being conceptually meaningful variables100 and proportion of occurrences to predictors obeying to the general rule of thumb of 10 to 1101 01 Table .Table 3Ebiomod2 package102 (v. 3.3–7) for R32. One of the main features of the BIOMOD2 software is the ability of producing an ensemble model by combining predictions from single models nine different methods: maximum entropy (MAXENT), general linear models (GLM), general boosted models , classification tree analysis (CTA), artificial neural networks (ANN), surface range envelope (SRE), flexible discriminant analysis (FDA), multiple adaptive regression splines (MARS), random forests (RF), with default settings39. Explicit D. bellottii absence records were not available so we used two sets of pseudo-absences (background data) extracted at random in a proportion of ten pseudo-absences per presence cell103. Data (presences and pseudo-absences) were split at random into a calibration (70%) and a validation set (30%). We evaluated 140 models: 2 pseudo-absence sets × 10 iterations × 7 models. Models’ performance was evaluated using two measures: the true skill statistic (TSS104) and the area under the receiver operating characteristic (ROC) curve (AUC)105. For the ensemble modeling, only those models with a TSS and ROC scores above 0.8, and 0.9, respectively  were considered106.We produced an ensemble approach to obtain predictions for the current distribution implemented in raster107, rgdal108, dismo109, vegan110, sp111, rJava112, performed in R32 using the R2C2 computational facility (https://rcastilho.pt/R2C2/R2C2_cluster.html).All calculations and analyses were performed with R version 3.0.3, with the following R packages Supplementary Table 1.


Demyelination and axon degeneration are major events in all neurodegenerative diseases, including multiple sclerosis. Intoxication of oligodendrocytes with lysophosphatidylcholine (LPC) is often used as a selective model of focal and reversible demyelination thought to have no incidence for neurons. To characterize the cascade of cellular events involved in LPC-induced demyelination, we have combined intravital coherent antistoke Raman scattering microscopy with intravital two-photon fluorescence microscopy in multicolor transgenic reporter mice. Moreover, taking advantage of a unique technique of spinal glass window implantation, we here provide the first longitudinal description of cell dynamics in the same volume of interest over weeks after insults. We have detected several patterns of axon–myelin interactions and classified them in early and advanced events. Unexpectedly, we have found that oligodendrocyte damages are followed by axon degeneration within 2 days after LPC incubation, and this degeneration is amplified after the recruitment of the peripheral proinflammatory cells at day 4. Beyond day 7, the recovery of axon number and myelin takes 3 more weeks postlesion and involves a new wave of anti-inflammatory innate immune cells at day 14. Therefore, recurrent imaging over several weeks suggests an important role of peripheral immune cells in regulating both the axonal and oligodendroglial fates and thereby the remyelination status. Better understanding the recruitment of peripheral immune cells during demyelinating events should help to improve diagnosis and therapy. Myelin sheath, generated by oligodendrocytes (OLs) in the central nervous system (CNS), is a multilayer membrane wrapping axons and providing electrical insulation, high-speed axonal conduction, and trophic support . Demyeli2  has been reported to trigger the selective death of spinal OLs but not the death of axons . Seven d+ myeloid cell contribution to axons and myelin reorganization. We report for the first time that initial intoxication of OLs is itself responsible for the early recruitment of proinflammatory immune cell coincident with the onset of Wallerian degeneration. Soon after, it, however, converts into a prohealing inflammation that is coincident with remyelination and prior to the delayed increase of fully remyelinated axons.To address this question, we have thus established a unique murine model of focal LPC incubation that does not sever axons mechanically. Using a combination of intravital two-photon microscopy and coherent antistoke Raman scattering (CARS) microscopy, we have then simultaneously obtained access in real-time to the label-free detection of myelin , as wellAll experimental and surgical protocols were performed following the guidelines established by the French Ministry of Agriculture . The architecture and functioning rules of our animal house, as well as our experimental procedures, have been approved by the “Direction Départementale des Services Vétérinaires” and the ethic committee .Seven- to Ten-week-old fluorescent reporter mice were used for spinal cord glass window implantation  and LPC The window was applied as described in –1 with a bandwidth 150 cm–1, dominated by the CH2 and CH3 stretching modes. Whereas nonlinear excitations of CFP fluorescence and EYFP fluorescence were optimal at λp = 806 and λs = 1,050 nm, respectively, wavelengths mixing between λp and λs provided the excitation of EGFP  with excitation wavelengths λp = 806 and λs = 1,050 nm, temporally synchronized and spatially overlapped on the sample plane. The corresponding excited resonances correspond to lipid vibrations approximately 2,850 cm of EGFP . The CAR+ cells were sorted using FACS (fluorescence-activated cell sorting) into lysis buffer (10:1 mix of Resuspension Buffer and Lysis Enhancer from Cells Direct one-step quantitative reverse transcription–polymerase chain reaction (RT-PCR) kit; Thermo Fisher Scientific, Waltham, MA, United States). Reverse transcription reactions were performed using 5 μg total RNA. Polymerase chain reaction reactions were performed in 20 μL of Superscript II reaction buffer  containing 0.01 M dithiothreitol, 7.5 ng/μL of dN6, 20 U of RNase inhibitor (Invitrogen), 10 mM dNTP and 200 U of Superscript II reverse transcriptase (Invitrogen) for 1 h at 42°C. Real-time PCR reactions on cDNA were performed using the LightCycler 480 system  using the SYBR Green I Master Kit  with 2 μL of cDNA and 200 nM of each PCR primer. Each reaction was performed in triplicate.Spinal cords were extracted 4, 7, or 14 days after LPC incubation. Spinal cords were dissected manually. Cells were dissociated using the MACs Adult Brain Dissociation Kit, mouse and rat . GFPMice were transcardially perfused with 4% paraformaldehyde. The spinal cords were removed, postfixed overnight, and cut into 100-μm coronal and sagittal sections using a vibratome . Immunofluorescent labeling was performed on sections fixed with paraformaldehyde 4%. The following antibody was used: anti-MBP . The sections and cells were incubated with appropriate Alexa-conjugated secondary antibodies and then counterstained with Hoechst 33258 .U tests for independent two-group comparison.Images were handled using ZEN 2.1  and ImageJ software . Axons and cells were counted manually. All the presented values are means ± SEM unless otherwise stated. Data were statistically processed with nonparametric Mann–Whitney n = 3 in transversal injection, vs. 57% ± 4.0%, n = 3 in vertical injection; n = 3), and axonal networks remained stable after 2 days    and reacn = 4) , we concThe average CARS signal in a region of interest reported the lipid density irrespective of their distribution into structured myelin sheath or into degenerative myelosomes . The lonConversely, axons regenerated linearly from D4 to D14, although CARS signal remained stuck at its minimal value in the first week and then promptly recovered during the second week . On D14,To clarify the sequence of cellular events responsible for the brutal loss of axons despite a progressive decline of CARS signal, we next looked for subtle changes of myelinated axon morphologies as readouts of the effect of LPC on OLs and their axonal counterpart. Eight typical morphological patterns of degeneration were established from the pool of images acquired from five animals . (1) BotTime-lapse images over hours conclusively demonstrated the possible evolution from one type of pattern to the next , suggestMost axons were in advanced degenerated states  by D2 F, a time + cells that peaked on D14 and completely resorbed by D28, time of full axonal recovery , CD86, CD32 CD16, NOS2, and interleukin 6 (IL-6)] and five anti-inflammatory genes , as well the expression of Ly6G as a marker of granulocytes and neutrophils. Proinflammatory genes  model of demyelination . We haveMyelosomes detection by CARS microscopy  contribuIndividual axon imaging confirmed that axonal regeneration started between D4 and D7, whereas, at D7, myelin coverage remaining at the minimal lipid density was present in the environment. Indeed, the ratio of myelinated axons was transiently low at D7, and it increases at D14. Our results therefore confirmed that the regenerative processes were differently and independently regulated by the postlesion physicochemical environment in the case of neurons and OLs. Different chemokines  and cyto+ neutrophils triggered the fast degeneration of axons (+ cell infiltration could be beneficial for axonal regeneration. Noteworthy, this second wave was coincident with the fast recovery of CARS signal, as expected from a beneficial impact on remyelination, and despite the only weak impact observed on demyelination for the first wave. As the clearance of myelin debris is a prerequisite for remyelination (Our results outlined two waves of innate immune cell infiltrations with different phenotypes, the first between D2 and D4 and the second between D7 and D14. The negative correlation between axons and innate immune cell densities observed on D4, as well as the significant secondary decrease of axon density between D2 and D4, supported the idea that the first wave of infiltration could be harmful to the axons. Similar conclusion was indeed made in an EAE model of inflammation where the initial infiltration of EGFPof axons . On the lination , we prop+ cells of the first wave expressed Nos2 and IL6 significantly more than EGFP+ cells at D14, and they were significantly more present medially next to the dorsal spinal vein despite a lateral location of the lesion. However, cells of the second wave expressed significantly more anti-inflammatory genes such as Tgf-beta, Igf1, Arg1 cd206, and IL10, and they were preferentially found on the DRG side and progressively invaded the lesion. A DRG resident pool of macrophages (Two characteristic distribution patterns were evidenced for these two waves supporting the existence of at least two different subpopulations. Indeed, EGFProphages  was likerophages .In conclusion, this study shows that LPC exposition can itself cause axon degeneration first as a consequence of oligodendroglial suffering and second due to innate immune cell infiltration. It emphasizes the importance of multimodal nonlinear optical microscopies to characterize the subcellular substrate of myelin plasticity. By pointing out the crucial role of dynamic inflammatory processes, it highlights the requirement of intravital studies to unravel multicomponents physiological response involved in neurodegenerative diseases. On the same animal over time, it finally illustrates the ambivalence of innate immune responses, thereby paving the way to immunomodulatory therapies as a strategy to improve assistance to neurodegenerative patients.All datasets generated for this study are included in the article/The animal study was reviewed and approved by the Comité d’éthique à l’expérimentation animale no 71.FD and BE designed the animal model, the imaging experiments, interpreted the results, and wrote the manuscript with inputs of all the authors. SB aligned and maintained the multimodal microscope. All authors contributed to image acquisition. BE, EB, and CK did the experiments, analyzed the images, and quantified all the data.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.


Curcuma longa of Indian Himalayas collected from different altitudes. For the same, turmeric rhizome extracts have been prepared from the aforesaid medicament and its anticancer activity and antimicrobial potential have been evaluated. Formation of Ag and Au nanoparticles was realized via UV–Vis spectroscopy and transmission electron microscope (TEM) confirmed size of the NPs. Antibacterial activity has been checked against Bacillus subtilis and Escherichia coli. The anticancer prospective has been observed against A549 and PC3 cell lines of both Au and Ag NPs and the cytotoxicity on PC3 and A549 cell lines was assessed using MTT assay. Results revealed higher amount of biochemicals, antibacterial and anticancer activity in Ag and Au NPs synthesized from rhizome extract collected from highest altitude. For the first time impact of altitudinal variations on phytochemicals and nanoparticles has been reported which have significant effect on its antimicrobial and anticancerous activity.Conservative remedies have a gray history worldwide and these provide productive and pertinent tools to tackle ailments. Also, the high altitude areas of Indian Himalayas with their wealthy biodiversity anchorage around 2000 plant species. Ensuing study demonstrates the synthesis of Silver (Ag) and gold (Au) nanoparticles (NPs) and utilizes one of the medicaments  In different ecological niches, plants behave differently in terms of biochemical aspects in order to better adapt to their environment. This broad range of environmental factors across altitude thus affects both, the chemical-composition and ultimately the survival of medicinal plants in such regions. Ecological conditions affect secondary metabolites or compounds that plants produce, which are habitually basis for their medicinal activity2. Plant adaptation at higher altitude is regulated by avoiding and overcoming stress conditions by means of various physiological and biochemical mechanism. The aforesaid mechanisms include production of secondary metabolites, oxidative stress and evolution of resistance-conferring genotype that produce ecologically adapted phenotypes. Altitudinal variation consequences on phytochemicals, secondary metabolites profiles and bioactivities of higher plants has not been well explored. Even though plant products in different forms such as pastes and powder, etc. from various species of Indian Himalayas are still frequently being used by rural habitants for treating various infirmities. However, scientific validation for probable use of these medicaments for treatments of various diseases is exceedingly significant and need more exploration.The altitudinal variations are one of the interesting aspects and may prove supportive in understanding the medicinal impacts of various plant species. Biosynthesis of the secondary metabolites in medicinal plants is not only genetically controlled, but is affected by biotic and abiotic stresses as well2. Green synthesis of nanoparticles assign improvement over chemical and physical methods as it is easy, cost effective, eco-friendly and there is no need to use high energy, pressure, temperature and harmful chemicals3. The plant mediated synthesis of nanoparticles found to be suitable among different green synthesis methods as it favors to the formation of stable nanoparticles in less time4. Medicinal plants contain various phytochemicals which possess high therapeutic values provide better platform for the synthesis of nanoparticles as they are nontoxic and also provide natural capping agent5. Among the inorganic nanoparticles, gold and silver nanoparticles provide superior material properties with functional versatility. Silver nanoparticles, in particular, have attracted increasing interest because of the unique properties , which can be explored in antimicrobial applications, biosensor materials, catalytic applications, cosmetic products, electronic components and non-linear optics7. Gold and silver nanoparticles (Au and Ag NPs) are commonly used in the fields of electronics, optics, and medicine8. Plant extracts are environmentally and economically friendly materials and have been explored in the synthesis of nanoparticles9. Turmeric (Curcuma longa L.) is widely used as a spice and in cosmetic products. Turmeric is now a popular medicinal plant worldwide and curcumin is the main component of turmeric which functions as a medicine with different properties like anti-oxidant, anti-bacterial, antifungal, anti-parasitic, anti-inflammatory, anti-mutagenic and anti-carcinogenic10.The chemical synthesis of nanoparticles is a preferred method, which encompasses the use of toxic chemicals as reducing, capping and stabilizing agents that may lead to the formation of harmful and unsafe non environmental friendly byproducts. The need for eco-friendly and non-toxic techniques for the synthesis of nanoparticles lead to an increasing interest in the biological protocols which require no use of harmful chemicals and lack in the formation of toxic byproductsCurcuma longa L.) were used for synthesis of Ag and Au NPs. This plant contains flavonoids, proteins, phenolic compound (such as curcumin) saponins and several mineral elements among others which could reduce the metal ions to their nanoparticles12.In the ensuing work, aqueous rhizome extracts of the turmeric plant  and K. pneumonia23. Antimicrobial activity of the Ag NPs has been found more as compared to the methanolic extract of turmeric and antibiotic24. Antibacterial activity of C. longa rhizome extract and synthesized Au NPs have been studied against two Gram positive bacteria  and two Gram negative bacteria 27.Antibacterial activity of biosynthesized Ag and Au NPs from rhizome extracts of different altitudes have been studied against two pathogenic bacteria using disc diffusion method and zone of inhibition as depicted in Figs. , 5. Discl Tables . The Au C. longa from different altitudes revealed anticancer activity against A549 (lung adenocarcinoma)  against A549 cells at concentration of 100 µg/ml. Maximum cytotoxic activity of Au NPs against PC3 cells was observed in the sample prepared from Shimla hills (36%) followed by the sample prepared from Mandi hills (35%) and 34% for hills Bilaspur  and gold (III) chloride hydrate (HAuCl4·xH2O) were obtained from Loba Chemie. Rhizomes of turmeric were collected from three hills of Himachal Pradesh. Identification was confirmed by a plant taxonomist at Botanical Survey of India, Dehradun. Rhizomes were washed to remove sand and debris and dried at room temperature under air for 1 week and powdered. An aqueous extract was prepared by adding 2.5 g of rhizome powder in 100 ml of distilled water and then kept inside orbital shaker for 48 h at room temperature. The filtered solution was cooled, dried and used for synthesis of NPs. The extract solution used for all the syntheses was stable but when in combination with the metal salts, changes in color were noticed9.Silver nitrate . There was a gradual color change during the course of the reaction. A similar procedure was applied for synthesis of Ag NPs by use of AgNO3 salt. In the case, about 5 mg of rhizome extract of C. longa was mixed with 30 ml, 0.06 M Na3 Cit solution16. After that freshly prepared 5 ml, 0.02 M AgNO3 solution was added drop wise at temperature 40–60 °C for 30–40 min on magnetic stirrer. There were periodic changes in color due to formation of nanoparticles as shown in Fig. For biosynthesis of Au NPs, about 5 mg of rhizome extract of 16.The appearance and stability of Ag and Au NPs were monitored and characterized by use of absorbance peaks (UV–Vis spectrophotometer)8. TEM was performed by use of Philips CM10 transmission electron microscope operating at 120 kV. The system was fitted an intensified video camera to assist the alignment and a slow scan CCD (charge-coupled device) camera. Final images were recorded on CCD. Fourier transform infrared (FTIR) spectra of NPs were determined by use of an ATR Cary 630 infrared spectrometer operated at a frequency range of 4,000–650 cm−1 at a resolution of 16 cm−1. Prior to the Fourier transform infrared spectroscopy (FTIR) analysis, the prepared NPs solution was centrifuged and dried.The particle size and morphological aspects of synthesized NPs were illustrated via transmission electron microscopy (TEM)13. The test organisms used were Bacillus subtilis (Gram positive) and Escherichia coli (Gram negative). Nutrient Agar was used as the media for the culturing of bacterial strains. Various concentrations of nanoparticles synthesized from rhizomes of C. longa from different altitudes have been tested against bacterial culture. Strains were swabbed on the surface of the Sabouraud agar plates and discs were prepared from Whatman No. 1 filter paper. For the sake of comparison, the anti-bacterial activities, blank disc impregnated with DMSO was used. Diverse concentrations Ag and Au nanoparticles  were added on the disc and the plates were incubated at 37 °C for 24 h. The antimicrobial potency of the test samples were measured by determining the diameter of the zones of inhibition in millimeter.Biosynthesized Ag and Au nanoparticles were studied for antibacterial activity against pathogenic bacteria using disc diffusion methodC. longa rhizomes from different altitudes at 100 µg/ml concentration14.Anticancer activity of Ag and Au NPs was tested against two cancer cell lines i.e. A549 (lung adenocarcinoma) and PC3 (prostate cancer). Cell lines were treated with different Ag and Au NPs synthesized from 30 whereby they studied phytochemicals in the rhizomes of C. longa and reported the presence of saponins, steroid, tannin, anthocyanin, coumarin, emodins, protein, amino acid, flavonoids, diterpenes, phytosterol, phenol, phlobatannin, leucoanthocyanin, anthroquinone, chalcones, glycosides and carbohydrates with different solvents . The presence of various phytochemicals  in different solvents  from C. longa rhizome extract37. Results from the quantitative analysis of aqueous extract of C. longa rhizomes reported maximum amount of carbohydrates, proteins, in the rhizomes collected from district Bilaspur and tannins, phenols and saponins flavonoids, terpenoids and alkaloids were found highest in the sample collected from district Shimla .In this study qualitative analysis of phytochemicals revealed the presence of different phytochemicals including carbohydrates, proteins, flavonoids, alkaloids, tannins and saponins. Our results are in agreement with the previous work doneC. longa from hills of Shimla, Mandi and Bilaspur showed maximum peak of absorption at 421 nm, 419 nm and 422 nm, respectively whereby for gold nanoparticles maximum peak of absorption were observed at 520 nm for Shimla, 519 nm for Mandi and for Bilaspur at 518 nm. Transmission electron microscopy (TEM) showed the formation spherical silver and gold nanoparticles in the size range of 2–60 nm. Smallest size nanoparticles were formed from the rhizome extract of C. longa collected from higher altitudes, where maximum amount of various phytochemicals  were observed, which showed important role in controlling the shape and size of the nanoparticles. It was studied that functional groups such as phenolic and alkaloids are responsible for capping, stabilizing and reduction of nanoparticles35. The reduction mechanism also capable to control the size and stability of the nanoparticles produced from plant extracts34. Polysaccharides have many functionalities including hydroxyl groups and a hemiacetal reducing end that are capable of reducing precursor salt. The oxidation of polysaccharides hydroxyl groups to carbonyl groups plays important role in the reduction of gold salts38.UV–Vis spectrophotometric analysis of silver nanoparticles synthesized from rhizome extract of C. longa from different altitudes showed maximum antimicrobial activity against B. subtilis and E. coli from district Shimla at concentration 300 μg/ml and minimum activity was observed at concentration 150 μg/ml from district Bilaspur. The gold nanoparticles of C. longa from district Shimla showed maximum antimicrobial activity at concentration 300 μg/ml against B. subtilis and E. coli minimum from district Bilaspur at concentration 150 μg/ml. This could be due to smaller size of nanoparticles and presence of variable amount of phytochemicals in the rhizome extract of C. longa collected from Shimla hills. Size and shape dependent antibacterial activity of silver nanoparticles against two Gram-negative bacteria Pseudomonas aeruginosa and Escherichia coli was checked by Amin et al.20. Antibacterial activity of the smallest-sized spherical silver nanoparticles was observed maximum against both bacterial strains as compared to the triangular and larger spherical shaped silver nanoparticles.The silver and gold nanoparticles synthesized from rhizome extract of C. longa collected from district Shimla. This can be due the presence of more amount of phenols, flavonoids and tannins etc. in the rhizome extract and smaller size of the nanoparticles. Similarly synthesized bio functionalized gold and silver nanoparticles using different plant extracts of guava and clove and checked anti-cancer efficacy against four different cancer cell lines human colorectal adenocarcinoma, human kidney, human chronic myelogenous, leukemia, bone marrow, and human cervix38. They concluded that flavonoids functionalized gold nanoparticles synthesized using aqueous clove buds extract are more effective than guava leaf extract towards anti-cancer activities.The maximum anticancer activity of silver and gold nanoparticles against A549 and PC3 cell lines was observed in the nanoparticles synthesized from rhizome extract of 37, although their biologically active compounds are unknown. The ensuing endeavor present an evaluation of Curcuma longa and antimicrobial and anticancer activity of Ag and Au NPs synthesized from rhizome extract of plant34 collected from varied altitudes. The nanoparticles demonstrated an intense antibacterial activity against E. coli and B. subtilis. Anticancer activity of the aforesaid nanoparticles has been checked against A549 and PC3 cell lines. The synthesized nanoparticles revealed variation in size with elevated altitude. Small size of nanoparticles has been reported at higher altitude (Shimla hills). Various phytochemicals including flavonoids, phenols, tannins, and terpenoids were found in maximum amount in rhizome extracts of higher altitude. The greater surface stabilization effect as a result of maximum phytochemicals presence could be a possible reason for reduction in the size of nanoparticles30. Ag and Au nanoparticles both inhibit the bacterial growth. The Ag nanoparticles synthesized from rhizome extract of hills Shimla with higher altitude showed maximum antimicrobial activity. These nanoparticles revealed percentage cell cytotoxicity against A549 and PC3 cell lines. Curcuma longa has been used in traditional medicine as a household remedy for different diseases in many parts of the world. Nanoparticles prepared from the rhizome extract of C. longa can be used to develop many medicines and may be supportive tool to cure many diseases. The use of such eco-friendly nanoparticles as an anticancer agent in medical applications, render this method potentially exciting for a large-scale synthesis of other inorganic materials . The effect of anticancer activity of rhizome extract of Curcumalonga can be enhanced via formation of the aforesaid nanoparticles by the projected method. Results revealed that Ag and Au NPs demonstrated effective anticancer activity en route for cancer cell lines. Size variation of nanoparticles varies with altitude and has publicized immense variation in cancer activity. Therefore, in conclusion we out to inference that altitude has major effect on size of nanoparticle and the same nanoparticles were more effective in cancer inhibition than the large sized NPs.Medicaments play important role in drug development as plants produce a wide range of phytochemicals. Herbal drugs are prescribed widely because of their effectiveness and no side effect in clinical experienceSupplementary file1 (DOCX 3990 kb)


Neopestalotiopsis isolates were obtained. Fungal identification of 20 representative isolates was performed using morphological characterization and phylogenetic analysis based on the internal transcribed spacer (ITS) region of ribosomal DNA, part of the translation elongation factor 1-alpha (TEF) and the β-tubulin (TUB) genes. Phylogenetic analysis indicated that the 20 isolates from this study formed four consistent groups, however, overall tree topologies do not consistently provide a stable and sufficient resolution. Therefore, even though morphological and phylogenetic separation is evident, these isolates were not assigned to any new taxa and were tentatively placed into four clades (clades A–D). Pathogenicity tests on detached mango leaves of cv. Kent showed that the 20 isolates that belong to the four Neopestalotiopsis clades from this study and induce lesions on mango leaves. This is the first report of species of Neopestalotiopsis causing mango grey leaf spot disease in Mexico.Mango is one of the most popular and nutritious fruits in the world and Mexico is the world’s largest exporter. There are many diseases that directly affect fruit yield and quality. During the period 2016–2017, leaves with grey leaf spots were collected from 28 commercial mango orchards distributed in two main production areas in Sinaloa State of Mexico, and 50  Mangifera indica L.; Anacardiaceae) is the fifth most economically important tropical fruit in the world [Mango , anthracnose (Colletotrichum spp.), mango malformation disease (Fusarium spp.), stem-end rot (Lasiodiplodia spp. and Neofusicoccum spp.) and grey leaf spot  are the most important diseases [Diseases caused by fungi are the main limiting factor for mango production worldwide . In Mexidiseases ,11,12,13diseases ,15,16. INeopestalotiopsis was segregated from Pestalotiopsis based on the conidial morphology and the phylogenetic analysis of the nuclear ribosomal RNA gene [Neopestalotiopsis, which seems to have evolved from the lineage of Pseudopestalotiopsis whose members have dark concolorous conidial median cells, while Pestalotiopsis presents the three light concolorous conidial median cells [The genus RNA gene . GeneralRNA gene . The conan cells . The moran cells .Neopestalotiopsis clavispora, N. egyptiaca, Pestalotiopsis anacardiacearum, P. mangiferae and P. uvicola [P. mangiferae as the causal agent of grey leaf spot in mango in Mexico, and the three median cells of the conidia of this species were observed as dark brown, which was not comparable with the characteristics of the genus Pestalotiopsis. However, the multi-locus sequence data proposed by Maharachchikumbura et al. [Neopestalotiopsis.Pestalotiopsis-like taxa are important plant pathogens in tropical and subtropical regions, infecting diverse crops and causing leaf spots, dry flowers, root rot, trunk diseases, fruit rot and fruit scab ,25,26,27 uvicola ,28,29,30 uvicola  reporteda et al.  were notDuring the period 2016–2017, symptoms of grey leaf spots were observed in commercial mango orchards distributed in the Sinaloa state of Mexico with high incidence and disease intensity up 8%. Therefore, the aims of this study were to (1) identify fungal taxa associated with grey leaf spot of mango in Sinaloa by a combination of morphological characterization and phylogenetic analyses and (2) determine their pathogenicity and virulence on detached mango leaves.Alternaria spp. and Colletotrichum spp. Based on the initial phenotypic characterization following the morphological traits described by Maharachchikumbura et al. [Neopestalotiopsis. Since our effort was on pestalotiopsis-like species, we excluded the remaining isolates from further analysis. A total of 50 Neopestalotiopsis isolates from symptomatic mango leaves were obtained. Initially observed symptoms were irregular brown lesions not delimited by the veins  between clades, Clade A was different from Clade B, but similar to Clade C and Clade D (p ≤ 0.05) to the rest of the clade, showing the largest conidia. The widest conidia were formed in the Clade B and Clade C with significant differences (p ≤ 0.05) when compared with the Clade A and Clade D (p ≤ 0.05) between clades, the longest apical appendages were observed in the Clade C. The Clade B presented the longest basal appendages and was significantly different  to the other clades. The most common number of apical appendages was three with no significant difference between clades   showed variation between different clades and isolates of the same clade . Colonie Clade D . Conidia Clade D . The con Clade D . In the  Clade D . The lenn clades .Neopestalotiopsis were selected as representative for phylogenetic analysis  as the outgroup taxon. The alignment contained 1500 characters  and the combined datasets resulted in a best scoring RAxML tree with a final maximum likelihood (ML) optimization value of likelihood of −6532.281881, shown in Twenty isolates of analysis . The comotiopsis . The comNeopestalotiopsis distributed in the four clades were pathogenic on detached mango leaves cv. Kent. Inoculated leaves developed irregular brown lesions . Additionally, the virulence of the 20 Neopestalotiopsis isolates did not show significant differences (p ≥ 0.05) and the growth rate was not correlated with the lesion area .All 20 tested isolates of  lesions  with abuNeopestalotiopsis and separate them from other pestalotiopsis-like genera [Neopestalotiopsis; however, using analysis of the combined ITS, TUB and TEF sequence data did not allow differentiation at the species level. The overall topology of our phylogenetic tree was not sufficiently supported, similar to that published in previous studies that maintained the Neopestalotiopsis isolates as Neopestalotiopsis spp. [Pestalotiopsis-like fungi are difficult to differentiate based on morphological characters, because they vary according to the nature of the isolation and environmental conditions . Based oe genera ,18. The sis spp. ,33,34,35sis spp. .Pestalotiopsis mangiferae, and Pestalotia anacardii in mango and Neopestalotiopsis cubana in rubber tree (Hevea brasilensis) [The pathogenicity test confirmed that all inoculated isolates were pathogenic on mango leaves cv. Kent, inducing brown and grey lesions with conidia exuding a cirrus from black acervuli. The symptoms were similar to those reported on mango grey leaf spot disease caused by pestalotiopsis-like fungi in Italy and China ,16,29,37ilensis) ,38,39. IJatropha curcas, Canthium dicoccum, Vigna unguiculata, Cocos nucifera and Vacciniea corymbosum [In this study, the incidence (% orchards infected) of mango grey leaf spot disease was high, with 100% in both municipalities of Sinaloa. The incidence at the field level (% tree infected) was not evaluated, but 70% to 100% have been reported in Italy . Similarrymbosum ,42,43,44rymbosum ,13 may hNeopestalotiopsis Clades A–D showed no significant differences in disease severity under controlled conditions (humidity and temperature), but this could be different under field conditions, as reported in mango orchards from Taiwan and mainland China infected with grey spot disease [ disease ,16. Ther disease . The gro disease ,37. Alth disease . The sym disease . TherefoNeopestalotiopsis species causing mango leaf spot in Mexico. The variation of the specific isolates in each of the clades described in this work, provides information on a threat to the mango industry, either due to its development of resistance to fungicides, as well as possible variations in virulence that it could have in the different cultivars and climatic conditions. Therefore, it is crucial to continue studying these clades in order to have knowledge of their epidemiology and their impact on mango productivity. To our knowledge, this is the first report of genus Neopestalotiopsis causing grey leaf spot of mango in Mexico.This study represents the first detailed investigation of morphology, phylogeny and pathogenicity of During the surveys carried out in 2016 and 2017, mango leaves exhibiting typical symptoms of grey spots  were col2) from the margins between the necrotic and healthy tissues were surface sterilized with 1% sodium hypochlorite for 60 s. The leaf pieces were washed with sterile distilled water and excess liquid was removed by sterile filter paper. The leaf pieces were placed in PDA plates and incubated at 25 °C for 3 days in darkness. Mycelial plugs from the edge of fungal hyphae developing from the tissues were aseptically transferred to fresh PDA and were incubated at 25 °C for 7 days in darkness. Monoconidial cultures were obtained using the method described by Zhang et al. [Symptomatic leaves were washed with neutral soap and surface sterilized with 90% ethanol for 30 s. The pieces of leaves  were taken from 3-days-old cultures and placed on PDA. The plates were incubated at 25 °C in darkness. Colony diameters of three biological replicates of each isolate were daily examined over 5 days. The growth rate (rk) was calculated with the linear growth function y = r xk + c  and was expressed in mm day−1 [n = 50) were visualized with a Nikon Eclipse Ni-U, microscope. All images were prepared using Adobe Photoshop CC 2018 and all measurements were made with NIS-element D software. Cultures of each isolate were deposited in the Culture Collection of Phytopathogenic Fungi of the Faculty of Agriculture from El Fuerte Valley (FAVF) at the Autonomous University of Sinaloa . Statistical analysis was performed with InfoStat® 2017 [p ≤ 0.05. The correlation between growth rate and lesion area was analyzed by Pearson correlation.Macroscopic and microscopic characteristics were examined for 20 representative isolates of mm day−1 . After 1at® 2017  by ANOVA−1 and stored at −20 °C. The ITS region of ribosomal DNA, part of TEF and TUB genes were amplified using the following pairs of primers: ITS4/ITS5 [2. Amplifications were carried out in a Bio-Rad CFX96 thermocycler  with the following profile: an initial denaturation step at 95 °C for 3 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for ITS, 54 °C for the TEF, and 58 °C for TUB for 30 s, extension at 72 °C for 60 s and a final extension at 72 °C for 10 min. The PCR products were visualized on 1% agarose gel electrophoresis stained with ethidium bromide. The amplified PCR products were purified using QIAquick PCR Purification Kit  and sequencing was carried out by the Macrogen .Total genomic DNA was extracted from fresh fungal mycelia, scraped from the margin of a colony grown on a PDA plate, incubated at 25 °C with a DNeasy Plant Mini Kit  following the manufacturer’s protocols. DNA concentrations were quantified using a Nano Drop One  and the samples were diluted in sterile water to 100 ng μLTS4/ITS5 , EF728F/TS4/ITS5  and BT2ATS4/ITS5 ,53. PCR http://tree.bio.ed.ac.uk/software/figtree/).DNASTAR Lasergene SeqMan Pro v.8.1.3 was used to obtain consensus sequences from sequences generated from forward and reverse primers and these were subsequently deposited in the GenBank database . MultiplNeopestalotiopsis isolate was inoculated on three leaves  and the experiment was repeated twice. Mango leaves inoculated with PDA plug without fungal mycelium were used as the control. Inoculated leaves were incubated at 25 °C under a 12/12 h light/darkness on plastic trays lined with two layers of paper towel moistened with sterile distilled water and enclosed in a plastic bag to maintain moisture. Ten DAI, the presence or absence of symptoms on the inoculated leaves served to determine the pathogenicity of the isolates, and the virulence of each isolate was evaluated by determining disease severity. Inoculated leaves were pressed in a botanical press for 15 min and after, digitalized in a scanner (HP Officejet Pro 6830) at 300 dpi resolution. Disease severity was evaluated using digital images and the affected area was calculated by measuring the lesion area (mm2) concerning the size of the leaf by digital image analysis, with ImageJ Software  [® 2017 [Pathogenicity test was carried out on 20 isolates selected based on an initial phylogenetic analysis which resulted in four clades. The pathogenicity of all isolates was tested on mango (cv. Kent) leaves collected from commercial fields at Ahome, Sinaloa. Detached healthy leaves were washed with neutral soap and rinsed with running tap water to remove dirt and were superficially disinfected with 70% ethanol on both sides . DisinfeMD, USA) . The sev [® 2017 .


Difficult geographic diversity and late presentation to medical attention often make the laparoscopic cholecystectomy difficult and chances of conversion and complication remains. Various preoperative grading scales have been developed for predicting the difficulty of surgery in cholecystitis patients; however, intraoperative assessment of anatomical status and inflammation of the gall bladder has not been assessed till date except for some guidelines like the Parkland grading scale (PGS). We aimed to utilise this guideline in patients undergoing laparoscopic cholecystectomy in rural community of the developing nation.  PGS was applied for all the patients undergoing laparoscopic cholecystectomy and laboratory and outcome factors like preoperative white blood cells (WBC), open conversion, subtotal cholecystectomy, length of surgery, and bile leaks postoperatively were assessed. th grade, and two as 3rd grade according to PGS system. Postoperative bile leak was seen in three patients among which two were grade five GBs and one was grade four. Preoperative WBC, conversion to open, subtotal cholecystectomy, length of surgery, and postoperative bile leak all significantly increased with increasing grades (p < 0.05).  Among 178 patients who underwent cholecystectomy, there were 40 grade one GBs, 90 grade two GBs, 26 grade three GBs, 16 grade four GBs, and six grade five GBs. With a conversion rate of 6.74%, eight patients underwent subtotal cholecystectomy. Among them, four patients were graded as 5th grade, two as 4 PGS can be applied in patients undergoing laparoscopic cholecystectomy in the rural setting of a developing nation. With its application, postoperative course could be predicted and adequate counselling can be done about the possibilities of the outcome. Laparoscopic cholecystectomy is one of the most common surgical procedures performed globally and is the treatment of choice for the management of symptomatic gall stone diseases . Owing tPGS is a five-tiered easily implementable grading system based on anatomy and inflammatory changes in the gall bladder due to persistent inflammatory processes that ensue when diseased . We aimeThis was a prospective cross-sectional study conducted over a period of six months from June 2019 to November 2019 in a tertiary hospital of western Nepal which includes all the patients undergoing laparoscopic cholecystectomy for symptomatic cholelithiasis. Ethical clearance was taken from Institutional Review Committee (Ref No: IRC-LMC 05-G/019). PGS was applied for all the patients, and outcome factors like preoperative white blood cells count, open conversion, subtotal cholecystectomy, length of surgery, and postoperative bile leaks were assessed. It was assumed that the above variables would increase as the severity of the disease increases that could be delineated by an increase in the severity grade. Four faculty surgeons were responsible for the surgery with minimum experience of performing more than 500 laparoscopic cholecystectomies while the PGS classification was done by the author himself in most of the cases wherever feasible. All the surgeries were performed electively whilst acute cases were performed early morning the next day with a maximum wait of around 12 hours as laparoscopic facilities were not available during night times. Intraoperative cholangiogram was not done in any of the cases. PGS is a five-tiered grading system developed by Parkland Memorial Hospital, Texas, USA, in 2017 which is based on a concept of having (1) a limited number of grades, (2) being easy to remember, and 3) having consistent assignment among users applying the anatomical and inflammatory aspects of the disease process [ having cp value less than 0.05 was considered significant. The Welch's one-way ANOVA was used to evaluate the relationship of the PGS for cholecystitis with the continuous outcomes of length of hospital stay while the Tukey–Kramer test was applied for all pairwise comparison of means between the grades.The laparoscopic cholecystectomy done at our institute has been a four-port surgery with umbilical port serving for camera, epigastric port, and two lateral 5 mm ports. The Hasson open technique was used to create the umbilical port, and all the patients were assigned as per PGS by the operating surgeon himself. For easily visualized gall bladder, initial view was considered the screen image which was seen once the gall bladder was grasped and retracted cephalad. In cases with severe inflammation and nonvisualized gall bladder, the initial view was described as the view of the inflamed area. A special pro forma was designed including PGS cholecystitis severity criteria and filled out by the operating surgeon himself. The pro forma included peri- and postoperative variables like age of the patient, sex, subtotal cholecystectomy, open conversion, complications like postoperative bile leak within two months, and wound infections. SPSS version 16 was used for statistical analysis, and a A total of 178 patients were enrolled in the study among which 146 were females and 32 were males. Mean age of female was 44.41 ± 15.47 years while males were 51.13 ± 12.72 years of age. There were 40 grade 1 GBs, 90 grade 2 GBs, 26 grade 3 GBs, 16 grade 4 GBs, and 6 grade 5 GBs. Mean level of ALT was 30.06 ± 32.82 units/L, while mean AST was 29.72 ± 25.50 units/L. Mean level of hemoglobin was 12.52 ± 1.19 g/dL. Gall bladder wall thickness was highest in grade 5 PGS, while pericholecystic collection was seen in only 10 patients in ultrasonography.  Similarlth grade, and two as 3rd grade according to the PGS system. Postoperative bile leak was seen in three patients among which two were grade five GBs and one was grade four. Preoperative white cell count increased with increasing Parkland grades. Subtotal cholecystectomy, conversion to open, postoperative bile leak, and preoperative WBC all significantly increased with increasing grades.p = 0.0001, Df = 4, and F = 6.937). The Tukey–Kramer test for all pairwise comparisons (p < 0.05) revealed Grade 1 significantly different from Grade 5, Grade 2 from 5, Grade 3 from 5, Grade 4 from 5, and Grade 5 from 1, 2, 3, and 4. Histological evaluation revealed 150 GBs to have chronic cholecystitis, 15 cases of acute cholecystitis which included 8 grade 2 GBs and 7 grade 3 GBs. Eight specimens turned to have acute on chronic cholecystitis while 3 patients had gangrenous cholecystitis who fall into grade 5 according to PGS (p = 0.0001).Similarly, the Welch one-way ANOVA was used to evaluate the relation between the Parkland grading scale for cholecystitis and continuous outcome of length of surgery which was statistically significant  had established a grading scale which gives 5 graded definitions for four categories that includes clinical, imaging, operative, and pathological diagnosis of the disease which requires extensive data collection and histology reports take days to be ascertained . In contLimitations of the following evaluation study on usefulness of PGS include limited sample size and single institutional study. Only four surgeons classified the intraoperative first view image according to grades though maximum number of cases were rated by the corresponding author which we believe could have subjective variation if assessments were done by a greater number of surgeons and biasness do exist when interobserver variability has not been assessed. More such studies assessment and validation along with multicentre data could actually delineate the potential of the PGS grade and check its reliability and applicability globally.PGS can be applied in patients undergoing laparoscopic cholecystectomy in the rural setting of a developing nation. With its application, postoperative course could be predicted and adequate counselling can be done about the possibilities of the outcome.


We provide a list of recommendations to improve pollen quantification.Although the use of metabarcoding to identify taxa in DNA mixtures is widely approved, its reliability in quantifying taxon abundance is still the subject of debate. In this study we investigated the relationships between the amount of pollen grains in mock solutions and the abundance of high-throughput sequence reads and how the relationship was affected by the pollen counting methodology, the number of PCR cycles, the type of markers and plant species whose pollen grains have different characteristics. We found a significant positive relationship between the number of DNA sequences and the number of pollen grains in the mock solutions. However, better relationships were obtained with light microscopy as a pollen grain counting method compared with flow cytometry, with the chloroplastic  Due to new sequencing technologies and bioinformatics tools, metabarcoding has been increasingly used to identify taxa in environmental samples1 to monitor biodiversity4, to investigate ecosystem functioning5 and interaction networks8, in both aquatic and terrestrial ecosystems. Nevertheless, its reliability in quantitative approaches, which depend on the match between counts of high-throughput sequence reads and the amount of sampled biological material2, is still the subject of debate10. While taxon identification can reveal individual diet breadth11, species richness, and the composition of habitats2, communities12 and ecological networks4, taxon quantification provides knowledge on species evenness in those habitats, communities and diets or on the level of individual or species specialization in networks, all of which is very useful in ecological studies. Research on pollination and knowledge of the quantities of pollen transported by pollinators allow for the estimation of plant-pollinator interaction strength and hence it gives a more realistic representations of networks than those made possible using traditional approaches such as observing visits to plants by pollinators13.Environmental DNA metabarcoding is a molecular method that consists of investigating environmental DNA samples made of complex mixtures of genomes from numerous organisms16, insect loads17, insect nests18, airborne samples19, and to quantify pollen abundance across various sample types. Several studies found significant positive relationships between pollen abundance (estimated using light microscopy) or pollen DNA quantities, and the abundance or the frequencies of high-throughput sequencing reads in experimental samples21, airborne samples23, insect pollen loads27 or in brood cells of solitary bees28. Conversely, other studies found low or no significant pollen-sequence abundance relationships when using ITS2 markers applied to pollen provision in bee corbicula containing huge amounts of pollen21.Metabarcoding has been used in pollen studies to identify pollen in honeyvs plastid), cannot be corrected whereas it may be possible to reduce the impact of others. Establishing relationships between pollen quantity and DNA sequence abundance requires accurate estimates of the concentration of pollen in the samples used for DNA extraction. However, pollen concentration measurements made using light microscopy, and pollen DNA isolation are usually performed on different subsamples which possibly result in differences in pollen concentrations as a result of pipetting artefacts. In addition to the possible artefacts that occur during PCR and preparing sequencing, these may cause significant variation among replicates and artificially affect the relationship between the concentration of pollen and sequence abundance. An alternative approach which, to our knowledge, has not yet been tested, is counting pollen grains in the DNA isolation pellets. Since at this stage of experimental processing there is no longer a risk of DNA contamination in the laboratory, pollen counting could be automated and applied to the whole pollen population, rather than to a small fraction, as is the case in traditional methods using microscopy. However, post-PCR counting would preclude crushing pollen grains for DNA extraction, which, in any case, is not required for efficient DNA isolation20. Efforts are also needed to reduce the accumulation of spurious sequences, chimeras and Taq polymerase inhibitors, during PCR and sequencing processes29, which are expected to fluctuate with the genetic markers, the PCR and sequencing conditions30 and the reagents used. Many studies which investigated artificial31 or natural pollen mixtures17 used samples with huge pollen concentrations  stored in a single bee corbicula31 or several grams of pollen27. Such large quantities of pollen could release large amounts of DNA polymerase inhibitors32 and cause PCR dysfunction. It would thus be useful to design experiments with lower pollen amounts, for instance, in the range of those involved in pollination, typically from a few to several thousand grains34. All these potential biases can be exponentially amplified during PCR, increasing the variability of the data set31, reducing the strength of statistical tests and affecting the relationship between pollen concentrations and sequence abundances.Many factors associated with handling samples, technical processes, or the biological material itself can affect the accuracy of metabarcoding quantification. Some factors, related to pollen , species DNA (gene copy number) and the characteristics of DNA markers  of which the pollen has very different characteristics 39. We analysed two plant specific loci, the internal transcribed spacer region 1  of the nuclear ribosomal region, and the plastidial P6-loop of trnL (UAA) intron (≈75 bp), efficient for investigating potential degraded DNA, respectively, hereafter referred to as ITS1 and trnL. The loci were amplified using three different PCR conditions . The number of pollen grains in each subsample was estimated before DNA isolation using light microscopy. Moreover, as we demonstrated in previous work20 that the mechanical pollen disruption did not give higher DNA yield, we used flow cytometry to count uncrushed pollen grains in the extraction pellets after enzymatic DNA isolation. Our main objectives were to investigate, for pollen quantities in the range of insect pollen loads involved in plant pollination: (1) whether using flow cytometry on extracted solutions provides more accurate estimates of pollen grain abundance than light microscopy, and consequently, a stronger relationship between the number of pollen grains and sequence abundance; (2) how the relationship is affected by the number of PCR cycles, the type of markers and plant species with different pollen characteristics.We investigated relationships between the number of pollen grains and sequence counts abundance obtained by high-throughput sequencing, using two species (trnL reads (360 samples), of which 56.68% were assigned to Hippeastrum sp. (HIP) and 43.32% to Chrysanthemum sp. (CHR). The total number of ITS1 reads (360 samples) reached 302,934, most of which (78.59%) were assigned to CHR.We obtained 2,699,831 trnL  and ITS1 reads . Compared to 25 PCR cycles, the numbers of trnL sequences at 30 and at 35 PCR cycles were 23 and 235 multiplied in HIP and 65 and 954 multiplied in CHR, respectively. Compared to 25 PCR cycles, the number of ITS1 sequences at 30 and 35 PCR cycles respectively, was multiplied by 24 and 56 in HIP and by 11 and 12 in CHR, respectively. Therefore, ITS1 amplification was lower than that of trnL and increased slightly with an increase in the number of PCR cycles.The 25 PCR cycle amplification conditions produced relatively few 2; line slope close to 1) than for CHR. Flow cytometry tended to detect fewer CHR pollen grains than microscopy, especially in samples with low pollen abundance  and less variable for trnL  than for ITS1 . For trnL, the slope of the relationship ranged from 0.61 to 0.85, that is a 1 log increase in the number of pollen grains, a 0.61-to-0.85 log increase in sequence quantity  or quite similar slopes (ITS1), except HIP at PCR35 for ITS1, which had a steeper slope than CHR.For both markers and regardless of the number of PCR cycles, ANCOVA linear model (lm) revealed highly significant positive relationships between the number of sequences and the number of pollen grains Table ; Fig. 2.trnL  or quite similar pollen-sequence relationship slopes (ITS1), except HIP at PCR35 for ITS1 which had a steeper slope than CHR. Overall, there was no pollen quantity–plant species interaction, except for ITS1 at PCR35.There was also a significant species effect in each PCR cycle for ITS1 but only at PCR25 for trnL sequences tended to steadily increase with increasing PCR cycles  a species effect, mostly for ITS1; (iii) generally no pollen grain x plant species interactions, and (iv) less variability in R2 and slope for trnL than for ITS1. On the other hand, flow cytometry pollen estimates gave weaker predictions (lower R2 and line slopes) of trnL sequence quantities, thus showing that flow cytometry was generally less efficient than microscopy in counting pollen grains.The ANCOVA linear model highlighted very similar trends in the relationship as microcopy estimates Table ; Fig. 3 40 but in contrast to others31, we found a significant, and often strong positive relationship between the number of DNA sequences and the number of pollen grains in the mock solutions. However, the strength of the relationship was influenced by the pollen counting methodology, the marker, the species and the number of PCR cycles.In this study, we investigated whether it is possible to predict the abundance of DNA sequencing reads from the quantities of pollen grains and if so, how accurately, and how the prediction is affected by the methodology used to estimate pollen abundances in mock solutions, PCR conditions, plant species and the type of markers (nuclear versus plastid). In agreement with previous studies40, resulting in unavoidable variation in pollen concentrations. Moreover, since counting pollen grains under a light microscope is very time consuming and as mock solutions often have high concentrations of pollen, only small subsamples and a small proportion of microscope slides (and consequently of pollen population) are usually inspected23. Such methodological problems may then be further amplified by PCR, thus blurring the relationship between sequence and pollen abundance. In an attempt to reduce these biases, in every pollen sample based on microscopy counting, we counted pollen after DNA isolation using flow cytometry. While DNA isolation and flow cytometry were performed on the same mock solution and since more than 80% of the stock solution was counted, we expected flow cytometry to provide the best estimates of pollen grain quantity, and therefore, the best sequence-pollen grain abundance relationships. Unexpectedly, stronger relationships were found using light microscopy, showing that it provided better estimates of pollen abundance than flow cytometry. Moreover, the efficiency of flow cytometry appeared highly species-dependant. Indeed, both microscopy and flow cytometry provided similar estimates of HIP pollen grain abundance, but flow cytometry largely underestimated the abundance of the smaller CHR pollen grains, especially in the less concentrated mock solutions (50–250 grains). We observed that, despite the fact that pollen grains were not crushed before DNA extraction many CHR grains were in fact fragmented after DNA extraction. Fragments were then likely confused in spectrograms with the uncounted myriad of tiny biological and mineral particles present in the mock solutions. On the other hand, even though partially destroyed, the bigger pollen grains of HIP could still have been detected and counted. Moreover, due to the absence of lacunae and only one aperture, HIP pollen grains were possibly less sensitive to fragmentation than CHR pollen grains.Usually, in experiments which aim to evaluate the potential of metabarcoding for pollen quantification, the estimates of pollen abundance in suspension and the DNA extractions have been performed using different subsamplestrnL, very accurate prediction of DNA read abundance was obtained from pollen grain quantities estimated by light microscopy, with determination coefficients and slopes generally higher than those found in the literature with the same markers40 or with other markers31. Moreover, except at PCR25, the good relationship was conserved across species and PCR cycles and trnL sequence abundance increased steadily with increasing PCR cycles. These positive results confirmed that: (i) light microscopy counting of pollen grains after DNA extraction was a suitable method to prepare mock solutions and replicates, (ii) trnL amplification was not subject to important PCR biases, and (iii) the degree of repeatability of high-throughput sequencing was high41.We found that for 27 but with less accuracy (lower determination coefficients and slopes) and higher variability across species and PCR conditions than with trnL. Although HIP has higher pollen DNA content42 than CHR36, it produced fewer ITS1 sequences than CHR, whereas both species produced similar quantities of trnL sequences regardless of the quantity of pollen and PCR conditions. Furthermore, in contrast to what we observed for trnL, ITS1 abundance did not increase steadily with the number of PCR cycles and remained almost stable between PCR30 and PCR35, although with a modification in the relationship between the pollen and sequence abundances. These results suggest that: (i) the amplification efficiency and copies of trnL in pollen from the two species is comparable despite the fact that the number of plastids may vary across species43, (ii) HIP had fewer ITS1 copies in its nuclear genome, lower amplification or sequencing efficiency than CHR; (iii) the processes involved in these alternative causes would have been primarily influenced by DNA characteristics rather than by DNA quantities per se.The relationship between pollen–DNA sequence counts and ITS1 was also highly and positively significant, in the same range as that obtained by other authorstrnL and ITS1 sequences. Indeed, high GC contents can inhibit amplification44 due to primer mis-annealing, Taq DNA polymerase errors, gene mis-amplification and the synthesis of chimeras45. High GC-richness can also alter sequence reading during sequencing46. Analysing rDNA genes, Pinto & Raskin (2012) found that sequence counts were inversely proportional to the GC content of the samples (R = 0.78). We consistently observed that CHR had 60% GC in ITS1 and only 30% in trnL and that HIP had 10% more GC in ITS1 but only 1% more GC in trnL than CHR. Furthermore, amplification and sequencing using Illumina technologies are more effective for short DNA fragments and cause less variability in amplicon length than for longer fragments47. Together, the difference in GC-richness between markers and species and in sequence length probably explained the lower yield of ITS1 sequences compared to yields of trnL sequences in general and for HIP compared to CHR in particular, while both species had almost equal quantities of trnL sequences. Moreover, the accumulation of GC-induced inhibitors with PCR cycles45 is likely responsible for the fact that the number of ITS1 sequences did not increase beyond PCR30, a trend that was not observed in trnL.The observed differences between species, markers and PCR conditions could be related to the GC content in trnL in pollen quantification40. Therefore, our findings call for further studies to determine whether trnL can be applied to many other plant species and routinely used to take greater account of interaction strength in pollination network studies. Kraaijeveld et al., (2015) noted that trnL also had higher discriminatory power than other standard markers such as ITS, rbcL and matK. It is worth noting that, probably due to their relatively low GC content, plastid loci such as trnL40 (≈35% GC content), rbcL40 (≈42% GC content), matK (≈35% GC content) or trnT48 (≈25% GC content) may provide a better estimation of pollen number than nuclear markers (ITS1 and ITS2 ≈ 60% GC content). However, the drawbacks of ITS could to some extent be alleviated49 by using high-fidelity DNA polymerases such Phusion High-Fidelity DNA polymerase40 or Herculase II Fusion DNA polymerase (the present study), 3% (our study) or 5% DMSO50 and by applying low primer annealing temperatures45. The detrimental impact of the accumulation of polymerase inhibitors could be reduced by increasing the number of PCR steps (3 to 5 successive PCRs) and by diluting a subsample of the previously obtained amplicons in fresh reagent mixtures at each step45. However, this method would be costly and time-consuming and not practical for high input studies.Despite its high potential, we know only three other studies that (successfully) used 31 or studies whose goal was to understand patterns of floral resource use in bees analysed huge quantities of pollen40 sometimes corresponding to several pollen pellets. The resulting high DNA amounts could have led polymerase inhibitors to accumulate in solutions with increasingly negative consequences for PCR amplification and thus explain the low correlation or the lack of a correlation between sequence counts and pollen quantities reported in some studies31.Some experimental studiestrnL and ITS1 markers provided the highest relationship slope and the best sequence-to-pollen prediction (trnL) while the effects of plant species either alone (PCR25/trnL) or in interaction with pollen amounts (PCR35/ITS1) were minimised. Moreover, for the low-pollen amount samples, PCR25 had the disadvantage of producing few sequences, which could be confused with contamination and be removed from the data set by protocols designed to remove eDNA contamination (airborne pollen or non-pollen plant tissues deposited on insect bodies8). A dual-indexing strategy  has sometimes been used to identify pollen by metabarcoding40, but this approach is not recommended for pollen quantification28, especially if the single–run sequencing involves multi-locus analysis. In practice, better quantification results have often been obtained with single-indexing PCR amplification and single-locus sequencing48.With 30 PCR cycles, both 51; (3) to use a multi-locus approach including short plastid markers with low GC content, and (4) to use high-fidelity Taq DNA polymerases (such as Phusion High-Fidelity or Herculase II Fusion DNA polymerases), 3% or 5% DMSO, and a low primer annealing temperature to reduce some of ITS marker weaknesses; (5) to apply, at least for trnL and ITS1, a 30 cycle PCR; (6) to use single-locus sequence analysis rather than multi-locus sequencing; and finally, (7) to normalize the amplicon concentration before sequencing in order to reduce sequencing artefacts due to very different DNA amplicon concentrations52.To sum up, to improve pollen quantification, we recommend that: (1) unless special care has been taken to prevent pollen break out, to avoid using flow cytometry, or trying to perform flow cytometric counting before DNA extraction; (2) in the case of very high pollen abundances, as typical in bee corbicula, to either dilute samples to obtain solutions with a few thousand pollen grains or to use an alternative method such as PCR-free genome-skimming22 but others did. For instance, Bell et al. (2018) and Richardson et al. (2015) found under- or over-representation of some species in sequencing products compared to pollen populations estimated using light microscopy. In studies using plant mixtures, an effect of species identity on the sequence-pollen DNA abundance correlation has been observed, but without deleting the sequence-pollen counts correlation20.While we only investigated two plant species, additional studies are required to determine whether our findings can be generalised to many other species. Moreover, we did not consider species mixtures. Some studies did not detect any pollen-mixture effecttrnL, Pornon et al.20 found highly significant positive correlations between the number of insect visits to plant species (more than 23 species in mixtures) and the number of their sequences in sequencing products. This occurred even though visits to flowers do not usually accurately reflect pollen transport by insects53. So, we believe our findings are not an exception but that the question of pollen quantification in mixtures deserves further investigations that take all the recommendations we suggest here into account.Furthermore, using ITS1 and Hippeastrum sp.  and Chrysanthemum sp. . We were unable to identify the species to which the plants belonged, likely because they were the products of many species and cultivar crossings . The plants were chosen because they are widely sold in gardening stores, they produce large amounts of pollen  in winter when native species are not in flower and because their pollen has strikingly different characteristics. CHR has small (20 µm to 35 µm diameter), echinate pollen grains with a tectate ectoexine with large lacunae and three apertures38, and a 2 C value of 11.87 pg DNA36. HIP pollen grains are bigger (50 µm to 100 µm in diameter), with a semitectate ectoexine and one aperture35. Their DNA content ranges between 2 C = 13.35 pg and 2 C = 17.09 pg42. These differences between and within species in pollen grain structure and DNA content may potentially influence the amount of DNA extracted and the number of sequencing reads produced by each pollen grain.We investigated variation in the number of reads produced by 50, 250, 1,200, 6,000 pollen grains belonging to the ornamental cultivars, trnL are not the same, two batches of 40 pellets were counted for the two markers, containing respectively the two types and the four quantities of pollen. To prevent rapid pollen sedimentation, extraction pellets with empty pollen grains were re-suspended in 1 ml of 60% glycerol before counting using flow cytometry.Pollen stock solutions were obtained for each species by vigorously shaking fresh stamens in 10 mL sterilized tubes, kept sealed at 4 °C, then adding 3 mL of lysis buffer CF solution  to each tube and mixing thoroughly. The concentration of pollen grains in each stock suspension was estimated (pre-extraction estimations) by counting pollen grains in 10 μl (HIP) or 2 μl subsamples  under a light microscope, with seven replicates per stock suspension. Based on the known pollen concentration of the stock suspensions, we took subsamples of each species each containing 50, 250, 1,200, 6,000 pollen grains with five replicates . After DNA extraction and isolation, we performed an additional pollen grain count (post-extraction estimations) of each mock solution using flow cytometry (CyFlowSpace Sysmex). As the samples with ITS1 and et al.20. trnL and ITS1 markers were then amplified at 25, 30, 35 PCR cycles with three replicates per PCR cycle condition . For both trnL and ITS1 amplification, PCR reactions were performed in a 25 µl reaction volume containing 5 µl 5x Herculase II reaction Buffer, 25 mM each dNTP, 0.4 µM of each tagged PCR primer, 0.25 µl Herculase II fusion DNA polymerase, 2 µl DNA and distilled water. After reviewing the PCR protocol from Pornon et al.20 and optimizing the annealing temperatures at 55 °C and 50 °C for trnL and ITS1 respectively, the PCR programs were: 2 min denaturation at 95 °C; followed by 25, 30 or 35 cycles , 30 s elongation at 72 °C) and a final elongation at 72°C for 3 min. For ITS1, 3% DMSO was added in the reaction solution to increase Taq polymerase specificity50. PCRs were performed in the Thermal Cycler GeneAmp PCR System 9700 (Applied Biosystems) and each PCR product was visualized on 1% agarose in TAE 0.5X buffer and quantified on the QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems). Two libraries (one for each marker) were generated using 2 µl of each PCR product following the manufacturer’s guidelines for the Illumina TruSeq Nano kit, except that no sonication was performed. Libraries were sequenced using the MiSeq Illumina technology, 2 × 250 paired-end run for ITS1 and 2 × 75 paired-end run for P6-loop of trnL, using the NGS core facility at the Génopole Toulouse Midi-Pyrénées (www.get.genotoul.fr).Total DNA extraction of mock solutions was performed with the DNeasy Plant Mini kit (Qiagen) according to the protocol of Pornon et al.20 and completed with barcodes of all species belonging to the Chysanthemum and Hippeastrum genera obtained from the EMBL database, using the ecoPCR function of the OBITOOL package54 and following the OBITOOL pipelines (git.metabarcoding.org/obitools/ecopcr/). Sequence treatment followed a step-by-step analysis procedure of the OBITOOL protocol. Paired-end reads were assembled using the Illumina paired-end utility that aligns the two reads and returns the reconstructed sequences. Sequences of low alignment quality (<40%) were discarded. Each sequence was affiliated to its corresponding sample using the ngsfilter command and dereplicated into unique sequences using the obiuniq command. As some of these sequences may contain PCR and/or sequencing errors, as well as chimeras, they have to be discarded using the obigrep command while keeping sequences more than 20 bp long and with a count equal to or greater than 10 sequences. As a final denoising step, we kept sequences with no variants, with a count greater than 5% of their own count (command obiclean). Then, a single taxon was assigned to each sequence using the ecoTag program that compared the sequences produced to our taxonomic reference library. When assigning our two target plant species, a best match score >95% was allowed for each marker.We built our own barcoding reference library using barcodes from Pornon 55. Independent statistical analyses were performed for each combination of markers (ITS1 or trnL) and PCR conditions . We tested the fixed effects of the logarithm of the pollen quantity, plant species (HIP and CHR) and their interaction on the mean number of sequence reads averaged over the three PCR replicates. Different analyses were performed for either pre-extraction or post-extraction estimates of pollen grain quantities. When the interaction between plant species and pollen amount was not significant with respect to sequence abundance, it was removed from the model and simple effects of either plant species or pollen quantity were tested (type II sum of squares). Normality of the residual distribution was systematically checked and, if necessary, a log transformation was applied to the response variable.To compare the two methods of estimating the number of pollen grains in HIP and CHR, we fitted a linear regression model to the number of pollen grains estimated by flow cytometry using the number of pollen grains estimated by cytometry as predictive variable. To analyse the relationship between the number of pollen grains  and the number of sequence counts, we fitted an ANCOVA linear model with the lm function of the R base package