dataesr/scientific-paragraphs-categorization huggingface dataset:
Phylogeographic reconstruction Phylogeographic reconstruction was done using the georeferenced 'Asian genotype' dataset (748 sequences). PastML results on the ML rooted tree, as well as the LSD2 time tree, showed generally high probability values for the ancestral state (geographical region) assigned to each node (Fig. 6 ; Supplementary Fig. S7 ). A large group of South-East Asian sequences was found to be ancestral to the others, which spread towards Polynesia and from there gave rise to the American lineage. From French Polynesia, the virus likely spread to other islands located in the Pacific and also reached the South American continent, more particularly Brazil. Another route going from Polynesia to Haiti (Caribbean) and then to Brazil (South America) was also suggested but with a smaller expression.
Mutation tracking and selection analyses In all three datasets analysed for selection ('Afro-Asian' dataset-759 sequences, 'Asian genotype' dataset-752 sequences, and 'early Am' dataset-68 sequences), the FUBAR method detected evidence of negative/purifying selection for many codon sites, uniformly distributed across the genome (Supplementary Fig. S8 ). Positive selection was detected by MEME for a total of 50 sites (Supplementary Table  S8 ), two of which were also detected by FUBAR. In 36 of these sites, the alternative AA(s) were caused by mutations in at least two positions in the codon. Seven codon sites with signals of positive selection showed high frequencies of the alternative AA in at least one of the genetic groups defined in the previous section K101R (C_101), M2074L (NS3_572), I2445L/M/T (NS4B_176), V2449I/A/F/T (NS4B_180), Y2594H (NS5_74), S3162P (NS5_642), and D3223S/V (NS5_703) (Supplementary Fig.
2. 1 (except in one sequence) and is absent in all the remaining sequences (except in one sequence from ZB. 1. 0). The alternative AA in M2074L (NS3_572) occurs in a large proportion of the sequences of ZB. 2. 1, while in the remaining sequences only the consensus M AA occurs. The alternative AA in R3045C/S (NS5_525) is present in all ZB. 2. 1 (except in 3 sequences) and in two sequences of ZB. 2. 0 and is absent in all others (Supplementary Fig. S9 ). We also analysed the variation in the proteins that have been targets of vaccine development, E, prM, and NS1  (Pattnaik et al. 2020 ). The envelope (E) protein in this dataset presented 112 nonsynonymous mutations, but all below the 5 per cent frequency threshold. Five of these had mutually exclusive AAs between ZA ('African genotype') and ZB ('Asian genotype') (Supplementary Table  S8 ).
Discussion Herein, we analyse a curated dataset of ZIKV near-complete genomes, which is representative of the global ZIKV genetic diversity. The detailed genome-wide diversity and differentiation analyses of ZIKV carried out here with a larger number of sequences than in previous studies have allowed identifying the genetic groups and the most relevant mutations to classify and propose a dynamic nomenclature system, as well as to reconstruct the most likely dispersion routes and explore the putative evolutionary processes involved.
Genome-wide diversity and differentiation patterns The consensus sequences obtained from this dataset have very few differences from the reference genome KJ776791 proposed by  Theys et al. (2017)  (only one AA change, and 10 NT changes), which confirms this sequence as a good reference for ZIKV for future studies. Most of the AA positions were highly conserved. The sites with the highest NT and AA diversity were in gene regions C, prM, NS1, NS3, NS4B, and NS5. Previous studies have described nonsynonymous mutations that only occurred in certain populations. However, with the larger number of sequences used in this study, we have found some occurring in other previously unexplored populations. For example, the A148P AA change was previously reported to occur in human isolates from both 'African' and 'Asian' lineages but not in African mosquitoes, suggesting potential relevance in human infectivity  (Wang et al.
2016 ). However, we found it present in mosquito samples from the 'Asian genotype', potentially implying a different interpretation concerning infectivity. Few ZIKV genomes from mosquito host and from nonhuman primates are available, which prevents thorough analyses of the differentiation between viral sequences coming from different host species. The older sequences available in our dataset, from Africa and Malaysia, are from mosquito host species and no viral sequences from humans are available from that time for comparison. In our curated dataset, the recent sample from Guinea (MN025403_Guinea_2018-08) was the only one from the 'African genotype' collected in human host and it groups with the older mosquito African sequences. Also, the remaining sequences obtained from mosquito hosts are scattered across the phylogenetic tree and haplotype network and are very similar to the ones sampled in humans, which suggests small between-host viral genetic differentiation.
5 per cent for Pre-Am-ZIKV (obtained from three sequences from Southeast Asia and one from Micronesia), which is coherent with the older and ancestral nature of Pre-Am-ZIKV. The genetic diversity values that we obtained (mean entropy at NT level of 0. 021) are high when compared to those reported by  Collins et al. (2019). However, in this case, the authors calculated within-lineage entropy based on single-NT variants found within the sequencing assemblies of each sequenced strain, producing very low entropy values (<0. 004). Contrastingly, here we used consensus sequences available in GenBank and do not have information about the within-host variability, which would also certainly be interesting to look at and identify potential associations with pathogenicity  (Rossi 2018). When establishing the classification and nomenclature proposal here presented, we also took into consideration the diversification events and the most likely dispersion routes of ZIKV.
This analysis also supported the naming of the other subgroups within ZB. 2 (ZB. 2. 1 and ZB. 2. 2) as both derived from the basal ZB. 2. 0. The dispersion of the pandemic within the 'Asian genotype' (ZB) was South-Eastern Asia > Polynesia > South America > the Caribbean and Central America > Northern America. Although the method used in PastML is considered robust to phylogenetic uncertainty and sampling bias  (Ishikawa et al. 2019) , it still depends on sampling availability and other countries that are not in this dataset may have also contributed to the pandemic. The dispersion patterns indicated by these reconstructions were consistent when using the rooted tree and when using the time tree, but revealed some uncertainties at particular nodes of the tree, in particular involving sequences from Haiti and Cuba.
For example,  Faye et al. (2014)  found evidence for 13 recombination events between African strains when sequencing partial E and NS5 regions. Han et al. (2016)  reported a recombinant sequence from Brazil having two genomic regions more similar to a Suriname strain, while the remaining genome was more similar to a French Polynesia strain. Shrinet et al. (2016)  found evidence of recombination in African isolates and not in the Asian lineage. Shrivastava et al. (2018)  found evidence of one recombination event but noticed that both parents and daughter strains were very similar to each other and thus concluded that this was not a real recombination event. Simón et al. (2018)  did not find any evidence of recombination in the strains from Latin America. These discrepancies between studies are likely related to the different datasets and methods used.
Tracking mutations We have identified the mutations that were segregated into different groups. Seven sites with evidence of positive selection showed a high frequency of the alternative AA in particular lineages/genetic groups (R101K, M2074L, I2445L/M/T, V2449I/F/T, Y2594H, S3162P, D3223S/V), which may point to a role in the adaptive process. In the dataset that we analysed, the substitution S139N (prM) occurred in all members of the Am-ZIKV clade and not in the other clades, except for one sequence in the ZB1. 1 clade. This mutation has been reported as being exclusive to the Am-ZIKV clade (e. g. Pettersson et al. 2016). Another interesting mutation is the A982V (188 V in  Liu et al. 2017, referring  to the position within the NS1 gene). The V AA confers enhanced mosquito infectivity  (Liu et al.
2021) , and the epidemiological and genomic situation of ZIKV should be continuously monitored. In conclusion, we used a large dataset of near full-genome ZIKV sequences to analyse its genetic diversity, phylogenetics, and phylodynamics and understand its differentiation patterns and guide the development of a dynamic classification system. Our results are consistent and extend the results of previous studies. Based on those, we develop a classification system that avoids geographical references and is flexible to accommodate potential future lineages. It will be a helpful tool for studies that involve ZIKV genomic variation and its association with pathogenicity. Furthermore, it will serve as a starting point to study on-going ZIKV epidemics and outbreaks that lead to the emergence of new variants.
Landmark configuration and specific leaf area estimation For leaf GMM analyses, we flattened, dried and scanned mature leaves at 600 dpi resolution using a CanoScan 5600 F scanner (Canon, Tokyo, Japan). In total, we used 3391 leaves from 683 individuals (four or five leaves per individual). We used the scanned images to extract landmarks and leaf contours with IMAGEJ v. 1. 5 (Abr amoff et al. , 2005). For each leaf, we selected 13 landmarks, focusing on the primary veins, symmetric structures, and homologous parts. Three landmarks were distributed along the middle axis of leaves (LM 1-LM 3) and nine others were symmetrically distributed in other parts of the leaves (LM 4-LM 13)  (Viscosi et al. , 2009; Savriama & Klingenberg, 2011; Viscosi & Fortini, 2011; Liu et al. , 2018)  (Fig. 4a ). We organized the raw data for all leaf landmark configurations into 13 pairs of Cartesian coordinates (x, y) using IMAGEJ v.
Tree phenology We selected one sympatric site on Shangfang Mountain located in the southwest of Beijing Municipality, China (115°48 0 E, 39°39 0 N) to observe phenological characters of the two oak species. In this site, Q. dentata is growing in open conditions on the top of the hills, whereas Q. aliena is growing in the bottom and on the hillside, at higher density, resulting in straighter trees than Q. dentata. We recorded bud burst, leaf-expansion, flowering, and fruit stage periods of 15 Q. aliena and 10 Q. dentata trees, as described by  Crawley & Akhteruzzaman (1988). During the 2018 flowering period, we observed nearly daily almost all flowers on each tree and recorded the duration of pollen release. We used SIGMAPLOT v. 12. 5 (SYSTAT Software GmbH, Erkrath, Germany) to illustrate the flowering phenology of the two oak species.
Data analysis Genetic diversity, population cluster analysis and gene flow We estimated the average expected heterozygosity (H E ), observed heterozygosity (H O ) and fixation index (F) for each population across all 12 nrSSR loci with GENALEX v. 6. 5  (Peakall & Smouse, 2012). We then evaluated the significance of the differences in diversity between the allopatric and sympatric populations with two-group Mann-Whitney U tests implemented in SPSS v. 22 (SPSS Inc. , Chicago, IL, USA). We estimated genetic differentiation among populations and between species of sympatric and allopatric populations using hierarchical analysis of molecular variance (AMOVA), implemented in ARLEQUIN v. 3. 5  (Excoffier & Lischer, 2010). We then evaluated the significance of genetic differentiation using 10 000 permutations in ARLEQUIN v.
3. 5. We assigned individuals to homogenous clusters, without consideration of sampling information, by model-based Bayesian clustering analysis using STRUCTURE v. 2. 3. 4  (Pritchard et al. , 2000). We used 20 independent replicates for each value of K (1-10), where K is the (unknown) number of genetic clusters, and 100 000 Markov Chain Monte Carlo (MCMC) repetitions followed by 100 000 burn-in iterations for each run. We subsequently estimated the most likely number of clusters with Pr(X¦K) and DK implemented in the program STRUCTURE HARVESTER  (Earl & VonHoldt, 2012) , as described by  Evanno et al. (2005). We used admixture coefficient (Q) values to distinguish whether the sampled individuals were purebreds or hybrids with a threshold value of 0. 9, as suggested in other oak population studies  (Lepais et al.
, 2009; Peñaloza-Ram ırez et al. , 2010). To visualize the genetic clusters, we used DISTRUCT software (Rosenberg, 2004) and plotted the admixture proportion of each individual on a map based on the best K value. We also performed a single principal coordinate analysis (PCoA) with all genotypes together based on the genetic distance matrix with GENALEX v. 6. 5  (Peakall & Smouse, 2012)  and then plotted separately the sympatric and allopatric individuals to visualize individuals' genetic proximities. In addition, we used a new threshold-free approach based on the results of the PCoA between species in sympatry and allopatry to extract information on inter-specific divergence using the MIXSMSN package in R  (Prates et al. , 2013). This allows us to measure the degree of overlap of the two species in sympatry vs allopatry.
This was followed by a static heating with default temperatures. We assessed the mode and 95% highest posterior density after checking for data convergence. We selected the best model by comparing the marginal likelihoods from three models with different gene flow directions. To estimate asymmetrical gene flow, we used the posterior distribution estimates to compute the mean and standard deviation of mutation-scaled migration rate (M) for Q. aliena ? Q. dentata and Q. dentata ? Q. aliena in sympatry and in allopatry  (Beerli, 2006). Differences among these parameters were tested by analyses of variance (ANOVAs) followed by post hoc analyses using Tukey's HSD (honestly significant difference) test for multiple comparisons implemented in SPSS v. 22 (SPSS Inc. ).
Morphological analysis We first performed a generalized procrustes analysis (GPA)  (Rohlf & Slice, 1990)  to minimize differences between landmark configurations by translation, scaling and rotation to maximize the coincidence of leaf coordinate data using the MORPHOJ program  (Klingenberg, 2011). Next, we took the mean of the landmarks across four to five leaves of the same tree as suggested by  Klingenberg (2011). Finally, we exported the normalized matrix of 26 procrustes coordinates of mean landmarks per tree for leaf shape analyses  (Viscosi et al. , 2009). Using mean landmark values per tree rather than individual leaf landmark data does not result in significant loss of information in subsequent analyses, as shown by  Viscosi & Cardini (2011). Discriminant analysis is considered the most powerful statistical method for investigating taxonomic differences between groups  (Klingenberg, 2011).
In this study, we subjected procrustes coordinates of the 683 individuals to linear discriminant analysis (LDA), using the MASS package in R v. 4. 0. 2  (Venables & Ripley, 2002) , to determine the proportion of purebreds and hybrids in each population, based on the resulting linear discriminant (LD) scores (Albarr an-Lara et al. , 2018), and graphically visualized the results on a map. In addition, we performed a principal component analysis (PCA) and displayed the distribution frequency of principal component (PC) scores for all individuals to visualize the differences between species in leaf shape, using the VEGAN package in R  (Oksanen et al. , 2022). We also used the threshold-free approach on the results of PC1 for leaf shape between species in sympatry and allopatry using the MIXSMSN package in R, as described earlier for genetic markers  (Prates et al.
Patterns of inter-specific phenotypic divergence To characterize leaf traits' climatic responses, we first extracted 19 bioclimatic variables at 30 s (c. 1 km 2 ) resolution from WORLDCLIM v. 2 raster layers  (Fick & Hijmans, 2017). We transformed these variables using z-scores set to increase with temperature and precipitation (Table  S4 ), using a PCA in the VEGAN R package  (Oksanen et al. , 2022). We then studied SLA variation for all, sympatric and allopatric populations as a function of climatic factors (precipitation seasonality, annual precipitation, temperature seasonality and annual mean temperature). We also estimated the divergence of leaf shape by GMMs between species in sympatry and in allopatry using data acquired from all sampled individuals or only from the genetically and morphologically purebred individuals.
New Phytologist (2022) www. newphytologist. com as suggested by  Adams (2004)  and  Bates et al. (2015) , using the LME4 package in R v. 4. 0. 2. We assessed the significance of the effects using an ANOVA test implemented in the CAR R package  (Fox et al. , 2013). A significant interaction between species type and community implies species-specific trait divergence  (Adams, 2004). In addition, we used linear mixed modeling to test the hypotheses (1) that the trait's environmental response differs in sympatry and in allopatry and (2) that the divergence between the two species in sympatry and allopatry depends on environmental factors, focusing on purebred individuals (either morphologically purebred and genetically purebred). We also assessed the significance of the differences by ANOVA.
dentata to Q. aliena (63. 7 and 48. 7, respectively) (Table  1 ). The mutationscaled migration rates (M) was higher from Q. dentata to Q. aliena (Fig. S1b ) than in the reverse direction (Fig. S1a ; Table  S7 ), significantly so in sympatry (P = 0. 05, Table  S7 ). Furthermore, we inferred that the rate of historical gene flow between species has been significantly lower in sympatric than in allopatric populations (P < 0. 05, Table  S7 ; Fig. S1 ). Genetic structure The Pr(X¦K) and DK statistics obtained from Bayesian clustering strongly support the presence of two major clusters in the dataset, one corresponding to Q. aliena and the other to Q. dentata (Fig. S2 ). In addition, analysis with the threshold value Q set to 0. 9 indicated that there is a slightly lower percentage of admixed individuals in sympatric than in allopatric populations (29 and 33%, respectively, Table  S1 ).
Patterns of phenotypic divergence Leaf morphological variation As recommended by Klingenberg (  2011 ), we manually removed 28 leaf outliers, i. e. specimens deviating strongly from the average (Table  S1  column H-I). The final dataset included 3363 leaves corresponding to 99. 2% of the sampled leaves. For Q. aliena, we found a clear difference between mean landmark values in sympatry and in allopatry. For Q. dentata, the difference was not obvious (Fig. S3 ). The morphological data provided stronger discrimination between species than the genetic markers: 87% of the individuals were 'pure' according to leaf shape GMM analysis (with LD scores < À1 or > 1), compared to only 66% when genetic markers were used for assigning species (Q scores < 0. 1 or > 0. 9) (Figs 5, 6; Table  S1 ). Overall, morphological and genetic taxonomic criteria matched for 401 (65%) of the individuals (Table  S8 ).
Acknowledgements The authors thank  Yang Xu ,  Wei Su , Ping liu from  Beijing Forestry University  ( BFU ) for leaf sampling, landmarking and flowering phenology record;  Dr Sun Han  from  BFU  for discussion on SLA analysis. Prof. Weining Bai  from  Beijing Normal University  for gene flow analysis. The authors thank the Associate Editor,  Prof. David Ackerly  and two anonymous reviewers for their helpful comments on the manuscript. This research was supported by a grant from the  National Science Foundation of China  (no. 42071060 ) to FKD.
Viscosity, pH and visual appearance of AmB-NE The viscosity of the AmB-NE (F7) was measured using Brookfield Rheometer equipped with a spindle 00 UL adaptor [Brookfield Engineering Labs. , Inc. , USA], and the pH of the nanoemulsion was determined using a pH meter [Systronics Ltd. , India] at 25 C. The average of three data points was obtained. Nanoemulsions with low mean globule size may look transparent, and the slightest evidence of destabilization can be easily recognized by holding the nanoemulsion against a readable background. In such case, the transparency of the nanoemulsion should not change before and after drug loading  [40].
The mucosal sections were then mounted onto diffusion cells. The receiver compartment was filled with citro-phosphate buffer (pH 4. 5), with 2% DMSO and methanol (1:1) used as the diffusion medium. The donor compartment was filled with one gram of AmB-NE gel, and to achieve occlusive conditions, the compartments were sealed with paraffin film. At regular intervals (0, 2, 4, 6, 24 h), 1 mL aliquots were collected, filtered through a 0. 45μ membrane filter, and evaluated by HPLC. The experiment was performed in triplicates. 2. 8. 6. 2. Permeation data analysis. The total amount of drug penetrating through the skin (g/cm 2 ) was plotted against time (min). The two crucial permeation study parameters, i. e. , drug flux (permeation rate) at steady state (J ss ), were determined by dividing the slope by the area of the Franz diffusion cell.
2 , but the changes were not substantial. However, in the case of AmB-NE stored at 25 AE 2 C, 60 AE 5% RH, there was a considerable increase in the globule size (p > 0. 05). The purpose of studying the rheology of the CA and AmB-NE loaded gel was to check the behavioural change in the gel base's viscosity after adding the nanoemulsion to the gel base. It was observed from multipoint viscosity measurements that the CA gel's viscosity was reduced significantly on dilution with nanoemulsions. Also, both the CA and AmB-NE loaded gel demonstrated pseudo-plastic behaviour (data not shown). Texture profile analysis (TPA) was used to determine the mechanical (sensory) properties of the AmB-NE gel. The output of the TPA analysis, penetrometry and spreadability studies are shown in Table  3. The TPA test suggested that the cohesiveness of the AmB-NE gel was sufficient, the work of adhesion was moderate, and the gel exhibited non-drip nature.
2021). Hence contagious asexuality and/or sexual reproduction may occur at small rates within Ap2n asexuals in natura. In addition, previous studies also suffered from a number of limitations. The first limitation is the lack of reliable nuclear markers available across asexual and sexual taxa. The most informative dataset (23 allozyme markers) dates back to the 80's, but shows a limited resolution with few alleles per marker (Abreu-Grobois and Beardmore 1982). Similarly, nuclear sequences (ITS1, Na + /K + ATPase, etc. ) show limited diversity across asexual and sexual taxa  (Baxevanis et al. 2006; Asem et al. 2016). Microsatellite markers described in  Muñoz et al. (2008)  present null alleles and fail to amplify in some sexual species  (Maccari et al. 2013b). Mitochondrial data also present limitations due to the potential co-amplification of nuclear pseudogenes  (Wang et al.
Ploidy We characterized the ploidy of putatively diploid and polyploid asexual females, as well as of males and females of each sexual species (147 individuals in total) using flow cytometry as described in  (Nougué et al. 2015b ) and sup. mat. 1. Data from  (Nougué et al. 2015b)  were added to the dataset, resulting in a total sample size of 206 individuals (Table  S4 ). We tested for difference in genome size between asexual and sexual lineages using t-tests. All significant P-values remained significant after Bonferroni correction, so we present only the uncorrected values for simplicity.