dataesr/scientific-paragraphs-categorization huggingface dataset:
8% identity with the sequence amplified from the S. latifolia sample collected from the same plot. Likewise, the sequence amplified from the C. viscosa sample collected in Le Petit Portland was 100% identical to a sequence originating from a D. alata plant sampled in Guadeloupe in 2002 (isolate YMMV-Guad2, Genbank accession number AF548501; Figure  7 ). The detection of YMMV in weeds provides evidence that YMMV has a wider host range than previously thought and that several weed species could serve as reservoirs of this virus. Viruses 2022, 14, x FOR PEER REVIEW 10 of 19 a lower occurrence of DMaV and potexviruses, and to a lesser extent, with that of CoV1. Likewise, DMaV and potexviruses had a lower occurrence in Grande-Terre. Weed management practices affected the occurrence of CoV1, potexviruses and YMV.
Viruses 2022, 14, x FOR PEER REVIEW 11 of 19 from a D. alata plant sampled in Guadeloupe in 2002 (isolate YMMV-Guad2, Genbank accession number AF548501; Figure  7 ). The detection of YMMV in weeds provides evidence that YMMV has a wider host range than previously thought and that several weed species could serve as reservoirs of this virus.
Data Availability Statement: The data presented in this study are available in Tables  S1-S3. The full dataset is openly available in Recherche Data Gouv at https://doi. org/10. 57745/KD1GEB, UNF:6:l34HgP31WUFIMsliouqJtQ==. The nucleotide sequences reported in this work have been deposited in the GenBank database under accession numbers OP131412-OP131422, OP353605-OP353613 and OP561793.
Collecting quantitative data on insect species occurrence and abundance is a major concern to document population trends. This is especially the case to assess the conservation status of species listed in the European Habitats Directive and to assess the efficiency of mitigation measures with a view to achieve the "no net loss of biodiversity" goal for protected species. However, at present, populations of riverine dragonflies listed in the Habitats Directive and protected under French national law are poorly quantified and monitored. Exuviae collection could be used for such monitoring but a standardised protocol is lacking. We here proposed and tested such a protocol to monitor riverine dragonfly populations through exhaustive exuviae collection along river bank transects. To define the optimal transect size and number of visits, ninety-eight 100 m-long transects divided into 10 m-long plots were monitored on three rivers in southern France.
Introduction Among the ongoing challenges in conservation entomology stands the necessity to acquire quantitative data to document the trends of insect populations  [1, 2]. Based on longterm quantitative studies, alerts on massive decline of insect populations have multiplied in recent years  [3] [4] [5] [6] [7]. This situation clearly urges the need for quantitative datasets allowing (i) to detect and monitor local populations, particularly those of threatened or protected species and (ii) to monitor national or supra-national population trends. However, for several dragonfly species, protocols suitable for the production of such quantitative data are simply lacking. This is the case in south-western Europe of the riverine community composed of the Splendid Cruiser Macromia splendens (Pictet, 1843), the Pronged Clubtail Gomphus graslinii (Rambur, 1842) and the Orange-spotted Emerald Oxygastra curtisii (Dale, 1834)  [8].
Data Analysis For each 10 m plot, 100 m transect, or site (left plus right river bank transects), species richness and abundance were calculated. We then calibrated the sampling effort (i. e. , number of visits and length of transects) that would be required to maximise abundance, species richness, and detection of the target species (i. e. , M. splendens, O. curtisii, and G. graslinii). We calculated the number of new species detected and the abundance of exuviae collected at each visit to assess the efficient number of visits. We compared them using a Friedman rank sum test, followed by a non-parametric Wilcoxon signed-rank test with Bonferroni adjustment to find post-hoc statistical differences. We also calculated the cumulated richness detected by plots. Cumulated species richness was plotted to calibrate the effective length of transects.
In the future, they should be used to test and develop other methods such as quantitative environmental DNA approaches. Efforts to produce genetic barcodes for species identification  [58, 59]  and to understand the persistence and accumulation of dragonflies DNA in their ecosystems  [60]  would probably soon make quantitative environmental DNA approaches available. Using standardised exuviae collection with such genetic methods will thus be useful to calibrate and validate the use of genetic methods to monitor odonate populations in nature. Finally, we should emphasise that this protocol has been used every year since 2018. Except for the impossibility to quantify how much storms may impact exuviae densities, no particular drawback has been identified since, and the dataset obtained is being analysed in order to describe temporal and spatial variability for these three targeted species and identify rivers and rivers portion of highest conservation value.
We analysed a curated dataset of 762 publicly available sequences spanning the full-length coding region of ZIKV from across its geographical span and collected between 1947 and 2021. The definition of genetic groups was based on comprehensive evolutionary dynamics analyses, which included recombination and phylogenetic analyses, within-and between-group pairwise genetic distances comparison, detection of selective pressure, and clustering analyses. Evidence for potential recombination events was detected in a few sequences. However, we argue that these events are likely due to sequencing errors as proposed in previous studies. There was evidence of strong purifying selection, widespread across the genome, as also detected for other arboviruses. A total of 50 sites showed evidence of positive selection, and for a few of these sites, there was amino acid (AA) differentiation between genetic clusters.
The full-length RNA genome sequence of ZIKV, composed of 10. 8 kb, was first published by  Kuno and Chang (2007)  (GenBank accession number [GAN] AY632535). It is based on a strain isolated from a sentinel rhesus monkey (Macaca mulatta) and lacks a N-glycosylation motif (VNDT) in the envelope region that is present in all human ZIKV genomes  (Theys et al. 2017). In 2016, a candidate WHO reference strain (GAN KX369547), isolated from serum from a French Polynesian patient in 2013, was proposed as being representative of clinically significant viruses with widespread distribution  (Trösemeier et al. 2016 ). However, this sequence does not cover the full length of the genome and in 2017 another sequence (GAN KJ776791) was proposed as the ZIKV reference  (Theys et al. 2017) , along with the correct positioning of the mature proteins in the polyprotein (https://rega.
2021 ), available at https://covlineages. org/resources/pangolin. html (last accessed: 20 January 2022). In this study, we present an in-depth genome-wide diversity analysis of ZIKV from over 800 complete coding sequences available in GenBank (NCBI), including strains covering the geographical distribution of ZIKV. We propose a classification and a new naming system for ZIKV lineages, inspired by the above-mentioned SARS-CoV-2 nomenclature system and based on phylogenetic analyses, clustering techniques, within-and between-group pairwise genetic distances, and evolutionary analyses to define genetic groups and subgroups. This nomenclature proposal avoids geographical terminology, using instead alphanumerical labels, and provides a dynamic system that can be rapidly updated as new lineages are identified.
Dataset compilation and sequence alignment A total of 2179 sequences of ZIKV were downloaded from the Gen-Bank database  (Benson et al. 2013 ) in December 2021. The filtering steps to select the sequences for analyses are detailed in Supplementary Fig. S1 , and the complete list of sequences and exclusion criteria are found in Supplementary Table  S1. Step 1: Before alignment-Criteria: size and nature of the sequence Sequences shorter than 7000 bp were excluded, after which 1377 sequences remained. From these, the ones identified as patent, recombinant, clone, synthetic construct, or mutant were eliminated, leaving 966 sequences that had been isolated from natural hosts. Step 2: After NT alignment-Criteria: removal of identical sequences and quality control (ACTG content, presence of stop codons in coding region) The 966 sequences were aligned against the curated ZIKV reference sequence with GenBank accession number (GAN) KJ776791 as described in  Theys et al.
(2017)  using the codon-correct alignment tool VIRULIGN v1. 0. 1  (Libin et al. 2019). The codon-correct NT alignment was then cleaned to: 1. Remove sequences with ACTG content lower or equal to 70 per cent of the complete coding region, 2. Remove sequences harbouring a stop codon within the coding region, which resulted in 874 sequences, 3. Remove identical sequences (with 100 per cent NT identity) using a custom script (available at https://github. com/seabrasg/zika_diversity-last accessed: 23 March 2022). We kept only the most recent sequence (and the first in alphabetical order, by accession id, when several sequences were encountered from the same date) resulting in 770 sequences. With one exception, all sequences that were excluded for being identical belonged to the same country (Supplementary Table  S2 ).
Sequences that had several of these issues along the alignment and originated from the same study were excluded, resulting in a final dataset of 762 sequences. In this final alignment (762 sequences), all sequences had collection date information and only four sequences did not have information about the collection geographical location (i. e. three imported cases in China and one in Japan). The classification of ZIKV sequences as 'African' or 'Asian' genotypes was done using the Genome Detective ZIKV typing tool  (Alcantara et al. 2009; Fonseca et al. 2019). Recombination was assessed for this full curated dataset using the Phi-test  (Bruen, Philippe, and Bryant 2006)  available in SPLIT-STREE version 4. 17. 0  (Huson and Bryant 2006)  and using the methods RDP  (Martin and Rybicki 2000) , GENECONV  (Padidam, Sawyer, and Fauquet 1999) , BOOTSCAN  (Salminen et al.
1995) , Maxchi  (Smith 1992) , CHIMAERA  (Posada and Crandall 2001) , SISscan  (Gibbs, Armstrong, and Gibbs 2000) , PhylPro  (Weiller 1998) , LARD  (Holmes, Worobey, and Rambaut 1999) , and 3SEQ  (Boni, Posada, and Feldman 2007) , implemented in RDP4  (Martin et al. 2015). Sequences with potential recombinant regions were identified in RDP4 when at least four methods had a Bonferroni-corrected P-value lower than 0. 05. Downloaded from https://academic. oup. com/ve/article/8/1/veac029/6555351 by KU Leuven Libraries user on 04 May 2022 From the 'Full curated dataset' (762 sequences) we excluded the sequences with potential recombinant regions ('Afro-Asian' dataset-759 sequences). This dataset included the 'African' and 'Asian' genotypes and was used in the phylogenetic, genetic diversity, clustering, and selection analyses.
We also did the clustering and selection analyses after excluding the African genotype ('Asian genotype' dataset-752 sequences). For the phylogeographical reconstruction, we excluded the sequences that did not have information about the geographical location of collection (Geo-referenced 'Asian' dataset-748 sequences). A subset of sequences from the early American expansion and the 5 years before that (2010-2015) ('Early Am' dataset-68 sequences) was also used in the selection analyses (Supplementary Fig. S1 ).
Phylogenetic reconstruction The tree reconstruction for the full dataset of ZIKV was done to confirm the separation of the 'African genotype' and 'Asian genotype' clades. The maximum-likelihood (ML) method was applied using the software package IQ-TREE version 2. 0. 3  (Nguyen et al. 2015)  with the GTR + F + G4 substitution model according to the best fit models identified by IQ-TREE ModelFinder. We evaluated the robustness of the tree with 1000 ultrafast bootstraps (UFBoot)  (Hoang et al. 2018 ) and 1000 SH-like approximate likelihood ratio tests (aLRTs)  (Guindon et al. 2010). The obtained ML tree was annotated using the online tool iTOL version 6. 3  (Letunic and Bork 2021)  according to the variables: host (human, monkey, and mosquito) and geographical regions based on the United Nations M49 coding classification of geographic regions (https://unstats.
Genetic diversity and distances AA and NT mutation tables were obtained from VIRULIGN and were used for the genetic diversity analyses using custom R scripts (available at https://github. com/seabrasg/zika_diversitylast accessed: 23 March 2022). The mutation tables were filtered to set the ambiguities as missing values. The proportion of missing data was analysed per AA position and per NT position. The most frequent AA and NT at each position for the entire dataset of sequences (consensus sequence) were compared with two reference genomes, the curated reference genome GAN KJ776791  (Theys et al. 2017 ) and the NCBI reference Natal genome GAN KU527068, sequenced from brain tissue of an autopsied microcephalic foetus  (Mlakar et al. 2016). For each position, AA and NT variation were characterized by estimating the frequency of the minor variants and the Shannon entropy diversity index, determined using the Chao-Shen estimator  (Chao and Shen 2003) , as implemented in the R software package entropy version 1.
3. 0. Considering that the present ZIKV nomenclature recognizing the 'African' and 'Asian' genotypes does no longer reflect the actual geography of the virus circulation for the vast Asian clade, we revisited the classification of these clades that was used to propose a new nomenclature. We used the Bayesian model-based hierarchical clustering algorithm hierBAPS  (Cheng et al. 2013)  as implemented in the software R package rhierbaps version 1. 1. 3 (Tonkin-Hill et al. 2018). This method combines model-based techniques with an initial fast distance-based complete-linkage agglomerative clustering  (Tonkin-Hill et al. 2019). Given an a priori upper bound for the number of genetic clusters, K, hierBAPS estimates K as part of the model fitting procedure and it finds the partition of the data (allocation of each sequence to one of K possible clusters) that maximizes the posterior probability of that allocation  (Tonkin-Hill et al.
2018). Two hierarchy levels of clustering were set with a prior upper boundary of 20 clusters. Ten independent runs were performed to assess the congruence of cluster assignment. This analysis was separately applied to genome-wide NT alignments of the 'Afro-Asian' and the 'Asian genotype' datasets. To visualize the relationships between haplotypes and cooccurrence in the different geographical areas, we obtained a TCS haplotype network  (Clement, Posada, and Crandall 2000) , as implemented in the software PopART  (Leigh and Bryant 2015). The TCS method starts by calculating pairwise distances between sequences and relies on statistical parsimony to find the connections between sequences  (Clement, Posada, and Crandall 2000). The method removes the sequences with more missing data than others and then masks the sites with gaps or characters other than NTs from the original alignment.
Mutation tracking and selection analyses We also investigated for evidence of selective pressure in the full and in the 'Asian genotype' datasets. We used two methods that detect selection by examining patterns of synonymous and non-synonymous substitutions on a per-site basis: (1) the mixed-effects model of evolution (MEME) that detects sites under pervasive or episodic positive/diversifying selection  (Murrell et al. 2012 ) and (2) the Fast, Unconstrained Bayesian AppRoximation (FUBAR) method that detects sites under pervasive positive/diversifying selection and also those under negative/purifying selection  (Murrell et al. 2013 ). Both methods were used as implemented in HyPhy version 2. 2 (Kosakovsky Pond, Frost, and Muse 2005) on the Datamonkey server  (Weaver et al. 2018). The MEME method uses phylogenetic models and an ML approach to describe the evolution of codons along tree branches by a continuous-time stationary Markov process.
9. With the aim of detecting the sites with evidence of selection during or immediately after the selective process  (Martin et al. 2021) , we also restricted the analysis to the period of the earlier Am-ZIKV expansion and the years just before that expansion by analysing sequences sampled from 2010 to 2015 (dataset 'early Am'). To understand the spread of the mutations on sites with evidence of positive selection, we analysed the AA frequencies at those sites for each genetic group obtained previously.
ZIKV full-genomes dataset The full curated dataset consisted of 762 ZIKV aligned genomes. The alignment had 10,269 NTs, corresponding to 3423 AA positions. Only the two terminal noncoding regions (5 ′ and 3 ′ -NCR) were not included in the analyses. The ZIKV genome encodes for three structural proteins: C (122 aa), Pr/M (168 aa, including 93 of the peptide Pr), E (504 aa), and seven non-structural proteins: NS1 (352 aa), NS2A (226 aa), NS2B (130 aa), NS3 (617 aa), NS4A (150 aa, including 23 of the peptide 2K), NS4B (251 aa), and NS5 (903 aa). Viral strains analysed here originated from 46 different countries across the world and have been sampled between 1947 and 2021 (Supplementary Table  S3 ). Genome Detective ZIKV typing tool indicated the 'African genotype' for 7 sequences, the 'Asian genotype' for 754 sequences, and signalled one sequence as 'Related to but not part of Asian' (OK054351_India_28 July 2021).
The distribution of the samples according to continent of collection was: Africa (1. 6 per cent), Asia (15 per cent), North America (57. 3 per cent), Oceania (2. 1 per cent), and South America (24. 0 per cent), and according to geographical region of collection: Eastern Africa (0. 3 per cent), Western Africa (0. 7 per cent), Middle Africa (0. 7 per cent), South-Eastern Asia (13. 3 per cent), Southern Asia (0. 8 per cent), Eastern Asia (0. 9 per cent), Melanesia (0. 1 per cent), Micronesia (0. 1 per cent), Polynesia (1. 9 per cent), South America (24. 0 per cent), Central America (28. 1 per cent), Caribbean (23. 0 per cent), and Northern America (6. 2 per cent) (Supplementary Table  S3 ). While a broad range of time, spanning 74 years of viral spread was covered in the dataset, the detection and public health impact of ZIKV outbreaks in the Americas, together with the availability of advanced next-generation (real time) sequencing methods, caused a dramatic sequencing scale-up effort in samples collected in 2016 (83.
Recombination analyses No evidence of recombination was found with Phi-test in SPLIT-STREE (P = 0. 1023806). Using the methods implemented in RDP4, three sequences were identified as having small genomic regions that presented evidence for potential recombinant history with African sequences: KY241700_Singapore_27 August 2016 (region 3950-4358 bp), KY241712_Singapore_30 August 2016 (region 52-269 bp) and EU545988_Micronesia_2007-06 (region 1-52 bp) (Supplementary Table  S4 ). We excluded these three sequences from the alignment to produce the 'Afro-Asian' dataset with 759 fullcoding sequences that was further analysed for the phylogenetic reconstruction and genetic diversity analyses.
Phylogenetic reconstruction The ML tree of the 'Afro-Asian' dataset of 759 sequences obtained in IQ-TREE confirmed the pattern of genetic differentiation of the 'African genotype' clade (n = 7) from the remaining samples (Fig. 1 ; Supplementary Fig. S3 ), forming the 'Asian genotype' clade. The sequence from the recent 2021 outbreak in India (GAN OK054351), previously identified by Genome Detective as 'Related to but not part of Asian', grouped with the early Asian Malaysia sequence. Sequences obtained from mosquito hosts formed clusters in African and early Asian clades, while they were dispersed in the rest of the tree.
Genetic diversity and genetic distances In the 'Afro-Asian' dataset of 759 sequences, the proportion of missing data per NT position was at most 15. 2 per cent (minimum coverage of 84. 8 per cent) (Supplementary Fig. S4A ). A total of 2663 parsimony-informative NT sites out of 10,269 total sites were found in the ZIKV alignment of the full dataset. The consensus NT genome sequence differed at 11 positions from the reference genome KJ776791, of which 3 showed a consensus NT with a prevalence above 95 per cent (Supplementary Table  S5 ). The consensus NT genome differed at 21 positions from the NCBI reference Natal genome KU527068, of which 17 positions had a consensus NT with a prevalence above 95 per cent. Overall, 99 per cent of the NT positions had a prevalence of the consensus NT above 95 per cent (Supplementary Table  S6 ).
The mean entropy at NT level considering the complete dataset was 0. 021 (95 per cent confidence interval: 0. 020-0. 022; range: 0-0. 69). The most variable positions were distributed across the genome and were present in every gene region (Fig. 2A ; Supplementary Fig. S4B ). From the 139 NT positions with frequency of alternative NT higher than 5 per cent, 18 were non-synonymous, located in proteins C (I80T, D107E), PR (N139S/K), E (V503A/E), NS1 (G894A, V988A, M1143V/T, T1145S), NS2A (I1162M), NS3 (M2074L, H2086Y/R), NS4B (I2367M/V), and NS5 (V2634M, I2842V, R3045C/S, Q3282W, T3328I/N, D3398E) (Supplementary Table  S6 ). The consensus AA genome sequence (Supplementary Fig. S5 ) differed at position 2634 (position 114 in the NS5 protein) from Downloaded from https://academic. oup. com/ve/article/8/1/veac029/6555351 by KU Leuven Libraries user on 04 May 2022    S5 ).
When considering the NCBI reference Natal genome KU527068, the consensus differed at four positions: position 940 (NS1_146) with AA K at 98. 8 per cent, instead of E; position 1027 (NS1_233) with AA T at 98. 9 per cent, instead of A; position 1143 (NS1_349) with AA M at 82. 9 per cent, instead of V; position 2509 (NS4B_240) with AA T at 99. 6 per cent, instead of I. Overall, 96. 9 per cent of the AA genome positions were highly conserved, showing a prevalence of the consensus AA above >99 per cent. The proteins with higher proportion of positions below 99 per cent consensus AA prevalence were PR (7. 6 per cent of positions) and C (4. 1 per cent of positions) (Supplementary Table  S6 ). The mean entropy at AA level considering the complete dataset was 0. 0097 (95 per cent confidence interval: 0.
ZIKV nomenclature The hierarchical clustering method implemented in hierBAPS identified four clusters for the full dataset in the first level of analysis, one composed of the African sequences and the others subdividing the 'Asian genotype' clade into three groups (Supplementary Table  S3 ). The second level of analysis on this dataset subdivided the African group into three groups: Western Africa (Guinea and Senegal), Eastern Africa (Uganda), and Middle Africa (Central African Republic). However, when analysing the ML tree, not all these groups had good phylogenetic support. Due to the small number of samples in this analysis (n = 7), we restricted the naming proposal to one group (new name: ZA). Conducting the hierBAPS analysis for the 'Asian genotype' dataset only (752 sequences), 6 clusters were obtained in the first level of analysis, while 12 nested subclusters were found in the second level of analysis.