dataesr/scientific-paragraphs-categorization huggingface dataset:
Statistical significance was assessed by a Monte-Carlo permutation test with 9999 simulations. The composition distance matrix between samples was computed with the bP measure applied on the presence-absence table of the P6 loop sequences in the dung piles. The seasonal variations of the occurrences of the P6 loop sequences identified at the genus level were then analysed in regard to the mean monthly abundance of macroscopic fruit residuals of the same genus found in the same dung piles  [18]. The coincidence of the sequence abundance peaks in the faeces with the fructification period was tested with a Pearson's correlation test after a log+1 transformation of the data. The statistical analyses were performed with the R software, using the ade4 package  [53]. The Mantel test was calculated with the function mantel.
Main identified taxa The barcode reference databases allowed to identify 113 (96. 6%) and 47 (40. 2%) of the 117 unique P6 loop sequences up to the family and genus levels, respectively. They were assigned to 41 families, 39 genera and 3 species (Table  S1 , Figure  3 ). In each Figure  1. Abundance distribution of the P6 loop sequences at different taxonomic levels. ", family": sequences identified at a finer taxonomic resolution than the family, "family": sequences identified at the family level only, ". family": sequences that could not be identified up to the family level. N = 5488 unique sequences longer than 10 bp and with occurrence higher or equal to 11. doi:10. 1371/journal. pone. 0060799. g001 Table  2. Number of sequences assembled by NGS of vegetal DNA extracted from tapir faeces (N = 39, 79660 mg of faecal product), using plant universal primers targeting the P6 loop region of the trnL (UAA) intron and primers specific for the Sapotaceae family, targeting the first internal transcribed spacer (ITS1) of nuclear ribosomal DNA.
62, p , 0. 001, Figure  3 ). As expected, the ITS1 primers specially designed to amplify the Sapotaceae family yielded a refined determination resolution, with 9 (75. 0%) and 8 (66. 7%) of the 12 unique sequences directly attributed to a genus and to a species, respectively, after confrontation to the EMBL database. Coupling with the local ITS database of the Nouragues allowed to further refine the identification of one unidentified sequence to one more species. The list of identified taxa is given in Table  3.
Only 40. 2% of the P6 loop sequences were identified to the genus level, which is much less well resolved than in the study by  Soininen et al. [29]  for the diet of Norwegian rodents for a comparison (they obtained. 75%). We checked that 87. 4% and 69. 2% of the 159 families and 643 genera, respectively, of the flowering photosynthetic plants listed in the Nouragues Reserve (http://www. nouragues. cnrs. fr/plant. html, accessed March 2011) had at least one species with the trnL (UAA) intron P6 loop sequenced in the EMBL database. Among the 20 non-referenced families, only four (Blechnaceae, Dichapetalaceae, Erythroxylaceae and Thelypteridaceae) have already been listed in the diet of tapirs  [18]. Most of the remaining families are small plants, that could hardly be a resource for the tapir, such as small, often epiphytic or epilithic, herbaceous ferns (Grammitidaceae, Hymenophyllaceae, Oleandraceae, Nephrolepidaceae, Schizaeaceae, Vittariaceae) and rare plants or localized trees or lianas (Agavaceae, Hernandiaceae, Thymeleaceae, Trigoniaceae, Ixonanthaceae, Rhabdodendraceae).
The local DNA barcoding database  [38]  allowed us to refine the determination of 14 sequences to the genus level. The incompleteness of the reference databases, however, explained only part of the low taxonomic resolution since among the 19 plant families with undetermined genus, 15 included genera from the Reserve with the trnL (UAA) intron sequenced in the EMBL. The low taxonomic resolution for 11 of these families, and for at least 6 more families (among 41), was explained by shared sequences between several genus. As an example, five genera of the Sapotaceae family carried the same P6 loop sequence. As a result, tapirs may consume plant species with a low or no variation of the P6 loop. In such plant groups, in temperate regions  [24] , only closely related species are usually not resolved  [34].
In our study, however, only three P6 loop sequence could be identified below the genus level by comparison with the EMBL database. We could check that both Spondias and Hieronyma had only one species listed in the Nouragues reserve. Ludwigia has several species in the reserve but two different species are sequenced in the EMBL and they differ, so the molecular marker appears to have a sufficient resolution to distinguish between the species in this genus. We believe that the species identification in these three cases can then be validated. The low taxonomic resolution seems not limited to our study since when testing their database developed for the tropical trees of the Nouragues Reserve, Gonzalez et al. [38]  could unambiguously identify only 63% of the genera using the full trnL intron.
This resolution power is much lower than expected for a comprehensive database in temperate regions (90% according to Valentini et al. [24] ). Consequently, it is likely, that the shorter P6 loop of the trnL intron is even less efficient in a tropical context to discriminate plant genera. The shared sequences between genera may be explained by a higher rate of lineage diversification and frequent explosive radiations in the tropics compare to temperate ecosystem  [38]. Our study stresses the interest of combining complementary markers (see the two-tiered approach of Newmaster et al. [54]  such as the internal transcribed spacer of the nuclear ribosomal genes to identify the consumed species (e. g. [33] ). Indeed, in our study 9 different Sapotaceae species could be attributed to 75% of the sequences targeted by the ITS marker.
Argostemma, however, is a genus naturally absent from the Guianas and this misidentification may result from the absence of the actually consumed Rubiaceae genus in the EMBL database that carries the same P6 loop. The same risk of misidentifation is possible for all the plant taxa that have not been sequenced in the EMBL yet and this is a major limitation of the approach using only this incomplete reference database. However and despite a relative low taxonomic resolution in our study case, the metabarcoding approach using the P6 loop approach was well validated by the high concordance with the literature on lowland tapir diet. Out of the families and genera identified here, 95. 1% (39/41) and 74. 4% (29/39), respectively, were already known to be consumed by tapirs  [18]. Moreover, our study allowed to retrieve not less than 41.
Conclusions The DNA of plants found in the faeces is known to be highly degraded by digestion and we opted for using the short P6 loop of the plastid trnL intron associated to high throughput sequencing in order to maximise the chance to capture short plant DNA fragments in the faeces. This was successful since we could retrieve DNA from more than 50 different plants in every faecal sample. However, a major limitation to this approach is that the taxonomic resolution is mainly limited to the family or the genus, both because several species can have the same sequence and because the reference database are not yet complete. As long as the sequence reference database will not be taxonomically covered, the best taxa candidates can not all be interpreted as strictly confident records of the diet.
We draw the attention of the reader that this approach, at this stage, only provides a list of hypothetical but likely plant taxa that can be eaten by tapirs. Nevertheless, our study case in the lowland tapir showed that the resulting information on the diet is well consistent with that from classical methods. It confirmed the consumption of many taxa, extended the range of use of some others and also allowed to discover new likely ones. Our study also showed that further development of the approach and refining of the taxonomical resolution is also possible thanks to the combination of complementary markers. The genetic reference databases are still under development, including DNA sequences from new organisms in new regions of the world (e. g. [57] ). As more taxa are being sequenced a refined appreciation of the composition and richness of the diets should be possible.
(A. L. De Candolle) Eymas N Presence of the taxa in the region was checked in Funk et al. (2007). Species names followed by a "*" and by a " N " are for sequences further identified at the species level thanks to the local database for the Nouragues Reserve (Gonzalez et al. 2009) and not yet listed in the diet of the lowland tapir throughout its range (comparison with review by Hibert et al. 2011), respectively. doi:10. 1371/journal. pone. 0060799. t003.
Acknowledgments We would like to warmly thank  Philippe Gaucher  and  Mael Dewynter  who encouraged and logistically supported the field work in the Nouragues Reserve and Cecile Vanpe,  Cyril Marmoex ,  Judith Andrivot ,  Stefan Icho ,  Pierre Terret ,  Wemo Betian  and  Helene Richard  for their precious help in data collection in the field. We thank the lab technician team of the  INRA in Kourou  for their technical advice for the lab and  Stephane Dray  for his advice on some of the statistical analyses. We also thank  Nigel Yoccoz  and two anonymous reviewers for their valuable comments on a previous draft. Last but not least, we are grateful to  Philippa Dancer  who kindly edited the English.
This work was funded by the  ONCFS (French Hunting and Wildlife Agency). It has also benefited from  Investissement d'Avenir grants  ( CEBA :  ANR-10-LABX-0025 ;  TULIP :  ANR-10-LABX-0041 ) and from grants from  ANR (METABAR). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
TnpA catalytic sites: cis/trans configuration We previously proposed that during the excision and integration steps in the transposition cycle, TnpA IS608 alternates between trans and cis configuration  (5). Although currently there are no crystallographic data demonstrating that TnpA IS608 , TnpA ISDra2 or a related TnpA from Sulfolobus can assume a cis configuration (6,7), an inactive TnpA from D. radiodurans has indeed been crystallographically captured in this configuration (Protein Data Bank: 2fyx). Comparison of these structures also suggests that a cis/trans transition would be structurally reasonable. In this study, we, therefore, addressed the potential conformation changes using other approaches. The rotation model raises the question of the catalytic properties of TnpA if it were to assume a cis configuration.
ACKNOWLEDGEMENTS The authors thank  L. Lavatine ,  G. Duval-Valentin  and  O. Barabas  for discussions. FUNDING Centre National de Recherche Scientifique (CNRS, France) ; by  ANR  [ ANR08   Blanc-0336 Mobigen  to M. C]; intramural Program of the  National Institute of Diabetes and Digestive and Kidney Diseases  (to F. D). Funding for open access charge: CNRS intramural funding.
The bias ranged from 55% to 75% (mean = 65. 3%, SD = 6%). The data of these eleven participants were analyzed together. The remaining three subjects were excluded from the analysis either because the bias was too extreme or absent. The basic features of the results of Experiment 2 are identical to Experiment 1 (see Fig. S2  in Text S1). We adapted our hybrid model to this second experiment. In the model described earlier, both sides were chosen with the same a priori probability. The response bias in the current experiment is modeled by introducing a bias a on the choice of the internal variable for the more frequent side. If participants did adapt perfectly to our experimental setup, the value of a should be 0. 65. The new hybrid model including this additional bias parameter was adjusted to the individual data; the average of the best fits for each subject is superimposed on the curves in Fig.
Material Visual stimuli were presented on a computer screen LIYAMA HM 903 DTA (19 in). The experiment was controlled using Matlab and the Psychtoolbox  [31, 32]. Visual stimuli were two colored dots (target and distractor), one red and one green presented each on one side of a third dot. The color of the central dot varied randomly from trial to trial. It was either red or green. Each dot had a radius of 44 pixels and the distance between the central dot and the peripheral dots was of 134 pixels, viewing distance was 50 cm. An auditory signal of 50 ms duration indicated the beginning of the task (Go signal). Its pitch varied randomly on each trial. It was either high (1500 Hz) or low (600 Hz).
Experimental Crystal data (i) Àx þ 1; Ày þ 1; Àz; (ii) Àx; Ày þ 1; Àz; (iii) Àx; y À 1 2 ; Àz À 1 2 ; (iv) x; Ày þ 1 2 ; z À 1 2 ; (v) Àx þ 1; y À 1 2 ; Àz þ 1 2. Data collection: COLLECT (Nonius, 1998); cell refinement: SCALEPACK  (Otwinowski & Minor, 1997) ; data reduction: DENZO  (Otwinowski & Minor, 1997)  and SCALEPACK; program(s) used to solve structure: SHELXS97 (Sheldrick, 2008); program(s) used to refine structure: SHELXL97 (Sheldrick, 2008); molecular graphics: DIAMOND  (Brandenburg & Berndt, 1999) ; software used to prepare material for publication: WinGX  (Farrugia, 2012). Grateful thanks are expressed to Dr T. Roisnel (Centre de Diffractometrie X, Universitede Rennes 1) for the X-ray data collection. Supplementary data and figures for this paper are available from the IUCr electronic archives (Reference: LH5619).
Computing details Data collection: COLLECT (Nonius, 1998); cell refinement: SCALEPACK  (Otwinowski & Minor, 1997) ; data reduction: DENZO and SCALEPACK  (Otwinowski & Minor, 1997 ); program(s) used to solve structure: SHELXS97 (Sheldrick, 2008); program(s) used to refine structure: SHELXL97 (Sheldrick, 2008); molecular graphics: DIAMOND  (Brandenburg & Berndt, 1999) ; software used to prepare material for publication: WinGX  (Farrugia, 2012).
Plasmid inserts were sequenced using the dideoxy-terminator reaction using the 907 reverse primer [59 CCG TCA ATT CCT TTG AGT TT 39]   45  and the BigDyeH Terminator v3. 1 Cycle Sequencing Kit (Life Technologies). Cycle sequencing products were cleaned using the AgencourtH CleanSeqH Dye-Terminator Removal kit (Beckman Coulter, Brea, CA) and run in an AB3130xl Genetic Analyzer (Life Technologies). Partial, raw sequences were assembled using 20 nucleotides minimum overlap and 99% maximum mismatches and visually edited using the gap4 program of the Staden package  46. Edited contigs were exported in fasta format including the number of reads per contig as weights, and chimeric sequences were eliminated by using a stringent chimera checking pipeline consisting of uchime  47  , Bellerophon  48  and by querying the sequences against a curated version of the Silva database SSURef_111_SILVA_ NR_98_26_07 (http://www.
arb-silva. de/) using blastn (see Supplementary Information for detailed parameters used). Non-chimeric sequences resulting from this pipeline were filtered for clones of planktonic origin by blastn queries against a local database including 1493 OTUs from 37, 860 16S rRNA PCR library sequences from the Global Ocean Sampling project plus 8,471 marine bacterial 16S rRNA sequences obtained from the ribosomal database project dataset, and graciously provided by S. Yooseph. The 16S rRNA sequences of the clones used in this study have been deposited in the GenBank database under accession numbers KC492587-KC492611 and KC492698-KC492704. Phylogenetic analysis. Clone sequences classified as non-chimeric and non-planktonic were fully sequenced using primers 907R and 785F (5' GGATTAGATACCCTGGTAGT 3'), individually assembled using gap4, and exported.
These sequences were aligned using the mothur  49  and imported into the SSURef_111_SILVA_18_07_12 database of the Silva project  50  using the ARB software  51. Phylogenetic trees were constructed by neighbor-joining using the phylip package V3. 6  52  and by Bayesian analysis using Mr Bayes v3. 2. 1364  53. For both methods, the aligned clone sequences and reference sequences from the Betaproteobacteria, Gammaproteobacteria and Alphaproteobacteria were filtered using an 1112 bp majority filter. 454-pyrosequencing analyses. The PCR products used to construct the clone library were used as template for 454 pyrosequencing by the Research and Testing Laboratory (Lubbock, TX) according to a standard multiple protocol of this sequencing facility and described by Dowd and coworkers  54. Briefly a PCR step (30 cycles) with a HotStart HiFidelity Polymerase and primers 28F (59 TTTGATCNTGGCTCAG 39) and 519R (59 GTNTTACNGCGGCKGCTG 39)  was performed in order to amplify the hypervariable V1-V3 region of the 16S rRNA gene.
Tag-encoded FLX amplicons were sequenced on the Roche 454 FLX sequencer using Titanium (Roche) reagents. Multiplex raw sff files were analyzed using a hybrid analysis pipeline. In brief, denoising was done by AmpliconNoise V1. 25  55  implemented in Qiime V1. 5  56  and rather than using chimera analysis by Perseus, de novo chimera detection and removal was done by using the uchime  47  module of Usearch V5. 0 57 followed by the same analyses as for the clone library (details of parameters and commands in Supplementary Information). Non-chimeric were unweighted and grouped into OTUs using uclust with 97% sequence identity. OTU representatives were selected based on abundance. OTUs of planktonic origin were identified by blastn searches as above. Pyrosequencing sequence reads representing the 86 OTUs have been deposited in the GenBank database under accession numbers KC492612-KC492697.
Oligonucleotide probe design. The specific oligonucleotide probe BET467 targeting the prevalent betaproteobacterial sequences in the clone library was designed using the probe design tool of the ARB software package  51. The specificity of the probe sequence was confirmed by the probe_match tool of ARB, the probe match tool of Ribosomal Database Project (http://rdp. cme. msu. edu/) and blastn searches. Only 3 non-target sequences in the probe_match tool of ARB database had less than 3 mismatches with this specific probe. As the 16S rRNA target region was potentially inaccessible  58  , two unlabeled helper oligonucleotides H-485 and H-446 were also designed. Oligonucleotide sequences of probes used, labeling and details are shown in Table  1. All probes were synthesized by ThermoFisher Scientific (Germany).
Images analyses were performed using a model Olympus DP72 (Olympus) camera system and Cell A (Olympus) imaging software. To obtain a rough estimate of bacterial cell numbers in the sponge tissue, 20 square zones of 20 mm 2 were selected. In each of these zones, the Betaproteobacteria signals were counted from the specific probe signals, and total bacterial cells and sponge nuclei were counted from the corresponding DAPI image. Transmission electron microscopy. TEM was performed according to Uriz and collaborators  61  with minor modifications. Briefly, small samples of sponge tissue (3-4 mm 3 ), were fixed overnight at 4uC in 2. 5% glutaraldehyde in 0. 1 M sodium cacodylate buffer pH 7. 4 (osmolarity adjusted to 980 mOsM with sucrose), washed in the same buffer at room temperature, and post-fixed for 1 hour in 1% osmium tetroxide.
Acknowledgements We thank the OOB diving team for collecting the sponge specimens and  Shibu Yooseph  ( JCVI ) for providing us with a database of planktonic rRNA genes associated with his previous publication. Molecular biology, and DNA sequencing analyses were performed using the  Marine Biodiversity and Biotechnology platform  ( Bio2Mar ) and microscopy analysis using the Cytometry and Imaging platform (http://www. obs-banyuls. fr/pci/index. php)both at OOB. We thank  Olivier Thomas  ( Univ. Nice ),  Detmer Sipkema  ( Univ Wageningen ),  Maria-Jesus Uriz  ( CSIC Blanes ),  Nataly Bontemps  and  Bernard Banaigs  ( Univ. Perpignan ) and  Thierry Perez  ( Univ. Marseille ) for suggestions to the experimental work and very fruitful discussions. We thank the  UPMC doctoral school 392  ( Diversity of Life) for its doctoral fellowship  to  Julie Croue.
5 and similarity of 0. 8). Some additional gaps were closed by Sanger sequencing of the PCR amplicons. The published sequence is composed of 9 contigs with a sequence length from 2 kbp to 1. 9 Mbp, grouped in 4 scaffolds. The P. atrosepticum CFBP6276 genome consists of one circular chromosome, containing 4,860,851 bp, and two circular plasmids, containing 5,876 bp and 2,681 bp, respectively. The percentages of GϩC content are 51% for the chromosome and 47% for the plasmids. A total of 4,430 coding sequences were predicted using the Rapid Annotations using Subsystems Technology (RAST) v4. 0 automated pipeline  (14). Nucleotide sequence accession number. The P. atrosepticum strain CFBP6276 genome sequence has been deposited in GenBank under the accession no. ASAB00000000.
ACKNOWLEDGMENTS This work was supported by the  Centre National de la Recherche Scientifique (CNRS, Gif -sur-yvette, France). We thank  Perrine Portier  ( Collection Française de Bactéries Associées aux Plantes [CFBP], Angers, France ), who supplied P. atrosepticum  CFBP6276 , and  Xavier Latour  ( Université de Rouen, Evreux ) and  Valérie Hélias (Fédération Nationale des Producteurs de Plants de Pomme de Terre   FN3PT/RD3PT ) for critical reading of the manuscript.
Transcriptomic Data The microarray used contained 992 gene-specific 50-mer oligonucleotides generated from 63 imprinted genes (See Table  S1 ), 256 genes encoding proteins involved in neurotransmission and 157 genes encoding proteins related to energy homeostasis  [23]. A direct, dye-swapping comparison strategy was used to compare conditions, and dye-swaps were replicated several times, with at least four animals/group. Heat maps were constructed by compiling the differentially expressed genes (DEG) in the various comparisons (OP1/CD1, OP2/CD2, OR2/CD2, OR2/OP2, OP2/OP1), facilitating further indirect comparison between groups. The heat maps shown in Figures  2  and 3  highlight the dysregulated expression of three categories of genes: genes relating to metabolism, neurotransmission and imprinted genes.
Arrays were normalized by LOESS regression, using the LIMMA library from the R/BIOCONDUCTOR packages to assess differential expression between conditions. Microarray experiments, described according to MIAME guidelines, have been deposited in NCBI's Gene expression Omnibus repository under accession number GSE30085 (http://www. ncbi. nlm. nih. gov/geo/query/acc. cgi?acc = GSE30085).
Microarray Analysis Heat maps were constructed by compiling the differentially expressed genes (DEG) in the various comparisons (OP1/CD1, OP2/CD2, OR2/CD2, OR2/OP2, OP2/OP1), thereby allowing further indirect comparison between groups. Biologically relevant pathways were identified after importing Entrez Gene IDs into IPA software (version 8. 7 IngenuityH Systems, http://www. ingenuity. com). Several analyses were carried out with the lists of DEG obtained for the OR2/CD2, OR2/OP2 and OP2/OP1 comparisons and the subset of genes displaying dysregulation in both OP1/CD1 and OP2/CD2 comparisons. All the spot entries were recognized by the program and included in network and function/pathway analyses. The DEG were mapped to the genetic networks available in the Ingenuity database and were then ranked on the basis of their score.