dataesr/scientific-paragraphs-categorization huggingface dataset:
4B ). Not all of the bacterial HGTs assessed either through in silico or GFP analysis localized to the endomembrane system, with other HGTs experimentally localized to the pyrenoid, mitochondria, and cytoplasm (SI Appendix, Fig. S23 ). We noted a weak enrichment in chloroplast-targeting predictions associated with bacterial genes in ochrophyte genomes. Within the genes manually identified from P. tricornutum, this was only found to be significant (P < 0. 01) with ASAFind but not HECTAR, and was much weaker than equivalent biases (χ 2 P < 10 -10 ) for genes inferred to have been horizontally transferred with eukaryotic algae (Dataset S1). Within the ochrophyte-wide bacterial HGT dataset, this enrichment was only detected in bacterial HGTs acquired at early time points in ochrophyte (Fig. 4A ).
S24A ). We found no obvious biases in the frequencies of intron retention or exon splicing  (17)  documented in bacterial HGTs compared to other genes (Dataset S1). We identified little preference in the distribution of HGT genes across P. tricornutum chromosomes, barring a possible (P < 0. 05, χ 2 ) enrichment on chromosomes 9 and 32 (Dataset S1). We noted that 14 of 298 (4. 8%) of the bacterial HGTs were adjacent, and in the same transcriptional orientation, to other bacterial HGTs, whereas only 6 (2. 1%) were adjacent to other bacterial HGTs but in opposing transcriptional orientation (SI Appendix, Fig. S24B  and Dataset S1). However, structural inspection of the bacterial HGTs that are adjacent to one another suggest that they predominantly possess similar functions to one another, suggesting that they are likely the result of recent paralogy events, as opposed to being a remnant of horizontally acquired bacterial operons (SI Appendix, Fig.
Both the taxonomically larger transcriptome-enriched dataset and the combination of automatically and manually resolved phylogenies allowed the detection of a wide range of different HGTs (e. g. , horizontal transfers involving other algae with secondary chloroplasts) (Fig. 1 ), and an improved resolution of the probable time points when each transfer occurred (Figs. 2  and 3 ). Our data underscore how HGTs from a range of different sources have impacted on the evolution of eukaryotic micro-organisms, a concept that has been the subject of extensive debate  (56) (57) (58). Further phylogenetic reconstructions, using denser taxonomic sampling strategies may detect even greater numbers of HGTs in P. tricornutum. We note the specific importance of bacterial HGTs in the recent evolution of diatoms.
3  and SI Appendix, Figs. S4 and S18-S21). Ultimately, our data point to a continuous bombardment of ochrophyte genomes with bacterial DNA, which may have occurred more substantially in diatoms than other ochrophytes. We note that large numbers of bacterial HGTs into the diatom backbone have been recently detected in a parallel study, using a dataset composed of genomic sequences only  (58) , underscoring that this result is unlikely to be artifactual. The discrepancy between the number of bacterial HGTs attributed to early points in diatom evolution (e. g. , >450 potential HGTs occurring in a common ancestor of polar centric and pennate diatoms) (Fig. 2 ) and the number of bacterial genes detectable in extant diatom genomes (e. g. , 295 retrieved in P. tricornutum) (Fig. 1 ) is intriguing.
4  and SI Appendix, Figs. S22  and S25 ). A secretory bias  (7)  has been previously inferred for horizontally acquired genes in the basal stramenopile Blastocystis, and diminished expression levels of horizontally acquired genes (as also inferable for bacterial HGTs compared to algal HGTs in our dataset) has previously been shown in many microbial eukaryotes  (8, 62) , suggesting that these may be general functional features of HGT. It remains to be determined why large numbers of bacterial HGTs have occurred into diatoms and, indeed, how bacterial HGT has facilitated the success of diatoms in the contemporary ocean. We note that diatoms in the wild typically coexist with bacterial exo-and endosymbionts, with which they are particularly dependent for both nutrient uptake  (21, 63, 64) , and which may even attach to diatom frustules  (65).
These interactions may provide a potential route for the horizontal exchanges of genes that facilitate symbiotic interactions. We detect a small number of genes with closest evolutionary affinity to known bacterial symbionts of algae in our data, albeit without obvious quantitative enrichment (Dataset S1). Further studies, with greater taxonomic sampling of ochrophyte lineages known to engage in phagotrophy [e. g. , chrysophytes  (11, 66) ], will be invaluable for confirming whether there is a specifically higher frequency of bacterial HGT into diatoms compared to other ochrophytes. More broadly, it remains to be determined how HGT has allowed diatoms to explore different niches. This may be accomplished via the functional characterization of individual genes-for example, using transformable model species such as P.
Materials and Methods Transcriptome Data. Transcriptome libraries were generated for seven diatom species: These were Guinardia flaccida RCC3088, Haslea sp. RCC2677, Minidiscus sp. RCC1721, Navicula sp. RCC3092, Pleurosigma sp. RCC3090, Shionodiscus bioculatus RCC1991, and Synedra sp. RCC2510 (Datasets S1 and S3). These species were selected based on their high abundances in Tara Oceans metabarcode data  (10, 69). Cultures were grown in f/2 medium enriched with silicate, at a 19 °C constant temperature, in a 12-h 30-μE m -2 s -1 :12-h dark light cycle. Total cellular RNA was harvested from exponential phase cultures using TRIzol reagent (Invivogen) and precipitated in RNase-free isopropanol, as previously described  (5). One microgram RNA, as quantified with a nanodrop, was treated with one U RNase-free DNase (Promega) following the manufacturer's instructions, and repurified with isopropanol.
Each library was sequenced using 101-bp paired-end reads chemistry on a HiSeq 2000 Illumina sequencer and, on average, 25 billion paired-end raw reads were obtained for each sample. Trimming was performed using an in-house software based on the FastX package (hannonlab. cshl. edu/fastx_toolkit/index. html). Raw data were filtered to remove clusters with excessive intensity in bases other than the base called. Adapters and primers were removed, along with all nucleotides after the first nucleotide with quality value lower than 20. Reads shorter than 30 nucleotides after trimming, and reads and their mates that mapped onto run quality-control sequences (PhiX genome) were serially removed. Finally, ribosomal RNA-like reads were excluded using SortMeRNA  (70). Assembly was performed by Genoscope using trinity-v2.
4. 0 (71) under default parameters. Sequencing data are archived in the European Nuclear Archive (ENA) https://www. ebi. ac. uk/ena/data/view/PRJEB34158 under the accession numbers ERR3497271 to ERR3497276. Peptide sequences for de novo transcriptomes sequenced for this study were generated using TransDecoder under default conditions. Peptide sequences for transcriptome data for which translations have not been made publicly available [e. g. , independent chrysophyte transcriptomes (24, 66)] were generated using getorf using -table 0, -minsize 100, -find 1 as default settings. Identification of all Genes of Horizontal Origin in the P. tricornutum Genome. Possible HGTs in the P. tricornutum genome since the radiation of the ochrophytes were manually identified using a pipeline adapted from previous studies  (5, 17, 24).
A list of the categories considered, and the libraries from which they are constructed, is provided in Dataset S1. The FASTA sequence of the single top-hits for each query gene against each reference subcategory, with a threshold e-value of 10 -05 , were extracted and compiled to form a local reference library of up to 151 closest homologs of the query sequence across the tree of life. The query sequence was then searched again by BLASTp against this local reference library, and the best-scoring hits were ranked. Genes that retrieved a best BLASTp hit, excluding ochrophyte orthologs, to a nonphotosynthetic stramenopile category (either oomycetes, labyrinthulomycetes, Blastocystis, or bicosoecids) (Dataset S1), were inferred to have vertical origin. Exactly 8,000 genes, which retrieved a best-scoring hit, excluding all ochrophyte orthologs, to a nonstramenopile lineage were retained for subsequent phylogenetic inspection.
Manual Verification of P. tricornutum HGTs. Alignments were generated for each retained gene using the local reference library previously assembled, MAFFT v7. 271 (74) using the --auto option. These alignments were subsequently refined using the in-built alignment program in GeneIOUS v10. 0. 9 (75) under highly stringent settings (gap open penalty 12, gap extension penalty 3), and were manually curated to remove poorly aligned sequences (defined as sequences with <50% pairwise coverage with the P. tricornutum query). A small number of genes  (21)  were split into separate N-and C-terminal domain alignments, based on the alignment of nonoverlapping sets of reference sequences to different regions of the P. tricornutum query. Alignments for which fewer than 2 nonochrophyte sequences could be aligned to the P.
An exemplar series of tree topologies are provided in SI Appendix, Fig. S1 , and full assignations are tabulated in Dataset S1. All BLAST outputs, alignments, trees, and internal BLAST search outputs are publically accessible from the osf. io library https://osf. io/t52wk/, in the folder "All HGTs into P. tricornutum genome. " Bootstrap replicate files are provided in this folder for trees resolved with the JTT matrix (as annotated above), along with tree reconciliations with LG and WAG matrices, for user exploration. Modified versions of Dataset S1, retaining only HGTs supported by >50% and >80% bootstrap supported, are provided within the same osf. io library, in the folder "Supporting Datasets. " Automatic Annotation of all Bacterial HGTs in Ochrophytes. Bacterial HGTs were detected using an automated pipeline (SI Appendix, Fig.
S3 ) across 162 ochrophyte genomes and transcriptomes (Dataset S3). Transcriptome libraries studied were decontaminated using a previously defined pipeline, comparing the BLAST similarity scores between pairs of transcriptome libraries as described elsewhere  (5, 70, 78). Each ochrophyte protein dataset was clustered at a 90% similarity cutoff with CD-HIT  (79)  to reduce sequence redundancy. The longest protein sequence of each cluster was retained, leaving a total of 2,677,637 nonredundant ochrophyte proteins. These proteins were searched against a modified version of NR GenBank using BLASTP (e-value < 10 -05 ), excluding all ochrophyte sequences and separately clustering bacterial, archaeal, viral, and the remaining eukaryotic sequences at 90% similarity with CD-HIT, and the best 5 overall hits, best 30 hits among nonochrophyte eukaryotes, and the best 30 hits among noneukaryotes were recorded.
Cleaning was performed using the longest ochrophyte sequence in the family as reference. Automated parsing of the resulting trees identified 2,653 multispecies HGTs (i. e. , sequences originating from the same HGT event found in multiple ochrophytes) and 7,426 single-species HGTs (i. e. , sequences originating from an HGT event found in a single ochrophyte). All alignments and trees are publically accessible from the osf. io library https://osf. io/t52wk/, in the folder "Quantification of bacterial HGTs. " Trees for exemplar HGT clusters (SI Appendix, Fig. S7  and Dataset S6) were realized with MrBayes 3. 2. 6 hosted on the CIPRES Science Gateway web portal, using three substitution matrices (GTR, Jones, and WAG)  (84, 85). For each dataset, two runs were performed, each comprising four chains for 600,000 Markhov Chain Monte Carlo generations, sampling at every 5,000 points, with the first 50% used as burn-in for the consensus.
Analyses were carried out using RAxML v8. 1. 17 (77), using three substitution matrices (GTR, JTT, and WAG), with the Γ correction and automatic bootstrapping. Identification of Composite Bacterial/Nonbacterial Genes in HGT Data. Bacterial HGTs identified in the above dataset, with at least one eukaryotic detected homolog, and >70% bootstrap support for the placement of ochrophytes and bacteria, were searched for composite domain origin based on a previously published pipeline (5, 32) using CompositeSearch  (86). Each protein sequence from each bacterial HGT group was added to a previously published library of bacterial, archaeal, viral, plasmid, and unicellular eukaryote genomes  (32) , and used in an all-versus-all BLASTp search with an expect value of 1 × 10 -05 and a minimum identity of 30%.
Sequences were grouped into families if they overlapped by more than 80% of their lengths. Com-positeSearch was used to identify potential composite families and their components, and composite families including sequences from a bacterial HGT group were investigated further. The identity of each domain in a composite gene was assessed by profiling the consensus origin (either bacteria, archaea, or ochrophytes) of the top 10 BLAST hits identified between the ochrophyte protein domain and their components in the library (Dataset S5). All BLAST search outputs are publically accessible from the osf. io library https://osf. io/t52wk/, in the folder "Identification of chimeric sequences. " Construction of a Multigene Reference Tree. The P. tricornutum genome  (17)  was searched by BLASTp against a uniref library (71) from which all lineages that currently or may have historically possessed plastids of secondary or higher endosymbiotic derivation (i.
e. , ochrophytes, cryptomonads, dinoflagellates, haptophytes, euglenids, and plastid-bearing rhizarians and apicomplexans) had been removed  (5) , and in each case the ID and expect value of the top BLAST hit were extracted. Similar BLAST searches were performed using the P. tricornutum query protein sequences against the 162 ochrophyte genomes and transcriptomes inspected for bacterial HGTs (Dataset S3), plus a further 42 reference eukaryotic genomes (Dataset S4). Manual alignments were constructed for 65 genes, for which the top-hit in >90% of the ochrophyte libraries inspected had an expect value that was smaller than the expect value associated with the uniref top-hit sequence, using MAFFT  (74)  and GeneIOUS (75), as above. Guide single-gene trees for each trimmed cluster were inferred using 100 bootstrap replicates of the neighbor-joining tree program built into GeneIOUS  (75).
Ochrophyte sequences that failed to resolve with other ochrophyte sequences were removed from each cluster, and two clusters which retained fewer than 80% occupancy of ochrophyte species were rejected (Dataset S1). Concatenated multigene sequences were built for each species using the 63 remaining clusters, and trimmed with trimAl using the -gt 0. 8 option to remove all residues that corresponded to gaps in >20% of the sequences  (76). Complete multispecies alignments are provided in Dataset S4. Bayesian analyses were performed using MrBayes 3. 2. 6 using two substitution matrices (GTR and WAG), and RAxML v 8 using three matrices (GTR, JTT, and WAG) hosted on the CIPRES Science Gateway web portal  (84, 85) , as above. Long-branch removal was performed by calculating the averaged branch length of each taxon in the concatenated tree, and serially removing the 10 longest diatom, nondiatom ochrophyte, or nonochrophyte branches from the alignment  (87).
Genes inferred to have either been horizontally transferred into or out of ochrophyte genomes through manual phylogenetic annotation of the P. tricornutum genome (Dataset S1), and P. tricornutum sequences identified in automated annotations of ochrophytewide HGTs (Dataset S3), were compared to functional annotations (biological processes, PFAM domains) assembled for the P. tricornutum version 3 genome annotation  (17) ; epigenetic marks and RPKM values for gene expression under replete media assembled from previous studies  (50, 51). Separate enrichment and expression values were calculated for P. tricornutum genes manually identified to be of bacterial origin, and for P. tricornutum genes acquired from a eukaryotic algal group (either cryptomonads, haptophytes, dinoflagellates, red algae, green algae, photosynthetic alveolates, or photosynthetic rhizaria).
Targeting predictions were obtained for all HGTs using ASAFind v2. 0 in conjunction with Signal P v3. 0  (41, 49)  MitoFates, with threshold prediction value 0. 35  (5, 39) ; HECTAR  (40, 97) ; and WolfPSort v 1. 1, taking the consensus prediction using animal, plant and fungi reference models  (42). Gene coregulation values were calculated for P. tricornutum genes in published diatom microarray metastudy data  (53) , and a novel dataset consisting of combat-normalized RNA-seq data from three previously published studies of P. tricornutum gene expression under N, P, and Fe stress  (26, 52, 54, 55). Coregulation values were calculated for bacterial HGTs identified by automated analysis, and attributed from MRCA estimations to have been transferred into P. tricornutum only, or an earlier ancestor of pennate diatoms, pennate and polar centric diatoms, basally divergent diatoms, Khakista and Hypogyristea, or all ochrophytes.
Targeting predictions, and gene coregulation outputs, are publically accessible within the Open Science Framework (OSF) library, https://osf. io/ t52wk/, in the folders "Localization of HGTs" and "Co-regulation of HGTs. " GFP Localization of Exemplar Bacterial HGTs. Ten bacterial HGTs identified in the P. tricornutum genome (SI Appendix, Fig. S4  and Dataset S6) were experimentally localized using full-length C-terminal GFP expression constructs. The full-length cDNA construct of the gene, amplified from total cellular RNA of P. tricornutum strain 1. 86 grown under replete enhanced seawater medium (ESAW) without silicate, in a 19 °C light:dark cycle as defined above, was cloned into a pPhat: extrabright GFP vector using HiFi assembly Mix (New England Biolabs), following the manufacturers' instructions.
Primers used for the GFP ligation are provided in Dataset S6. The complete GFP fusion vector was transformed into 5 × 10 7 late exponential phase P. tricornutum 1. 86 cells, plated on replete 1% agar ESAW plates without silicate, using biolistic transformation, as previously described  (98, 99). Transformant colonies were selected on agar ESAW plates supplemented with 100 μg mL -1 zeocin (New England Biolabs) and verified by Western blotting using anti-GFP antibodies (Agrisera). Expressing lines were visualized using a Leica SP5 (ER-tracked lines) or SP8 (brightfield lines) confocal microscope (Imaging Facility, Institut de Biologie de l'Ecole Normale Supérieure) using previously defined spectra  (5, 24). Where appropriate, cell lines were stained with either 0. 1 μg mL -1 Mito-Tracker Orange, or 0.
io/t52wk/. Each folder within the OSF portal contains a "readme" file detailing its contents; and a global inventory is provided on the portal wiki page. Data Availability. Osf library data have been deposited in the Diatom HGT paper dataset (10. 17605/OSF. IO/T52WK)  (100). Data pertaining to the construction and inspection of the stramenopile multigene tree are provided through the University of Cambridge Apollo Repository https://www. repository. cam. ac. uk/handle/1810/261420  (101). Sequencing data are archived in the ENA, https://www. ebi. ac. uk/ena/data/view/PRJEB34158 (accession nos. ERR3497271-ERR3497276).
Dorrell et al. PNAS | 9 of 12 Phylogenomic fingerprinting of tempo and functions of horizontal gene transfer within ochrophytes https://doi. org/10. 1073/pnas. 2009974118 EVOLUTION Nexus format outputs of the MrBayes trees, Newick format outputs of each RAxML tree, and support values obtained for the modified data and a comparison between the topologies obtained and previously published ochrophyte multigene trees (9, 33-35, 89-93) are shown in Dataset S4. Raw data, including single-gene and concatenated alignments and trees, within the Open Science Framework (OSF) library, https://osf. io/t52wk/, in the folder "Multigene trees. " ALE Inference of Bacterial HGT Data. ALE analysis.
ACKNOWLEDGMENTS. We thank  Fuhai Liu ,  Leïla Tirichine , and  Catherine Cantrel  ( Institut de Biologie de l'École Normale Supérieure ) for assistance with the maintenance of diatom cultures;  Guita Niang  and  Arnaud Meng  ( Genoscope ) for assistance with sequence assembly and data management;  Vincent Daubin  and  Bastien Boussau  for helpful discussions about the amalgamated likelihood estimation approach;  Marianne Jaubert  and  Angela Falciatore  ( Université Pierre et Marie Curie ) for assistance with biolistic transformations; and  Eric Bapteste  ( Université Pierre et Marie Curie ) for guidance in the identification of chimeric bacterial/nonbacterial proteins, respectively. R. G. D. was supported by a  European Molecular Biology Organization   Early Career Fellowship  ( ALTF 1124/2014 ) and a  CNRS Momentum Fellowship  (awarded  2019-2021 ).
C. B. acknowledges funding from the  European Research Council  under the European Union's  Horizon 2020 research and innovation programme  (Diatomic; Grant  835067 ),  Agence Nationale de Recherches (ANR)  Grant  ANR-19-CE20-0020  (BrownCut), and the  French Government Investissements d'Avenir programme OCEANOMICS  ( ANR-11-BTBR-0008 ). C. B. and H. M. acknowledge funding from  MEMO LIFE  ( ANR-10-LABX-54 ) and  Paris Sciences and Letters Research University  ( ANR-11-IDEX-0001-02 ). H. M. and B. P. -L. additionally acknowledge a PhD fellowship from the  Ecole Normale Supérieure  (to B. P. -L. ). A. V. and G. B. acknowledge funding from the  Excellence Initiative of Aix-Marseille University ,  A*MIDEX , a  French "Investissements d'Avenir" program  ( ANR-11-IDEX-0001-02 ) and the  MICROBIO-E program.
5%), but 4,786 genes exhibit a TE content in coding regions greater than 0. 25, including 1,512 genes predicted to be TE over the total length of the CDS (Fig. S2 ). Still more broadly, the level of expression is strongly negatively correlated with the within-gene TE content (r 2 =0. 038, p<2. 2x10 -16 , after excluding 709 genes with FPKM>100). Additionally, we generated a genome assembly of a female Orange River white-eye (Z. pallidus) using 10X mate-pair and 72X paired-end reads after cleaning. This assembly is much more fragmented (170,557 scaffolds and scaffold N50 = 375 Kb, Table  S1 ) than the Z. borbonicus. Both Z. borbonicus and Z. pallidus assemblies are available from Figshare repository URL: https://figshare. com/s/122efbec2e3632188674 (see also the data availability section).
MOLECULAR DATING The molecular phylogenetic analyses were aimed at estimating the divergence time of the Zosterops genus. Indeed, even if our focal dataset is composed of only three species, the divergence between Z. pallidus / Z. borbonicus and Z. lateralis represents the first split within the genus except Z. wallacei, i. e. the origin of clade B in  Moyle et al. (2009). As a consequence, our phylogeny (Fig. 3, S3 ) is expected to provide an accurate estimate for the onset of the diversification of the Zosterops lineage. For this molecular dating, we added three species to the 27 species used in the previous analyses and generated gene sequence alignments. Given that these newly added species -namely the silvereye, Orange River white-eye and willow warbler -have no gene models available yet, we used the AGILE pipeline  (Hughes & Teeling 2018 , see method) to obtain orthologous sequences.