dataesr/scientific-paragraphs-categorization huggingface dataset:
The optimal number of clusters was identified using the silhouette method in the dendextend package in R, and the dendrogram was subsequently cut into clusters based on groups of traits which exhibited consistently high average intra-group correlation. As an alternate measure of trait correlation which accounts for phylogenetic relatedness, we calculated phylogenetic independent contrasts 97 (PIC) on species-level average trait values using the ape package. PIC adjusts for the nonindependence of species due to their shared evolutionary history, and allows us to remove the effects of pseudo-replication (i. e. among closely related species within a genus) when calculating correlations. The corresponding correlations among these contrasts are shown in the bottom triangle of the correlation matrix in Fig.
4a. Species-level phylogenetic conservatism was calculated using the empirically measured (non-imputed) trait values via Pagel's λ  98, 99  which quantifies the extent to which trait correlations among species can be explained by their shared evolutionary history, with a value of one equivalent high phylogenetic signal (under Brownian motion), and a value of zero equivalent to no phylogenetic signal (a star phylogeny). All analyses were conducted in R v. 4. 2. 0, with the exception of the phylogenetic eigenvector calculations, which were obtained using the Arpack package in Julia v. 1. 6. 2. Open Access This article is licensed under a Creative Commons Attribution 4. 0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
Acknowledgements This work was primarily funded by the  Swiss National Science Foundation ,  Ambizione  grant # PZ00P3_193612  to D. S. M. Additional support was provided by  D. O. B. Ecology  to T. W. C. ;  SNSF Ambizione  grant # PZ00P3_193646  to C. M. Z. ;  SNSF Ambizione  grant #  PZ00P3_179900  to C. A. ;  U. S. National Science Foundation Graduate Research Fellowship and Embassy of Switzerland in the USA ThinkSwiss Research Fellowship  to G. R. S. and  UNAM-PAPIIT  grant # IN210220  to J. A. R. We thank  Ángel de Frutos ,  Gabriela Lopez-Gonzalez ,  Owen Atkin  and  Yongfu Chai  for sharing data, as well as the TRY database managers and numerous researchers who contributed open-access data.
Data availability The imputed trait data used in this study have been deposited in a dedicated GitHub repository with https://doi. org/10. 5281/zenodo. 6564051 (https://github. com/dsmaynard/ tree_traits), along with the cleaned phylogenetic eigenvectors from the seed plant phylogeny of Smith & Brown (2018) and corresponding tree species names matched using BGCI GlobalTreeSearch database v1. 3. The raw TRY trait data are not available due to data privacy and sharing restrictions, but can be obtained by making a data request to the TRY Plant Trait Database (https://www. try-db. org/TryWeb/).
In addition, DLGAP4 has a pLI score of 0. 992 in the Genome Aggregation Database (gno-mAD), suggesting that the gene is highly intolerant to loss of function mutations. In order to assess if the P616-3 mutation affects the overall predicted 3D structure and function, we took advantage of the I-TASSER iterative method  30. As well as the abnormal Cterminus, this method predicts changes in the overall structure of the protein (Supplementary Fig. 1a , DLGAP4 mutant). Thus, the seven nucleotide insertion affecting the C-terminus of DLGAP4 in patient P616-3 is predicted to lead to multiple changes and to impact overall protein function  31. Other cortical development gene mutations e. g. , in DCX, have also been shown to give rise to subcortical heterotopia or reduced cortical folding problems such as pachygyria, depending on the patient  6.
Immunoreactive bands were detected employing an infrared fluorescence system (Odyssey LiCor), similar as described in the western blot section. Quantitative changes in protein levels were evaluated with ImageJ software (NIH). Neuronal progenitor primary cultures. Neuronal progenitor primary cultures were generated from E12. 5 cortices maintained in a B27/N2 medium 86 containing a mixture (1:4) of Neurobasal/B27 medium without vitamin A and DMEM/F12 with Glutamax, supplemented with N2, 0. 1 mM nonessential amino acids, 1 mM sodium pyruvate, 500 μg/ml BSA, 0. 1 mM 2-mercaptoethanol and l00 μg/ml Primocin (Invivogen). Cells were placed on 14 mm poly-L-lysine (Sigma Aldrich) and laminin (Gibco) treated coverslips. After 24 h in culture, cells were transfected with Lipofectamine 2000 (Thermo Fisher) and 0.
Brain slices were extensively washed in blocking solution and incubated for 2 h at RT with secondary antibodies anti-mouse or anti-chicken Alexa 488, anti-rabbit Alexa 568, and antirabbit Alexa 633 (1:1000, ThermoFisher) and Hoechst (1:1000, ThermoFisher). Fluorescently labeled sections were imaged with confocal microscopes (Olympus Fluoview FV10i or Leica SP5) equipped with 10×, 20×, and 40× oil Plan-Neofluar and 63× oil Plan-Apochromat objectives. Negative controls were assessed in the absence of primary antibodies. Each individual image was taken every 0. 3-0. 5 µm, and a stack projection 10 µm thick was use for all the immunohistochemistry analyses. To obtain the whole z-stack data set, we used the "Z-projection" mode from ImageJ. Peptide competition assay. Briefly, two consecutive 70 μm brain slices from E14.
Dlgap4 RNA interference constructs and validation of shRNAs by RT-qPCR. Total RNA samples were extracted from Neuro2A cell cultures transfected with 5 µg DNA (pCAGMIR30-ShRNADlgap4) (Broad Institute, Supplementary Table  4 ) or a scrambled version (pCAGMIR30-ShRNA Ctl. ) designed with the siRNA Wizard Software (Invivogen). The ShRNA for Dlgap4 and the scrambled sequences were cloned in the pCAGMIR30 vector  87. Cultures were performed in quadruplicate. Real-time qPCRs were performed using the SYBR green method on total, RQ1 DNase-treated RNA samples, following MIQE guidelines  88. First-strand cDNA was synthesized using 50 ng/μl of total RNA, oligo (dT) and the Superscript III Reverse Transcriptase kit (Invitrogen). Gene-specific primers were designed using Primer Blast (NIH) from NM_146128.
In Dcx-DLGAP4 overexpression experiments, pDcx-DLGAP4WT-Flagires-GFP or pDcx-DLGAP4MUT-Flag-ires-GFP were each co-electroporated with 1:1 pDcx-GFP  89. The embryos were placed back in the abdominal cavity and development was allowed to continue until E15. 5, E16. 5, E18. 5, P0, or P8. Pregnant mice were sacrificed and the embryos were collected in cold dissecting medium (MEM, Gibco, with 15 mM glucose and 10 mM Tris, pH 7. 4) and brains were fixed in PFA 4%. Cell counting and quantifications. After immunohistochemistry experiments, z stacks were acquired for each coronal section in a multitrack mode, avoiding crosstalk artifacts for the fluorochromes. All z stacks and image processing were performed with ImageJ 1. 51 software (NIH). In general, image stacks contained between 25-40 confocal planes.
Counting was performed by assigning cells in 10 bins across the cortical wall (VZ, SVZ, IZ, CP) and relativizing the number of each bin to the total count. In % analyses, average control (ShmiRNACtl or in rescue experiments, ShmiRNACtl + Flag-empty) values were defined as 100% and the percentage of each individual value was established as relative to the average number. Each experimental condition was relativized to the control. In proliferation experiments, raw data expressed as % was established by counting the number of Ki67 + BrdU + Blbp-GFP + or Ki67 -BrdU + Blbp-GFP + cells and dividing them by the total number of BrdU + Blbp-GFP +. Labeled cells were counted in a Region Of Interest (ROI), a 100 or 200-μm-wide and 10-μm-deep stripe across the cortical wall divided by 10 bins. For shRNA and rescue experiments, Blbp-GFP + cells were counted in an ROI in sections of 70 μm, distinguishing cells restricted to or outside the VZ.
In all experiments, the total electroporated cells counted in each ROI ranged between 75 and 250 cells. Only the brains with <75 electroporated cells were excluded from data analysis. To assay spindle orientations, cortical sections were analyzed to determine anaphase-shaped nuclei lining the apical membrane after either N-cadherin immunolabeling or cell labeling with 1:200 Phalloidin in PBS (Molecular Probes, Alexa Fluor™ 633), in the VZ of electroporated ShRNACtl or ShRNADlgap4 brains at E15. 5. Anaphase nucleus angles were measured with the Image J software "angle tool". To assay cell morphology, Tbr2 + Blbp-GFP + soma surface were analyzed using the Image J software. BrdU injections and assessment of proliferation. Timed-pregnant mice were injected intraperitoneally with BrdU (BrdU, 99% Bioultra, Sigma-Aldrich, 50 μg per gram body weight in 0.
Sequences were aligned to hg19 by using BWA v. 0. 6. 1, and single nucleotide variants (SNVs) and indels were called by using GATK v. 1. 3. Annotation of variants was performed with GATK Unified Genotyper. All calls with read coverage of ≤2× or a Phredscaled SNP quality score of ≤20 were removed from consideration. The annotation process was based on the latest release of the Ensembl database. Variants were annotated and analyzed using the Polyweb software interface designed by the Bioinformatics platform of University Paris Descartes and Imagine Institute (see also Supplementary Tables  1 2 3 ). Filters used for variant screening were as following: (i) all variants previously observed (in dbSNP138 and/or in in-house projects) were excluded; (ii) only variants leading to abnormal protein sequence (splicing, non-synonymous, frameshift, stop) were retained; (iii) we considered the PolyPhen-2 and SIFT prediction status as informative but not restrictive.
2893T>G, p. Ser965Ala, exon 13; DLGAP1 c. 1397A>G, p. Asp466Gly, exon 8; see Supplementary information for clinical details and Supplementary Table  1 ). Genomic DNA amplifications were performed for DLGAP4 using standard procedures, and PCR products were analyzed by direct sequencing using an ABI3700 DNA analyzer (Applied Biosystems, Foster City, CA). See Supplementary information comparing clinical information of both P616 and P477. Further information are available upon request. En face imaging analysis. Confocal images were acquired with a 0. 17 μm z stack depth and the analyzed images had a total depth of 1 μm. At least three randomlychosen ROIs (246. 27 × 246. 27 μm) were analyzed per embryo (n = 2-3 embryos per condition). Images were analyzed using ImageJ and the software CellProfiler  24.
For pBlbp-GFP electroporations and cell count quantifications, data were subjected to t-test or analysis of variance (ANOVA). Significant main effects were analyzed further by post hoc comparisons of means using Tukey's multiple comparisons test. For cell distribution (or bin) analysis we performed Two-way ANOVA and Tukey's multiple comparisons tests. Pearson's correlation coefficient was calculated from colocalization of pixels images using the JACoP plugin and ImageJ. Pregnant mice/embryos were randomly assigned to the different DNA constructs. When possible, data were collected and analyzed in a blind manner to the experimenter.
Acknowledgements We thank  Dr. X. Morin  (LGN and DLG1 constructs),  Dr. R. Bayliss  ( EML1  constructs),  Dr. F. Watrin  (shRNA empty vector, pCAGMIR30), and  Dr. S. Etienne-Manneville  (Dlgap1 construct). We thank  Dr. A. Baffet  ( RPE1  cells). We are grateful to  S. Haddad ,  C. Chentouf , members of the  Yalcin lab  and  Dr. M. Penisson  for experimental help, and to  Dr. A. Uzquiano  and  Dr. K. Chinnappa  for feedback on the manuscript. We thank members of the lab for comments and discussions. We thank the IFM animal experimentation facility and cellular and tissue imaging platforms at the  Institut du Fer à Moulin , supported also by the  Région Ile de France  and the  FRC Rotary. D. M. R. and F. F. are associated with the  BioPsy Labex  project and the  Ecole des Neurosciences de Paris Ile-de-France (ENP)  network.
Our salaries and lab were supported by  Inserm ,  Centre national de la recherche scientifique (CNRS) ,  Sorbonne University. F. F. 's group obtained the following funding  French Agence National de la Recherche  ( ANR-13-BSV4-0008-01 ;  ANR-16-CE16-0011-03 , to F. F. and N. B. B. ),  Fondation Bettencourt Schueller  (F. F. ), the  European Union  ( EU-HEALTH-2013 , DESIRE, No  60253 , to F. F. and  N. B. B. ) , the  JTC 2015 Neurodevelopmental Disorders  affiliated with the  ANR  (for NEURON8-Full-815-006 STEM-MCD, to F. F. , N. B. ), the  Fondation Maladies Rares/Phenomin  (project  IR4995 , F. F. ), the  European Cooperation on Science and Technology (COST Action   CA16118 , including NBB and F. F. ). This project was also further supported by the  French ANR  under the frame of E-Rare-3, the  ERA-Net for Research on Rare Diseases  ( ERARE18-049 ) and the  Fondation pour la recherche medicale (FRM, Equipe FRM   2020  awarded to F.
Data availability Whole exome sequencing data are available from the authors upon reasonable request. The patient/parent consent does not cover the deposition of the full genetic data in a public database for privacy reasons; however, data is available for academic researchers upon request for academic research purposes and will be provided likely within one month of the request by email (corresponding author: fiona. francis@inserm. fr). The following databases and in silico softwares were used in the study: Human Gene Mutation Databases (http://www. hgmd. cf. ac. uk/ac/introduction. php?lang=english), NHLBI Exome Sequencing Project (ESP), Exome Variant Server (https://evs. gs. washington. edu/EVS/), the single Nucleotide Polymorphism database (http://ftp. ncbi. nih. gov/snp/), genome aggregation database (gnomAD, https://gnomad.
broadinstitute. org), 1000 genomes (https://www. internationalgenome. org/), ExAC Browser (http://exac. broadinstitute. org), PolyQuery (https://polyweb. fr/polyweb/index. html), Polyphen-2 (http://genetics. bwh. harvard. edu/pph2/), Mutation Taster (http://www. mutationtaster. org/), Sorting Intolerant from Tolerant (SIFT, https://sift. bii. a-star. edu. sg/). The genetic variant data generated in this study have been deposited in the dbSNP database under accession codes: rs761228547, rs1371852290, rs752804789, rs138812925, rs1663305011, rs750585274, rs72648943, rs72648278, rs747654057, rs55945684, rs112794616, rs200795874. The original pictures of co-immunoprecipitation and WB membranes and all immunofluorescence quantifications are available in the "Romero_et_al_ncommssource-data. xlsx" file, within the Source Data File.
Gene duplications increase genetic and phenotypic diversity and occur in complex genomic regions that are still difficult to sequence and assemble. PHD Finger Protein 7 (PHF7) acts during spermiogenesis for histone-tohistone protamine exchange and is a determinant of male fertility in Drosophila and the mouse. We aimed to explore and characterise in the chicken genome the expanding family of the numerous orthologues of the unique mouse Phf7 gene (highly expressed in the testis), observing the fact that this information is unclear and/or variable according to the versions of databases. We validated nine primer pairs by in silico PCR for their use in screening the chicken bacterial artificial chromosome (BAC) library to produce BAC-derived probes to detect and localise PHF7-like loci by fluorescence in situ hybridisation (FISH).
In a previous study published in 2019  [14] , we were interested in identifying testis genes conserved between invertebrates and vertebrates and exhibiting high relative mRNA expression in the testis of vertebrates, with a focus on chicken species. Indeed, this study highlighted a substantial list of uncharacterised genes for testis function in vertebrates, in particular in the chicken. Among the chicken testis-specific genes highlighted in this previous study, PHD Finger Protein 7 (Phf7) is indispensable for mouse and Drosophila male fertility  [15, 16] , encodes an actor in histone-to-protamine exchange during spermiogenesis and is highly expressed in the mammalian and chicken testis  [14, 17, 18]. We have been intrigued by the numerous bird orthologues of Phf7 gene  [14] , as shown in its phylogenetic tree of EnsEMBL database (63 paralogues in EnsEMBL release 88 [March 2017]), whereas the gene is present in a single copy in mammals.
Statement of the existence of an expanding family of PHF7 genes in the chicken genome In our 2019 study using data from the EnsEMBL 88 database released in March 2017, we noted that the mouse Phf7 gene (present in a single copy in mammalian genomes) exhibited 111 avian orthologs for only five bird species, as schematised in the trees in Supplemental Fig. 1(A/B ) (see also Supplemental Table  1 ), with 63 homologs in the chicken, and between 2 and 21 in the four other birds (flycatcher: 2; duck: 6; zebra finch: 19; turkey: 21). As seen in the tree in Supplemental Fig. 1B , the 63 predicted chicken genes were phylogenetically regrouped in nine subtrees. We arbitrarily named these subtrees: groups (Gr) A to I, for better clarification and further investigation. According to EnsEMBL 88, these genes are located on nine distinct loci on five chromosomes: Chr19 (GrA), ChrZ (GrB and GrC), Chr1 (GrD, GrE and GrI), Chr2 (GrF and GrH) and Chr12 (GrG).
Surprisingly, we have noticed that the number of chicken PHF7 genes annotated in EnsEMBL has fluctuated (from 0 to 68 genes) according to database versions (Supplemental Table  2 ), with only four orthologues described in the current version (release 105, [December 2021]) (Supplemental Table  2 , column K). Thus, we decided to evaluate precisely the number of PHF7 copies in the chicken genome by in silico PCR and genomic sequencing of bacteria artificial chromosomes (BAC) corresponding to genomic regions identified by fluorescence in situ hybridisation (FISH).
Characterisation of the chicken PHF7 loci by in silico PCR Primers designed in our previous study  [14]  and corresponding to the nine GrA to GrI PHF7 subtrees/groups described above are listed in Supplemental Table  3  (column B). We first verified that these primers amplified unique amplicons with the expected sizes (Supplemental Table  3 , column C) when used in genomic PCR with chicken DNA (Supplemental Fig. 2 ). We then used these primer pairs in in silico PCR (UCSC database). They highlighted nine distinct genomic loci described in Supplemental Table  3  (column D). These loci are on the same chromosome for each respective group of genes as those described in the EnsEMBL 88 release: two loci on Chr19 corresponding to GrA; two loci on ChrZ corresponding to GrB and GrC; three distinct loci on Chr1 corresponding to GrD, GrE, and GrI; two loci on Chr2 corresponding to GrF and GrH; and one locus on Chr12 corresponding to GrG.
Chromosomal localisation of chicken PHF7 loci by FISH and comparison with in silico PCR localisation For the nine groups, BAC clones were selected by PCR with primer pairs characterised above and as described in Materials and methods. We then used these specific BAC clones, listed in Table  1 , in FISH to map the nine groups of genes (GrA to GrI), after we validated the nucleotide sequence of each probe by sequencing (sequences obtained are in Supplemental Table  3 , column E) and submitted them to BLASTN analysis (Supplemental Table  3 , column F). The GrA and GrB sequence probes were associated with an accession number with the name 'PHD finger protein 7_like' (GrA: NC_006106. 5; GrB NC_006127. 5), this is why we use 'PHF7-like' for further designation in the text. The coordinates in BLASTN results were coherent with the loci delivered by in silico PCR (Supplemental Table  3 , column D).
Sequencing, assembling and gene annotation of BAC clones To further characterise the loci identified on the chicken genome by FISH, we sequenced the nine BAC clones targeted by the PHF7 screen (sequences are available in Genbank 1 ). The sequences contained between 87,395 bases for the smallest BAC (E) to 177,594 for the largest (I) (Table  2 ). For each of the nine nucleotide sequences, we performed BLASTN by using the NCBI database (GCRg7w -white Leghorn raceversion 106). The BLASTN match result was unique for each BAC, except for BAC G that first matched with Chr12 for almost its entire length and then with ChrZ for only 19% of its length (see Supplemental Table  3 , columns H and I). As excepted, the loci defined by these coordinates included the loci identified by in silico PCR for all BAC.
We noted that BAC E and BAC D, which both hybridised on GGA1p26 in FISH as stated above, are 2. 5 Mb apart and thus define two distinct PHF7-like loci on Chr1. Finally, these results confirm the chromosomal localisation and coordinates described above for the nine BAC as well as the existence of a tenth locus on ChrZ (GGAZ p21-22 corresponding to BAC G) that was not predicted by in silico PCR. We performed gene annotation of the nine sequences by using the FGENESH program (in Softberry) as described in the Materials and methods. We identified 116 predicted genes dispatched on the nine BAC, from 4 for BAC F to 28 for BAC I (Table  2 ). Details of each BAC annotation FGENESH outputs are listed in Supplemental Table  4. We submitted the corresponding 116 peptides to BLASTP to determine functional homology.
We retrieved a name and accession number for each of the 116 peptides (Supplemental Table  5 ). The annotation of each BAC is illustrated by gene maps in Fig. 2 (BAC I ) and Supplemental Fig. 3  (BAC A to H). There were a total of 39 PHF7-like proteins (in yellow) from 1 in BAC C, BAC F and BAC H to 10 in BAC I (see Supplemental Table  5  and Table 2 ). The BLASTP results for these 39 PHF7-like proteins (sequences in Supplemental Table  6 ) corresponded to an E-value equal or very close to zero (using 'by default' parameters) (Supplemental Table  5 ). Based on using ESPript for obtention of a consensus sequence, their sequence similarity was 30%-55%, with a consensus sequence of 265 amino acids (Supplemental Data; see also the phylogenetic tree Fig. 3 ).
Other duplicate gene families in the PHF7-like loci In each BAC, we noticed several groups of genes for which BLASTP results gave the same name and/or Genbank ID (Supplemental Table  5 , Fig. 2  and Supplemental Fig. 3 ), suggesting that they are phylogenetically related and are members of the same family. There are 1) families with two or three members: ras GTPase-activating protein 1 in BAC B, Cadherin-18 in BAC F, scm-like with four MBT domains protein 2 in BAC Fig. 1. (A) Fluorescence in situ hybridisation (FISH) localisations (white arrows) of bacterial artificial chromosome (BAC) clones (red signals) screened for groups of genes (GrA to GrI) based on the international standard of the chicken karyotype  [32]. Green signals are for microchromosome detection (GrA and GrG). GrA (A), WAG-038I15 (red) together with WAG-062P02 (green) on GGA19; GrB (B), WAG-119F08 on GGAZq12-13; GrC (C), WAG-041O23 on GGAZp12-21; GrD (D), WAG-023G20 on GGA1p26; GrE (E), WAG-038A02 on GGA1p26; GrF (F), WAG-037B06 on GGA2q11-21; GrG (G), WAG-035C03 on GGAZp21-22 and together with WAG-033 L02 (green) on GGA12; GrH (H), WAG-034E06 on GGA2p11-12; GrI (I), WAG-119 J04 on GGA1q35-41.