dataesr/scientific-paragraphs-categorization huggingface dataset:
To control for the multiple comparisons, we tested the differences of bat activity between the solar farms and the other habitats with Dunnett's post-hoc test using the glht function of the 'multcomp' package  (Hothorn et al. , 2008). The boxplots were generated by the ggplot2 package  (Wickham, 2016). Model validation was conducted using the 'DHARMa' package  (Hartig, 2021). We also examined spatial autocorrelation of model residuals using Moran's I statistic 'spdep' package;  (Bivand, 2020)  and found no significant spatial autocorrelation (P > 0. 10). To assess goodness-of-fit, we calculated marginal and conditional R 2 for each model using 'MuMin' R package  (Bartoń, 2020). We present the result of the full model and provide the Akaike information criterion (AIC) for both full and null models.
To explore the habitat use of bats on community level, we also conducted a multivariate exploratory analysis. With the help of the 'vegan' package  (Oksanen et al. , 2016) , we applied non-metric multidimensional scaling (NMDS) using the function metaMDS based on the logarithmic transformed data of the number of the sequences of the species and species groups (9 variables). Five NMDS dimensions were generated for which the calculated stress value was 0. 081, thus indicating good fit. Through this ordination, habitat types can be visualized in low-dimensional space according to their similarity in species composition. We evaluated visually the similarity of bat communities of the different habitats by the overlap of point clouds.
The B. nauphoetae genome was assembled into 879 contigs spanning 88,537,989 bp, with an N50 = 199,589 bp and a GC content of 44. 96%. Automatic and manual gene prediction resulted in 25,221 predicted protein-coding genes. The P. pyriformis genome was assembled into 633 scaffolds spanning 56,627,582 bp, with an N50 = 276,605 bp and a GC content of 60. 94%. Manual and automatic gene prediction resulted in 10,815 predicted protein-coding genes. Using BUSCO v3  [18]  with the eukaryota_odb9 dataset, the genome completeness was estimated to be 76. 3% and 76. 6% for P. pyriformis and B. nauphoetae, respectively (Table  1 ). BUSCO values, despite their wide usage, are not expected to reach 100% in lineages distant from model eukaryotes simply due to the true absence or high sequence divergence of some of the assessed marker genes.
e. , a composite dataset containing three oxymonad species and two other free-living Preaxostyla, the latter clearly possessing an MRO  [5, 17]. We used several methods to identify candidate mitochondrial proteins (results for all searches are shown in S2 File and summarized in Fig 1B  and 1C ). In most cases, proteins identified in the first step were searched against the custom mitochondrial protein sequence database based on the MitoMiner database  [22, 23]  (herein referred to as MitoMiner) and those with hits were considered as candidate mitochondrial proteins. In the last step the functional annotation of the candidates based on NCBI best hits or additional hint, co-occurrence across Preaxostyla, were considered to select the putative mitochondrial proteins from the candidates. For the purpose of the analyses the predicted proteins of Preaxostyla were clustered with predicted proteins of other Metamonada into orthologous group (S3 File).
As the most obvious approach, we searched all five investigated species for homologues of nuclear genome-encoded proteins typically associated with mitochondria or MROs in other eukaryotes including Paratrimastix and Trimastix. In the first step, we used profile Hidden Markov Models (HMMs) to search for components of the mitochondrion protein import and maturation machinery, considered one of the most conserved mitochondrial features  [24]. Homology searches resulted in 22 (T. marina) to 63 (M. exilis) hits, which were further evaluated by searches against the MitoMiner resulting in 57 candidates in total. Functional annotations indicated that all candidates recovered in oxymonad data sets are very likely false positives: mainly vaguely annotated kinases, peptidases, and chaperones (Fig 1B and S2 File).
For P. pyriformis and T. marina, the situation was different. Out of the 11 previously identified components of the P. pyriformis mitochondrion protein import and maturation machinery  [16, 17] , 10 were found in the predicted proteomes of these two species with this approach: the β-barrel outer membrane translocases Tom40 and Sam50 and the associated receptor Tom22, the inner membrane translocases Tim17, Tim22 and their associated protein Pam18 and Tim44, the α and β subunit of the mitochondrial processing peptidase (MPP), and the mitochondrial chaperone protein HSP70. Tim17 and mtHsp70 were also identified in the T. marina dataset, corroborating previous findings  [5]. The only exception was the previously detected Tim23 protein (PAPYR_1418;  [17] ), which was missed in our new search because of its high sequence divergence.
The successful identification of the protein translocon components in these two species validated our approach. The sensitive HMM searches were complemented by an extensive search for putative homologues of known mitochondrial proteins using a pipeline based on the MitoMiner database (S2 File). As already shown for M. exilis, the specificity of the pipeline in organisms with divergent mitochondria is low  [7]. The search recovered a similar number of hits in all investigated species, from 636 in S. strix up to 1,024 in P. pyriformis. An objective assessment of the localization of all these proteins is not realistic. Many of them are clearly not mitochondrial (e. g. , histones or ribosomal proteins), and others belong to very general gene ontology categories (e. g. , protein-binding) making the assessment impossible.
Homology-independent searches do not identify convincing candidates for mitochondrial proteins in the sequenced oxymonad genomes As an alternative to homology searches, we also searched our Preaxostyla sequence datasets for several types of signature sequences typical for mitochondrion-targeted proteins. The matrix proteins of mitochondria and MROs are expected to contain characteristic N-terminal targeting signals (NTS) needed for the targeted import into MROs  [30]. However, it has been previously recognized that the presence of a predicted NTS by itself does not prove the targeting, as such amino acid sequences can also appear by chance  [31]. Indeed, we have previously shown that 14 M. exilis proteins are imported into hydrogenosomes when heterologously expressed in T. vaginalis  [32].
Here we used TargetP v1. 1 and MitoFates v1. 1 for mitochondrial targeting signal prediction and identified from as little as two hits in S. strix up to 248 hits in B. nauphoteae (S2 File). Only 21 of these oxymonad sequences found hits in MitoMiner rendering them candidates which were considered further and annotated by BLAST searches against the NCBI nr database (Fig  1B ). None of these annotations strongly suggest a mitochondrial function. As an additional hint, we considered the conservation of NTS prediction across Preaxostyla. We reasoned that if the NTS in the protein is real it should be detected also in orthologues from other Preaxostyla species. Only four proteins from oxymonads (ribosomal protein L21, L23a, L34e, and tRNA-dihydrouridine synthase) fulfilled this criterion, none of them representing a reasonable mitochondrial protein in organisms that certainly lack a mitochondrial genome.
Depending on the species only five to 12 were considered as mitochondrial candidates (i. e. having hits in the MitoMiner database) (Fig  1B ) and the majority of these were Golgi apparatus/ER-related upon closer scrutiny. Like in the case of NTS-containing proteins, we assessed if the set of predicted TA proteins contains groups of orthologues containing more than a single Preaxostyla species. Indeed, 22 such orthologous groups were found. These were mostly without MitoMiner hits and in most cases annotated as SNARE or other proteins involved in vesicle trafficking outside mitochondria. This suggests that the TA prediction did produce true positives, but they most likely represent non-mitochondrial TA proteins. The only robust mitochondrial candidate from the whole list was Fis1 (mitochondrial fission protein 1) in T.
marina, which is a tailanchored (TA) protein mediating mitochondrial fission  [36]. Our search for proteins with MBOMP characteristics revealed six and 23 hits in T. marina and P. pyriformis, respectively, but only three of them had a hit in the MitoMiner database, including the P. pyriformis Tom40, the detection of which validated our approach (Fig  1B ). Among the hits from the oxymonad genomes, only four hit a protein in the MitoMiner database rendering them mitochondrial candidates, none of them being a known MBOMP (S2 File). Only 10 orthologous groups predicted as MBOMPs contained representatives of more than one Preaxostyla species. The annotations of none of them suggested a mitochondrial function: clathrin heavy chain, kelch-type beta-propeller, EF-hand domain-containing protein, eukaryotic translation initiation factor 3 subunit B, and BTB/POZ domain-containing protein.
Probing the oxymonad genomes with the T. brucei reference mitochondrial proteome provides further support for the absence of mitochondria in oxymonads Given the limitations of the previous searches, namely, high false-positive rates and uncertainty about mitochondrial localization of proteins included in the MitoMiner database, we have performed yet another analysis in which we used a carefully curated set of queries. In this analysis, we searched for Preaxostyla orthologues of proteins from the experimentally wellestablished mitochondrial proteome of Trypanosoma brucei  [37] [38] [39] [40]. The selection of this organism as a reference also considered the fact that it is a member of the eukaryotic supergroup Discoba, which frequently forms a sister group to Metamonada in phylogenomic analyses  [41] , making T.
Amino acid metabolism is richer in Trimastix and Paratrimastix than in oxymonads We noticed clear differences between oxymonads on one side and MRO-containing species T. marina and P. pyriformis on the other with respect to their amino acid metabolism. Hypothetical amino acid metabolism was reconstructed based on the metabolic maps in the KEGG database and catalytic activities of enzymes reported from other metamonads (S1-S3 Figs;  [57, 58] ). The capacity to de novo synthesize protein-building amino acids seems larger in P. pyriformis (cysteine, serine, glycine, threonine, and selenocysteine) than in oxymonads (cysteine, serine, and selenocysteine only). The inferred capacity of T. marina to synthesize only two amino acids (cysteine and selenocysteine) most likely reflects the incompleteness of the sequence data available.
exilis with a single exception of a divergent homologue of Pex19  [8]. This protein acts as a soluble cytoplasmic receptor for peroxisomal tail-anchored (TA) membrane proteins, which are inserted into the peroxisomal membrane with the help of the Pex3 anchor. The absence of a discernible Pex3 homologue in M. exilis indicates that the Pex19 homologue is involved in another cellular process. To map the situation in other Preaxostyla, we searched all datasets for homologues of human peroxins (S8 File). Besides putative homologues of Pex19, which were recovered in all species besides S. strix, and a Pex3 homologue recovered in P. pyriformis, no other peroxins were detected. The Pex3/Pex19 pair in P. pyriformis was already reported by Zı ´tek et al. [17] , who provided proteomic evidence for Pex3 being localized in the MRO and hypothesized that the Pex3/Pex19 system is involved in the targeting of TA proteins into the outer MRO membrane.
Altogether, the results of our searches do not indicate the presence of peroxisomes in Preaxostyla lineage. Thus, the apparent absence of Pex3 in oxymonads seems to be another trait linked to the loss of the mitochondrion in these organisms. The modification of mitochondria for life in low-oxygen environments typically leads to the loss of the capacity for the synthesis of heme  [78] , a cofactor of many proteins involved in redox reactions, namely the complexes of the respiratory chain. Preaxostyla are no exception in this respect as no homologues of these enzymes were identified in their datasets. Still, cytochrome b5 domain-containing proteins were recovered in all Preaxostyla (S6 File) indicating that they use hemoproteins and likely sequester heme from food.
This finding was most consistent with the cisternal adhesion model. Nonetheless, the organisms sampled with divergent Golgi morphologies were still quite distantly related and thus the evidence supporting the model was still relatively weak. By contrast, our dataset represents one of the tightest samplings to date where genomic data is available for the closest known species on both sides of the divide between stacked and unstacked Golgi morphology. Consequently, we searched in our datasets to assess the Golgi complement in the additional Preaxostyla representatives, particularly to see whether the complement was more extensive in the organisms possessing stacked Golgi bodies. The scope of our examination included proteins involved in vesicle formation, vesicle fusion, and the golgin proteins implicated in Golgi structure  [87] [88] [89] [90] [91] [92].
Genome assembly binning and decontamination The quality of sequencing data was assessed with FastQC (Babraham Bioinformatics, USA). For the Illumina data, adapter and quality trimming were performed using Trimmomatic 0. 36  [96] , with a quality threshold of 15. For the Nanopore data, trimming and removal of chimeric reads were performed using Porechop v0. 2. 3 (github. com/rrwick/Porechop). The initial assembly of the genomes was made only with the Nanopore and PacBio reads using Canu v1. 7. 1 assembler  [97] , with corMinCoverage and corOutCoverage set to zero and 100,000 respectively. After assembly, the data was binned using tetraESOM  [98]. The resulting eukaryotic bins were also checked using a combination of BLASTn and a scoring strategy based on the identity and coverage of the scaffold as described in Treitli et al.
The genome completeness for each genome was estimated using BUSCO v3 with the Eukaryota odb9 dataset and the genome completeness was estimated on the sets of EVM-polished protein sequences as the input. Reference-independent genome completeness estimation was performed using Merqury  [19]  with the Illumina decontaminated reads. To avoid bias in the results, the read decontamination was performed by removing only those reads that mapped to the contaminants identified during genome decontamination. Automatic functional annotation of the predicted genes was performed using the KEGG Automatic Annotation Server  [107] , in parallel to similarity searches against NCBI nr protein database using BLASTp. If both gave functional annotation, BLAST was preferred if the evalue was lower than 10 -30. Otherwise KEGG was used.
Targeted analyses of genes and gene families of specific interest were performed by manual searches of the predicted proteomes using BLASTp and HMMER [108], and complemented by tBLASTn searches of the genome and transcriptome assemblies to check for the presence of individual genes of interest that were potentially missed in the predicted protein sets (single digits of cases per set). Gene models were manually refined for genes of interest when necessary and possible. The annotated genomes were deposited in NCBI under accession number JAPMOS000000000 for P. pyriformis and JARBJD000000000 for B. nauphoetae.
Orthology grouping We combined the gene inventories of five Preaxostyla species (the two genomes presented here, the M. exilis genome GiardiaDB-46_MexilisPA203 in GiardiaDB release 46, the S. strix genome under NCBI BioProject PRJNA524138, and the T. marina transcriptome-derived predicted proteome in EukProt EP00771, [109]) with 14 other metamonads for which the genome or transcriptome drafts were available when the study was initiated (Sheet A in S3 File). To optimize the inflation parameter value of OrthoMCL, we performed multiple clustering runs with different inflation parameter values in the range 1. 01 to 30. 0 and calculated the number of OGs containing genes from all Preaxostyla but no other taxa and from all oxymonads but no other taxa. We chose to proceed with the value of inflation parameter = 4, because under this setting the sum of these two numbers was the highest, so the strength of the clustering should be optimal for our purpose.
Under this setting, the complete set of proteins from the 19 species (337,300 items) was assigned to 37,923 groups of genes or singletons (OGs) (Sheet B in S3 File). Proteins belonging to these OGs were automatically annotated using BLASTp searches against the NCBI nr protein database to acquire functional annotation (Sheet C in S3 File). Venn diagrams were generated using InteractiVenn  [110].
Search for mitochondrial proteins The comprehensive search for putative mitochondrial protein was performed for all five Preaxostyla species, including the previously analyzed M. exilis  [7]  as a control. The general design of the search followed the previously described methodology  [7]. Briefly, a custom mitochondrial protein sequence database was compiled using the MitoMiner v4. 0 database  [22, 23]  as the core and supplemented with MROs proteins from sixteen different organisms  [5, 50, 51, 63, [111] [112] [113] [114] [115] [116]. Redundant homologues (90% similarity threshold) were removed from the database using CD-HIT. The resulting non-redundant database, herein referred to as MitoMiner, contained 6,979 proteins. Most searches were divided in three phases. 1) hits generated by HMM searches, predictions of mitochondrial targeting sequence, predictions of transmembrane domains of TA proteins or predictions of mitochondrial β-barrel outer membrane proteins, were in (2) narrowed down to candidates, proteins with hits in the MitoMiner database.
3) All candidates were BLAST-searched against the NCBI nr database and the best hits with the descriptions not including the terms 'low quality protein', 'hypothetical', 'unknown', etc. were kept. Gene Ontology categories were assigned using InterProScan-5. 36-75. 0. If the annotations received from BLAST or InterProScan corresponded to the originally suggested reliably mitochondrial function, the candidates were considered as mitochondrial proteins. HMM searches using HMMER 3. 1b2 and profile HMMs previously employed by Karnkowska et al. [7]  were performed to specifically identify proteins involved in mitochondrial protein import and translocation, as these were shown to be often divergent  [51]. Mitochondrial targeting signals were detected using TargetP v1. 1 [117]  and MitoFates v1. 1 [118].
Significant hits, with at least 25% of the sequence predicted to form β-strands, no more than 10% assumed by an α-helical structure, and no more than 50% of the eight residues of the β-signal predicted as an α-helical structure, were analyzed further. On the top of the three-phase searches, we have also performed one extensive but low specificity homology search in which reciprocal BLAST analysis using the MitoMiner database was performed for each predicted proteome of Preaxostyla species with an e-value threshold of 0. 001. Functional annotation of the reciprocal hits was assessed by NCBI BLAST and items were inspected for cases of reliable mitochondrial proteins.
Trypanosoma brucei mitoproteome-guided comparative analyses Predicted proteomes of T. marina, P. pyriformis, S. strix, B. nauphoetae, and M. exilis were individually reverse BLAST-searched against the proteome of T. brucei (downloaded from Tritrypdb. org; November 2019; [122]). Only reciprocal best BLAST hits that were identified in any of the previously published mitochondrial proteomes of T. brucei  [37, 39]  were subjected to further phylogenetic analysis. For each such protein, the data set for the tree construction was composed of hits from a custom BLAST database of selected protist proteomes (downloaded from UniProt, November 2019; [123]). Protein sequence sets were automatically aligned with MAFFT v7. 453 using the L-INS-i refinement and a maximum of 1,000 iterations, followed by trimming of sites with >70% gaps.
Single gene phylogenies The phylogenies of the genes of interest were analyzed individually using the methodology described below. Eukaryotic homologues of the Preaxostyla genes were gathered by taxonomically constrained iterative BLAST search against publicly available sequence databases in order to sample as broad eukaryotic diversity as possible. In the cases of overrepresented taxa of low interest (e. g. Metazoa, land plants), only a small number of representative sequences were selected arbitrarily. In order to detect potential LGT from prokaryotes while keeping the number of included sequences manageable, prokaryotic homologues were gathered by a BLASTp search with each eukaryotic sequence against the NCBI nr database with an e-value cutoff of 10 -10 and max. 10 target sequences. The sequences were aligned using MAFFT and automatically trimmed using trimAl v1.
marina the transcriptome assembly was first translated in all six open reading frames using the ab initio gene prediction program GeneMarkS-T using the default parameters [135] and the longest resulting predicted protein sequences were used for the searches. Forward hits meeting an e-value cutoff of 0. 05 were subject to reciprocal BLASTp analyses against the Homo sapiens and M. exilis predicted proteomes as well as the NCBI nr database. Any absent components were also subject to additional tBLASTn searches in the nucleotide scaffolds. Hits were deemed positive if both forward hits and at least two of three reciprocal BLAST results retrieved the correct orthologue with an evalue cutoff of 0. 05. Paralogous gene families were subject to further phylogenetic analyses. Phylogenetic analyses were undertaken for the large, medium, and small subunits from identified AP1-4 and CopI families, as well as Syntaxin16 and Syntaxin5.
Resolved backbone alignments from the dataset curated for Karnkowska et al. [8]  were used and sequences from T. marina, P. pyriformis, B. nauphoetae, and S. strix were iteratively aligned with the backbone alignment using the profile option available through MUSCLE v3. 8. 31  [136]. All alignments were subsequently inspected and manually trimmed using Mesquite v. 3. 5 (mesquiteproject. org) to remove heterogeneous regions lacking obvious homology and partial or misaligned sequences. Maximum likelihood analyses were carried out using RAxML-HPC2 on XSEDE v. 8. 2. 10 for non-parametric bootstrap replicates  [137]. The best protein matrix model for RAxML was tested using ProtTest v. 3. 4. 2 [138] , set to account for Gamma rate variation (+G), invariant sites (+I), and observed frequency of amino acids (+F) with default tree and a consensus tree was obtained using the Consense package, available through the Phylip v.
Fe-S cluster containing proteins were predicted with MetalPredator webserver [125]. Predicted proteins were functionally annotated by BLAST searches against the NCBI nr database and by InterProScan against the InterPro database [126]. KEGG categories were assigned by Ghost-KOALA searches against the KEGG database [127]. Orthologous groups were created with the OrthoMCL software [128] and the Venn diagram of OG sharing among Preaxostyla was visualized using InteractiVenn.