dataesr/scientific-paragraphs-categorization huggingface dataset:
, 2014) , adapted for use in Mothur (https://mothur. org/w/ images/a/a4/Sylva. seed_v132. tgz), as the reference database for the 16S rRNA gene sequences. The sequences of plastids and mitochondria, and those not classified in the domain Bacteria (from the 16S rRNA gene sequence library), as well as sequences identified as homologs for hsp65 (groEL, in the database renamed as GROESL; see the hsp65 reference database design section; from the hsp65 sequence library), were discarded. The sequence library was clustered into operational taxonomic units (OTUs) using the Uparse pipeline in Usearch v10. 0. 240 software (OTUs at a 97% cutoff;  Edgar, 2013)  and Mothur (OTUs at a 99% cutoff). Two cutoffs were applied, as the 97% cutoff is used as a standard threshold for estimating bacterial diversity for the 16S rRNA gene.
The alpha diversity indices (richness, Chao-1; evenness, Simpson evenness; and diversity, inverse Simpson) were calculated in Mothur (97% and 99% OTU cutoff). Significant differences among samples were calculated by analysis of variance (ANOVA) with Tukey's post hoc tests (p < 0. 05; for caves or locations in Lascaux Cave), Student's t-tests, and F tests (p < 0. 05; for marked/unmarked areas) in the Past 4. 02 software  (Hammer et al. , 2001). Bray-Curtis distance matrices were calculated to describe differences in bacterial community composition among individual samples. Actinobacteria communities in the caves, in different locations of Lascaux Cave, and between marked and unmarked areas within each location were compared by nonmetric multidimensional scaling (NMDS) according to both gene markers (97% OTU cutoff).
e. , Sas-1, Passage, Apse, and Diaclase) in Lascaux Cave (99% OTU cutoff). Taxonomic classification of OTUs was performed with Mothur using the hsp65 and 16S rRNA gene SILVA V132 reference databases, and species-level identification with the hsp65 marker was verified by BLASTN in the online databases of the National Center for Biotechnology Information (NCBI; US National Library of Medicine). In parallel, a phylogenetic analysis based on hsp65 was performed using retrieved sequences from caves belonging to most representative OTUs and those from the GenBank database after using the nucleotide BLAST (BLASTN) tool. Retrieved sequences from GenBank were those with the highest similarity with selected OTUs. Multiple alignments were generated using ClustalX  (Thompson et al. , 2003). MEGA6 software was used to compute the maximum likelihood of neighbor-joining phylogenetic trees from the observed amino acid sequence divergence (%;  Tamura et al.
8/0. 8 and lower than -0. 35/-0. 5 for the hsp65/16S rRNA gene markers were visualized in Gephi 0. 9. 2. (M. Bastian et al. )  by Fruchterman-Reingold spatialization  (Fruchterman & Reingold, 1991). The average degree, eigenvector centrality, and modularity were computed, and the minimum number of links was filtered to five (using a degree range filter). Figures were plotted using the vegan package  (Oksanen et al. , 2018)  in the R computing environment (R Core  Team, 2018)  and Inkscape (v0. 92; http://www. inkscape. org).
| In silico analysis Differences in the variability of the hsp65 and 16S rRNA gene partial sequences between pairs of Actinobacteria strains at different taxonomical levels (e. g. , intraclass, intraorder, intrafamily, and intragenus) were determined using genomes retrieved from the Integrated Microbial Genomes and Microbiomes database (IMG, University of California, https://img. jgi. doe. gov/). For the analysis, sequences from the available genomes of 47 species from the order Streptomycetales (Streptomyces, Kitakatospora, Table  A2 ), and 58 species from the order Corynebacteriales (family Corynebacteriaceae: Corynebacterium; family Gordoniaceae: Gordonia; family Mycobacteriaceae: Mycobacterium; family Nocardiaceae: Nocardia, Rhodococcus; Table  A2 ) were used. Sequences were aligned and trimmed to the amplified length of both gene markers in BioEdit 7.
2. 5 software  (Hall et al. , 2011). Pairwise distances between sequences were calculated in Mothur v. 1. 39. 5 software  (Schloss et al. , 2009). Plots were created and the coefficient of determination (R 2 ) was calculated in Microsoft Excel (2016). F I G U R E 2 (a) Rarefaction curves for 37 common samples of the hsp65 and 16S rRNA genes at the 97% and 99% OTU cutoffs. The X-axis denotes the number of samples, and the Y-axis denotes the number of OTUs. (b) Pairwise molecular distances between sequences of the hsp65 (Y-axes) and 16S rRNA gene (X-axes) genes among Actinobacteria species from different taxonomical levels with the correlation coefficient (R 2 ) for each equation. OTU, operational taxonomic unit.
| hsp65 reference database design For taxonomic assignment using the hsp65 marker, the reference database of the Actinobacteria hsp65 gene was constructed similarly to that of the rpoB marker by  (Ogier et al. , 2019). Here, we built a new reference library based on the hsp65 gene encompassing both newly obtained and public sequences from the most important Actinobacteria taxa. Part of this new reference library was constructed with Nocardia and Gordonia sequences from all type strains, from strains from clinical collections (supplied by the French Observatory for Nocardiosis, OFN http://ofn. univ-lyon1. fr), and from environmental strains (supplied by CRB-EML http://eml-brc. org/) sequenced by our means for which sequence quality was verified by double sense sequencing. For the Mycobacterium genus, hsp65 sequences were available via the BIBI database (https:// umr5558-bibiserv.
univ-lyon1. fr/lebibi/lebibi. cgi), which is fairly robust as it contains all described Mycobacterium species. The rest of the sequences was collected from the online IMG and NCBI databases using sequences homologous to the 65 kDa heat-shock protein (hsp65) of the reference strain Nocardia asteroides ATCC 14759  (Rodríguez-Nava et al. , 2006)  amplified by the TB11 and TB12 primers  (Telenti et al. , 1993). Paralogs of heat-shock proteins, such as groEL genes from Escherichia coli  (Colaco & MacDougall, 2014; Duchêne et al. , 1994 ; C. M. S. Kumar et al. , 2015) , were identified using maximum likelihood, FastTree 2. 1  (Price et al. , 2010) , and were retained in the reference database (renamed GROESL sequences) as an outgroup that enabled amplified sequences belonging to this variant to be discarded.
This database was named "ACTIhsp65_V1. 0. 0. fas" and is available at https://doi. org/10. 5281/zenodo. 5576073 The hsp65 database contained 198 genera and 1066 unique taxa, the whole represented by 5165 sequences, of which 1782 were denoted as GROESL at the time of the analysis. TB11 and TB12 primer complementarity with sequences from the reference database was evaluated using the OligoAnalyzer 3. 1 tool (http:// www. idtdna. com/calc/analyzer). Percent coverage of primers for Actinobacteria genomes from different classes and orders was determined with primer BLAST using the RefSeq reference genomes (NCBI). Only targets with no mismatches for the last three nucleotides at the 3′ end and no more than four overall mismatches were included.
| Sequence polymorphism of hsp65 versus 16S rRNA genes for cave Actinobacteria The rarefaction curves of 16S rRNA genes for both 97% and 99% cutoffs were more tilted, indicating an accumulation of identical OTUs due to their repeated sampling, which did not increase with a stricter cutoff, in contrast to hsp65 (Figure  2a ). These results are consistent with the in silico analysis of hsp65 and 16S rRNA gene partial sequences obtained from 105 genomes (47 species from the order Streptomycetales and 58 species from the order Corynebacteriales) available on IMG. Pairwise distances between hsp65 sequences, even for closely related species, were higher than those between 16S rRNA genes (Figure  2b ). The hsp65 primers targeted hsp65 sequences from the reference database with lower numbers of mismatches compared with the outgroup (i.
e. , distant homologs renamed in the database as GROESL, GroEL of E. coli, and Chloroflexi members; Table  A3 ). However, the primers only targeted select groups from the class Actinobacteria, as demonstrated in the analysis with representative genomes from online databases of the NCBI (US National Library of Medicine; Table  A4 ). The number of taxa identified at class or lower taxonomic levels is shown in Figure  A1. Data indicated that 20% of the classes, 32% of the orders, 36% of the families, and 25% of the genus-level taxa detected with hsp65 were also recovered through 16S rRNA gene analysis. Although there was a trend for a higher number of taxa uniquely detected by the hsp65 marker at lower taxonomic ranks, contrary to the 16S rRNA gene marker, hsp65 sequences detected a lower number of taxa overall.
, 2012). However, a higher number of higherrank taxa were identified with the 16S rRNA gene compared with the hsp65 marker, which highlights gaps in the hsp65 reference database, preventing the complete taxonomic assignment of sequences. Another reason might be related to primer mismatches. Indeed, in silico analysis revealed that the primers amplified the gene from diverse Actinobacteria genomes; however, targets without perfect homology might be amplified with lower efficiency, leading to the underestimation of some taxa  (Deagle et al. , 2014). Overall, the data indicated that hsp65 is a suitable complement for the 16S rRNA gene marker in high-throughput sequencing methods for Actinobacteria, especially for taxonomic analysis at the species level; however, primer bias may need to be considered for community analyses at higher taxonomic ranks.
Moreover, the hsp65 reference database is biased against representation in nature, due to uneven selection of restricted taxa, especially compared with those available in online databases. The 16S rRNA gene is available to the scientific community for all species because it must be mandatorily supplied as part of the submission. However, hsp65 gene submission is not necessary when describing a new species, explaining the lack of databases. The availability of bacterial genomes is increasing and in the future, many more sequences will be added that will increase the coverage of our database, allowing for more complete detection of taxa within the Actinobacteria. In this study, the combination of hsp65 and 16S rRNA gene markers revealed that Actinobacteria diversity could vary according to anthropization in caves, on both higher (i.
, 2012). Mouflon and Reille are pristine caves with evenly distributed taxa in the Actinobacteria community, possibly as a result of their stable environments without man-made disturbances  (Mammola, 2019). Typical pristine cave taxa that were identified included Gaiellales  (Zhu et al. , 2019)  and Actinobacteria related to the environmental clone MB-A2-108_ge  (De, 2019; B. Zhang et al. , 2019) , according to the 16S rRNA gene marker. High proportions of Streptomyces and unclassified Actinobacteria based on hsp65 data suggest that each marker identifies particular Actinobacteria community members. Less anthropized areas, such as the pristine caves of Reille and Mouflon, and the Diaclase area of Lascaux, yielded a higher proportion of "Actinobacteria unclassified" when using the hsp65 gene compared with the 16S rRNA gene in the same zones.
Thus, these areas host species of families whose hsp65 gene sequences are missing in our database. Species may already be described for which the hsp65 sequence is not available, or they may simply represent new and as yet undescribed species. Therefore, our database, which was conceived to reach the species level, seems to be more suitable to identify sequences derived from anthropized environments. Environmental selection in oligotrophic habitats may promote rapid molecular evolution  (Kuo & Ochman, 2009; Sagova-Mareckova et al. , 2015)  and result in a high number of novel species isolated from caves  (De, 2019). A similar result was also found for the Streptomyces genus  (Hamm et al. , 2017) , whose genomes may undergo a high rate of evolution  (Cheng et al. , 2015). For pristine caves, high proportions of Streptomyces were obtained from the Diaclase, which is an isolated compartment that has been exposed to little human impact within the anthropized Lascaux Cave.
The OTUs associated with this species were very close to the GenBank sequences obtained from a patient with pulmonary coinfection with M. tuberculosis (KF432743. 1) and Madura foot (CP031418. 1; Y. Y. Zhang et al. , 2013). Other opportunistic pathogenic species of Streptomyces genera such as S. albus could also be found, at a low proportion, in Rouffignac cave, which may also be a consequence of human presence  (Martín et al. , 2004). However, pathogenic species were also found in pristine caves, including N. cyriacigeorgica, which is one of the most frequent Nocardia opportunistic pathogenic species in France. The finding in Mouflon represents the first detection of this species in caves. Thus, this species may belong to the autochthonous core microbiome of the caves. The OTUs associated with this species were close to the GenBank sequences obtained from patients (EF127505.
The sequences found in this cave may belong to the less virulent and most preserved phylogroups, unlike phylogroup II (the most virulent of this species), which was not identified here. Other opportunistic pathogens belonging to the Mycobacteriaceae family were also identified in Lascaux Cave. The two most proportionally abundant species, M. lentiflavum and M. algericum, are causal agents of pulmonary infections in humans  (Chida et al. , 2021)  and animals  (Sahraoui et al. , 2011) , respectively. In addition, the presence of these species has been reported in aquatic mediums  (Makovcova et al. , 2015; Moradi et al. , 2019; Mrlik et al. , 2012). OTUs obtained in our study that belonged to these two species were close to the GenBank sequences; one of them was obtained from Pinna nobilis (GenBank submission code MN854410.
The effect of the geological substrate could also be observed when comparing Diaclase and Apse, because these two locations displayed different Actinobacteria communities, especially in unmarked areas, as shown by the network analysis. Relative isolation and distance from the cave entrance may represent efficient filters for alien microorganisms and enable the maintenance of the cave oligotrophic community, typical for unmarked areas  (Cuezva et al. , 2009; Mammola, 2019). Our results showed that the Actinobacteria community, mainly Streptomyces taxa, varied according to the presence of visual marks. Marked areas, where Streptomycetaceae dominated, exhibited much lower diversity compared with unmarked zones, where Pseudonocardiaceae were prevalent. Unfortunately, comparisons of retrieved sequences with those in public databases yield low similarity scores that do not permit species associations to be made; therefore, we cannot hypothesize whether the presence of these OTUs is due to human disturbance.
, 2014; Kominek, 1972; Myronovskyi et al. , 2013; Saberi-Riseh & Moradi-Pour, 2021; Schütze et al. , 2014). Regarding S. fulvissimus and S. niveus, the OTUs belonging to these species were close to the GenBank sequences obtained from torrent muddy soil (CP071044. 1), rhizosphere (CP048397. 1), and deep-sea sediment (CP018047. 1). This seemed to confirm the ability of these species to survive in extreme environments away from human activity. Regarding the S. mirabilis-like grouping, the most proportionally abundant species in Lascaux stains found in this study, OTUs were close to the GenBank sequences obtained from fresh creek bank soil (CP074102. 1). The set of OTUs associated with this group is positioned in a phylogenetic clade with only 60% bootstrap, which suggests that they could represent a new taxon within Actinobacteria communities, producers of stains in Lascaux Cave.
| CONCLUSION Despite the limitations of our database, which yielded a high number of unclassified sequences, the hsp65 gene demonstrated its utility to complement the 16S rRNA gene which cannot resolve species but encompasses a larger part of the actinobacterial community. The use of hsp65 gene allowed us to detect, with a robust bootstrap, several species not previously reported in caves, demonstrating that it can be used reliably to differentiate Actinobacteria species in such environments. We expect these limitations to be addressed with the enrollment of new Actinobacteria sequences. This can be performed    F I G U R E A5 Phylogenetic tree showing the taxonomic diversity and allocation of hsp65 sequences from caves retrieved by a metabarcoding approach. OTUs were used to build this figure.
The evolutionary history was inferred using the neighbor-joining method  (Saitou & Nei, 1987)  and reveals the relationship of sequences from caves obtained in this study with sequences belonging to type and reference strains classified within the Actinobacteria class. The tree is based on the Kimura two-parameter method  (Kimura, 1980)  with the confidence values of the branches determined by bootstrap analyses  (Felsenstein, 1985)  based on 1000 replicates. Only values >80% are shown at the nodes. This analysis involved 116 nucleotide sequences. The scale bar represents 0. 020 substitutions per nucleotide position. Evolutionary analyses were conducted in MEGA 6 (S. Kumar et al. , 2018). OTU, operational taxonomic unit T A B L E A3 Percentage complementarity between the hsp65specific forward (TB11) and reverse (TB12) primers and the hsp65 sequence database (hsp65 sequences and GROESL sequences, paralogs to hsp65) with respect to the number of mismatches Note: Only matches with no mismatches on the last three nucleotides of the 3′ end and no more than four overall mismatches were included.
jejuensis were found in Lascaux and Mouflon compared with N. cummidelens in Rouffignac, Apse, and Reille (Figure A8). N. cyriacigeorgica was only found in Mouflon Cave and N. farcinica only in SAS-1. Both pristine caves presented similar compositions of Actinobacteria communities, especially Gaiellaceae, taxa related to the environmental clones MB-A2-108_fa and IMCC26256_fa (16S rRNA gene marker), and high proportions of unclassified Actinobacteria (hsp65). High proportions of unclassified Actinobacteria were found in Lascaux's Diaclase, as found in the pristine caves (Figure 5). The phylogenetic tree encompassing sequences of the most representative OTUs (99% cutoff) obtained in this study, together with their corresponding closest sequences obtained from GenBank is shown in Figure A5. This analysis revealed that the most proportionally abundant Mycobacterium (M.
algericum and M. lentiflavum), Streptomyces (S. mirabilis-like grouping, S. niveus and S. fulvissimus), Nocardia (N. jejuensis, N. salmonicida/cummidelens clade, N. cyriacigeorgica, and N. farcinica), and Pseudonocardia species are supported by a bootstrap >80%. Less represented species were also identified; for example, M. fluoranthenivorans, M. mucogenicum, and M. stephanolepidis/salmoniphilum clade, M. gallinarum (bootstrap > 80%), S. pratensis, and S. albus/sampsonii clade (bootstrap > 90%); and N. ninae (bootstrap = 98%). Identification of these OTUs according to the hsp65 reference database and corresponding closest sequences obtained from GenBank are listed in the Supplementary Table.
https://doi. org/10. 5281/zenodo. 6312516. 3. 6 | Co-occurrence of Actinobacteria in Lascaux Cave based on hsp65 versus 16S rRNA genes Co-occurrence networks revealed Spearman correlations between the relative abundances of Actinobacteria OTUs in Lascaux Cave (Figure 6), for both Lascaux locations (calculated by LEfSe; Segata et al. , 2011) or marked/unmarked areas (calculated by Metastats; White et al. , 2009). More co-occurrence connections were identified for hsp65 than for the 16S rRNA gene. For the hsp65 marker, no more correlated OTUs were indicated by LEfSe or Metastats, as typical for the Lascaux locations or marked/unmarked areas, respectively. Negative correlations between the OTUs were only identified with the 16S rRNA gene marker. Clusters of correlated OTUs dominated the unmarked areas, which also differed significantly in the Apse and Diaclase based on.
periodically by requesting other public databases, such as BLAST, or by enrolling sequences of previously identified strains isolated from environmental studies. The Actinobacteria community at different spatial levels reflected the natural quality of the caves and different locations within Lascaux Cave. Anthropization was shown to shape Actinobacteria communities, which altered typical cave communities and was exhibited by the Actinobacteria indicator taxa. Even if rare opportunistic pathogens from the Actinobacteria phylum were detected in anthropized caves, it is unknown whether these detected microorganisms represent an important health risk for visitors. We also found that marked areas on the surfaces of Lascaux Cave are important for shaping Actinobacteria communities, with particular reference to Streptomyces species, whose role in the formation of visual marks requires further research.
I G U R E A7 Average proportions of genus Mycobacterium in the Actinobacteria hsp65 gene amplicon sequence libraries in different caves (Rouffignac, Lascaux, Mouflon, and Reille) and different locations within Lascaux Cave (Sas-1, Passage B, Passage IP, Apse, and Diaclase). F I G U R E A8 Average proportions of genus Nocardia in the Actinobacteria hsp65 gene amplicon sequence libraries in different caves (Rouffignac, Lascaux, Mouflon, and Reille) and different locations within Lascaux Cave (Sas-1, Passage B, Passage IP, Apse, and Diaclase). T A B L E A2 Genomes of Actinobacteria reference strains from the orders Streptomycetales and Corynebacteriales retrieved from the Integrated Microbial Genome and Microbiome database and used for in silico analyses: comparison of the hsp65 and 16S rRNA gene partial sequences.
Grant for Cotutelle Studies from the  French Government (Campus France) , and the  Josef Hlavka Foundation and Charles University in Prague. We are grateful for being permitted to sample  Rouffignac Cave  ( F. Plassard ) and  La Reille  (Gaillard family) and to  G. Delorme †  and  Compagnie des Beunes  potholers for help with pristine caves. We thank  S. Géraud ,  J. C. Portais ,  A. Rieu , and  M. Mauriac  ( DRAC Nouvelle Aquitaine ) for providing key information and assistance, and  D. Henry-Lormelle  and her restoration team for providing technical help with Lascaux sampling, and the  Lascaux Scientific Board  for their helpful discussions. We are grateful to  E. Bergeron  and  D. Mouniée  for providing technical assistance with building the hsp65-based reference taxonomic database used in this study, especially with its enlargement by obtaining sequences from strains of different origins.
DATA AVAILABILITY STATEMENT The Illumina MiSeq amplicon sequences have been deposited in the NCBI Sequence Read Archive under BioProject PRJNA695576 (hsp65; https://www. ncbi. nlm. nih. gov/bioproject/PRJNA695576) and PRJNA694921 (16S rRNA gene; https://www. ncbi. nlm. nih. gov/bioproject/PRJNA694921). The Supplementary Table is available in Zenodo at https://doi. org/10. 5281/zenodo. 6312516 (hsp65 OTUs, cutoff 99%-their inferred taxonomic allocations according to the hsp65 database and, for selected OTUs, closest species obtained from GenBank BLAST with percent identity). The hsp65 metabarcoding DNA sequence database for taxonomic allocations using Mothur is available in Zenodo at https://doi. org/10. 5281/zenodo. 5576073. ETHICS STATEMENT None required.