dataesr/scientific-paragraphs-categorization huggingface dataset:
864. Resulting alignments were curated in BioEdit v. 7. 2. 0. [27]. The phylogenetic signal in the alignment of leptocylindracean sequences was assessed by comparing the skewedness of the treelength distribution of 100,000 random trees calculated under parsimony settings in PAUP (Phylogenetic Analyses Using Parsimony; version 4. 0 and other methods)  [28]  with threshold values for 4-state data in  [29] , given the number of parsimony informative sites and OTUs ( = distinct sequences) in the alignment. A maximum likelihood (ML) approach was used to infer the phylogenetic position of each of the OTUs and GenBank sequences, and to determine which of these resolved within -or as nearest sister to -Leptocylindraceae. Trees were inferred from the three aligned datasets using RAxML-VI-HPC  [30]  on the T-REX web server  [25].
Statistical analyses of the dataset To compare the total diversity across the stations, an OTUbased analysis was performed irrespective of the phylogenetic results using the whole dataset. A consensus taxonomic identity for the 454-V4 and Illumina-V9 sequences was obtained with ggsearch at a consensus confidence threshold of 80  [32]. Sequences belonging to Leptocylindraceae were gathered into a V4 and a V9 dataset. The two datasets were aligned with MSA program, MAFFT v6. 864, using FFT-NS-i on the Trex web server and the obtained alignments were checked manually in BioEdit v. 7. 2. 0. Pairwise similarities among the aligned sequences were calculated using Mothur (http://ww. mothur. org/). Based on the similarity matrix, sequences were clustered into OTUs at different clustering similarities, applying the rule of furthest neighbor and the highest precision (P = 1000).
Rarefaction curves to relate numbers of harvested sequences with number of retrieved OTUs were obtained using Mothur. After removing OTUs that included only one or two sequences, with the remove. rare function, Venn diagrams were constructed to assess the diversity across the sampling sites using Mothur. The abundance of sequences assignable to leptocylindraceans was compared between datasets of different sizes gathered from the same environmental sample, i. e. , V9 versus V4 and cDNA versus DNA datasets, by calculating the expected number of sequences of one species in a sample downsampling each sequence samples to the size of the corresponding sample in the other dataset. The expected abundance, expressed as a range, was computed at a 0. 05 significance level with the statistics proposed by Audic & Claverie  [33].
The datasets An overview of sequence numbers for the BioMarKs datasets is presented in Table  1. The complete BioMarks Illumina V9 dataset (including both DNA and cDNA sequences from all fractions, sites and depths, also including metazoans) contained ca. 130 times more sequences than the 454 V4 dataset, and ca. 70 times more diatom sequences. Rarefaction curves constructed with the total Leptocylindraceae dataset (Figure  S1  A & B) showed V4 OTUs reaching a plateau at a 97% similarity while V9 reached a plateau at 95%. Sequences initially assigned to Leptocylindraceae in the V9 dataset (using a 90% similarity threshold) were 30 times more abundant than in the V4 dataset. However, more than 2/3 of those sequences were removed because they either were more similar to other genera or could not be clearly assigned to Leptocylindraceae (false positives).
Taxonomic analysis The V4 dataset. A total of 51 OTUs were identified among the 51,378 putative leptocylindracean V4 sequences from Bio-MarKs. Following singleton and doubleton removal, 50,718 sequences in 12 OTUs remained (Table  1 ). These sequences were aligned into 412 positions of which 144 were parsimony informative sites. Within the ML phylogeny (Figure  1 , all sequences of nonleptocylindracean taxa pruned away; Figure  S2 , outgroups included) all putative leptocylindracean sequences formed a clade as sister to a clade with all other diatoms, though without bootstrap support. Pennate diatoms formed a clade inside a grade of centric diatoms. In general, topology of the V4 tree resembled that of the SSU rDNA tree (Figure  S3 ; constructed with complete or available information for the SSU rDNA region).
Within the leptocylindraceans, six terminal clades were identified, i. e. , clades that resolved internally into a polytomy. Five of these clades included reference sequences of known Leptocylindraceae species. Sequences in the clade including the reference sequence of L. aporus largely dominated the dataset (Table  1 ). References sequences of L. danicus and L. hargravesii were in the same clade, and the two taxa are considered together in the following, although several NGS V4 sequences were assigned to one or the other of the two species, given the higher uncertainty of these results. The sixth clade, without a reference sequence, included a single BioMarKs' OTU (with four sequences) and 22 GenBank sequences from Baffin Bay (Canada)  [34]. Sequences in this clade, herewith defined as the 'Baffin Bay Clade', differed from those of L.
danicus/hargravesii in 29 substitutions and seven insertiondeletions. The other 24 GenBank sequences belonged to clades including the reference sequences of L. aporus  (10) , L. convexus  (7) , L. danicus/hargravesii (6) and L. minimus (1), respectively. The V9 dataset. A total of 844 OTUs were identified among the 1,526,145 putatively leptocylindracean V9 sequences from BioMarKs. Following removal of singletons and doubletons 1,525,527 sequences in 403 OTUs remained (Table  1 ). Alignment of these 403 representative sequences with six sequences from GenBank and the 97 reference sequences of bolidomonads, Leptocylindraceae and other diatoms resulted in a dataset of 224 positions. An initial ML-phylogeny resulted in non-monophyly for Leptocylindraceae as well as non-monophyly for the genus Leptocylindrus.
Following removal of the false positives, the V9 alignment contained 165 OTUs and 188 positions, of which 76 parsimony-informative. The ML V9 phylogeny was inferred from the 165 OTUs, six leptocylindracean sequences from GenBank, and 97 reference sequences of bolidomonads, leptocylindraceans and other diatoms (Figure  2 , all sequences of non-leptocylindracean taxa pruned away; Figure  S4 , outgroups included). In this phylogeny, the branching pattern lacked any bootstrap support and Leptocylindraceae were not monophyletic, because the clade including T. belgicus was not sister to Leptocylindrus, thus deviating from the phylogeny shown from both the SSU rDNA (Figure  S3 ) and V4 trees (Figure  S2 ). Among the outgroups, neither pennates nor centrics were monophyletic (Figure  S3 ). Nonetheless, five clades could be delineated for Leptocylindraceae, one of which included the V9 reference sequence of L.
danicus and L. hargravesii (indistinguishable in this region) and the remaining four included each one reference sequence of the other known species in the family. Sequences in the clades including the reference sequences of L. danicus-hargravesii and L. aporus dominated the dataset (Table  1 ). GenBank returned six V9 sequences, four of which belonged to the clade including the reference sequence of L. convexus and two to that of L. danicus/hargravesii (Table  S2  in Tables  S1 ). We were unable to identify a clade corresponding to the V4 Baffin Bay Clade because the GenBank sequences from this locality did not include the V9 region.
Surface plankton samples usually contained a higher proportion of leptocylindracean V4 sequences as well as a higher taxonomic diversity than plankton samples taken at depth (Figure  S6  A-C, Table  S3  in Tables  S1 ). The V9 dataset, which was mainly obtained from cDNA templates from surface samples and cDNA and DNA templates from sediments, showed variable abundance in the three plankton size-fractions (Figure  S8  A-C, Table  S4  in Tables S1), except for sequences attributed to L. minimus and T. belgicus, which were abundant in the 0. 8-3 mm fraction (Figure  S8  E & F, Table  S4  in Tables  S1 ). In both the V4 and the V9 datasets, species detected in sediment samples were in general the same as those recovered in the plankton at the same site, but not all species found in the plankton were retrieved in the sediments at the same site.
Geographic allocation of sequences Clustering of MSA sequences (all size fractions and two depths, cDNA and DNA) into OTUs at 97% showed 187 and 1307 OTUs for the V4 and V9 datasets, respectively (Figure  3 A-C ). Upon removal of singletons and doubletons 150 and 1027 OTUs were retained. Venn diagrams for the V4 dataset showed that the Oslo Fjord and Naples samples shared the highest numbers of OTUs, and also exhibited many site-specific OTUs (Figure  3A ). The samples from Gijon and Blanes contained 7 and 5 OTUs only, which were shared with the samples from Oslo Fjord and Naples (Figure  3A ). Venn diagrams for the V9 data showed that, again, Oslo Fjord and Naples exhibited the highest numbers of OTUs, shared the maximum number of OTUs and contained many sitespecific OTUs (Figure  3B ). Of the 28 OTUs found at Varna, 15 were shared only with Oslo Fjord and 12 with both Naples and Roscoff (Figure  3C ).
V4 sequences belonging to leptocylindracean taxa were observed in the plankton samples of four of the six sites, and in benthos samples of two of the five sites (Figure  4 , Table  S3  in Tables S1, all size fractions, cDNA and DNA). Sequences of individual species at each station were generally uncommon or rare, constituting ,0. 5% of the total BioMarKs sequences (also including metazoans), except for L. aporus, which was abundant in the 2009 plankton sample of Naples. This species provided the bulk of the leptocylindracean sequences, constituting in all ca 25% of the diatom sequences in the overall V4 dataset (Table  1 ). Among the stations, the autumn 2009 plankton samples obtained from Naples and Oslo Fjord and the autumn 2009 sediment sample from Oslo Fjord were the most diverse, with V4 sequences close or identical to reference sequences of four species.
This result may depend on the fact that those samples were sequenced multiple times, and a higher number of sequences probably resulted in a better coverage of the diversity at those sites. Based on the V4 dataset, sequences attributed to L. aporus were present at many stations, while those of L. convexus were found only in the 2009 plankton sample of Naples. Leptocylindrus minimus V4 sequences were also rare and found only in Oslo Fjord samples. Sequences belonging to the Baffin Bay Clade were encountered only in the sediment samples of Oslo Fjord, and even there they were exceedingly rare (two in the 2009 sample, and two in the 2010 sample). With higher sequencing depth, V9 sequences attributable to Leptocylindraceae were present at all six stations, notably also in the plankton and sediment samples from Varna (May 2010) and Roscoff (April 2010) as well as in the sediment sample of Blanes (Figure  5 , Table  S4  in Tables  S1 ), where the V4 dataset did not show any leptocylindracean sequences.
Leptocylindrus convexus was conspicuous among the V9 sequences from plankton samples gathered at Blanes, Naples, Gijon and Varna (all in 2010), but basically absent from the V4 sequences obtained from the same samples or any other sample (Figure  4  & 5 ). The same applies to Tenuicylindrus belgicus, which was relatively abundant in the V9 samples from Blanes plankton samples but was not detected in the V4 dataset at that site (Table  S5  in Tables S1; at ,0. 05 significance). The far larger number of leptocylindracean sequences, or diatom sequences in general, in the V9-versus the V4dataset could not account for this difference in all cases (Table  S5  in Tables S1; at ,0. 05 significance). Like for the V4 dataset, V9 sequences of individual species in each site were rare (,0. 2% of total sequences in respective samples), with the exception of a few samples from Naples and Oslo Fjord, where they attained more than 1.
5%, providing most of the fraction of leptocylindracean sequences (3. 3%) in the total diatom V9 dataset (Table  1 ). Among the stations, the autumn 2009 plankton sample of Oslo Fjord was the most diverse, with representatives of all the known species, while Blanes, Naples, Roscoff and Varna samples were equally diverse, with V9 sequences of four of the five species. V9 sequences attributed to L. aporus and L. danicus/hargravesii were found at all sites, while sequences of the latter species were found in all samples and dominated in several of them. V9 sequences of L. minimus and T. belgicus were less widely distributed, being found only at four sites (Figure  5 ). Sequences belonging to clades including L. aporus, L. convexus and T. belgicus reference sequences were detected in plankton samples obtained when seawater temperatures ranged between 12.
5 and 22. 8uC (Table  S1  in Tables S1). However, they were absent from the June 2010 water column samples from Oslo Fjord, where the temperature was within this range as well. Sequences attributable to T. belgicus were present in the autumn 2009 samples from Naples (22. 8uC) and from Oslo Fjord (15. 5uC). A Blast search in the GenBank database for leptocylindracean sequences retrieved a restricted number of sequences and added only a few geographic locations at which these sequences were recorded (Table  S4  in Tables  S1 ). Sequences of L. aporus were detected in the North Atlantic Ocean, the Western Pacific Ocean and in Sagami Bay, Japan; those belonging to L. convexus were also detected in Sagami Bay, as well as in Monterey Bay and in the Caribbean Sea; those of L. danicus were found in the Caribbean Sea and near Honolulu, Hawaii, and those of L.
Discussion In this study we explored the diversity and distribution of individual species of a recently re-investigated diatom family, the Leptocylindraceae, by tracing their characteristic SSU rDNA sequences in environmental DNA datasets. A high number of NGS reads ascribed to Leptocylindraceae were recovered at six coastal European sites sampled during the BioMarKs project, and several sequences were also retrieved from other locations around the world, using geo-localized sequence data from GenBank. Most leptocylindracean sequences were identical or similar to the reference sequences of the six described species, corroborating the species diversity assessment performed with culture-based methods in the Gulf of Naples (GoN)  [7]. At least one novel group of sequences was identified, which probably belongs to an undescribed Leptocylindrus species.
danicus/ hargravesii -reference sequences of known species. Each of these clades exhibited no further internal phylogenetic or geographic Figure  1. RAxML tree inferred from the alignment of 12 representative V4 sequences of leptocylindracean OTUs from the BioMarKs data, 46 leptocylindracean sequences from GenBank, and 134 reference sequences of bolidomonads, Leptocylindraceae and other diatoms, utilizing the GTRGAMMA base substitution model and Hill Climbing algorithm. Bolidomonas pacifica and B. mediterranea were designated as outgroups. All non-leptocylindracean reference sequences were pruned from the tree following tree construction (see Figure  S1  for tree with outgroups included). Bootstrap values were inferred from 100 distinct alternative runs and values ,50 are deleted. Each OTU is labelled as follows: the first letter denotes the first letter of the genus, the second letter, the first one of the species; the number denotes the cluster number (numbering starts from zero); the number after the underscore denotes the abundance of the OTU.
doi:10. 1371/journal. pone. 0103810. g001 structure and the number of OTUs recovered within each of these clades was low (1 to 4). Notably, the reference sequences of the closely related species L. danicus and L. hargravesii were resolved in different OTUs, supporting the notion that these two OTUs represent different species. Basically, the leptocylindracean diversity in the European-wide BioMarKs V4 dataset corroborates the conclusion in Nanjappa et al. [7]  inferred from the sequence diversity among Neapolitan strains that leptocylindracean species diversity is low. The GenBank V4 sequences from sites in and outside Europe further support this conclusion because all were identical to or a few base pairs different from those already revealed among the BioMarKs sequences. Additionally, the GenBank sequences provided further support to the Baffin Bay Clade, which likely represents an undescribed species.
In the BioMarKs V9 sequences, the 97% cut-off showed massive micro-variation and a far higher number of OTUs than the sequences in the V4 dataset. Moreover, bootstrap support for clades with these OTUs was generally poor or non-existent and neither did they show any geographic structure. Nevertheless, the reference sequences were resolved close to an OTU or a set of OTUs containing high numbers of sequences with an unresolved sprawl of OTUs containing low numbers of sequences. Extensive micro-variability seems to be a characteristic of NGS data, showing up even within individual species  [38]. As a matter of fact, the Sanger SSU rDNA sequencing of clonal cultures from the GoN belonging to each of the known species showed little or no internal variation in the V4 and V9 region  [7]. Yet the documented OTU diversity, especially in the V9 dataset, could reflect a) rare intra-individual or intra-population sequence variation, and/or b) species that are exceedingly rare, inconspicuous or difficult to maintain in culture and are, therefore, unlikely to be detected using cell isolation and cultivation methods, and/or c) sequencing errors.
The higher micro-variation in the V9 dataset could in theory result from the fact that most V9 sequences were generated from cDNA, which requires an extra processing step, namely reverse transcription of rRNA into cDNA. The existence of the closely related pseudo-cryptic species L. danicus and L. hargravesii demonstrates that groups of closely related species do exist in Leptocylindraceae and may escape detection, even using the V4 marker. Therefore, the use of more variable markers in DNA metabarcoding is needed to check our inference that the diversity in the family Leptocylindraceae is low. To the aims of this paper, in further course of the discussion each clade as delineated in Figure  1  and 2 is considered as a species, and if it contains reference sequences of one or more species, then it belongs to those particular species.
Detection of Leptocylindraceae in the BioMarKs datasets At the outset, the whole BioMarKs Illumina V9 dataset for protists was 130 times larger than the corresponding 454 V4 dataset, but the ratio decreased to ca. 92 times when only leptocylindracean sequences were taken into account. This difference could be due to a minor bias of the V9 primers against amplifying leptocylindracean sequences. Neither the V4 nor the V9 region of Leptocylindraceae is markedly longer than those of other stramenopiles, and no inserts have been detected  [7] , rendering amplification bias improbable. A possible explanation for the observed difference could be exclusion of a high number of leptocylindracean V9 sequences (false negatives) due to the relatively low phylogenetic resolution offered by the shorter V9 region.
Despite the lower proportion of leptocylindracean sequences in the V9 as compared to the V4 protist datasets, the Illumina V9 dataset was still much larger than the 454 V4 dataset, allowing the detection of known leptocylindracean species in more plankton and sediment samples from the various stations. In fact assignable V9 sequences were present in samples in which not even a single sequence attributable to the family could be detected in the V4 dataset. In addition, in some samples the high numbers of V9 sequences attributed to species missing in the V4 dataset indicate that those species were abundant enough to be detected also at the sequencing depth of the 454 approach (results drawn within the purview of single replicate but with statistical support). Bias of the V4 primers against amplifying the V4 region in certain leptocylindracean species does not explain the absence of sequences because, at least in silico, all the primers fitted perfectly the target sites in the reference SSU rDNA sequences of all the leptocylindracean species, including those of L.
convexus, which was virtually absent from the V4 dataset. The advantages of the V9 region in terms of species detection should however be weighed against the lower resolution allowed by such a short SSU rDNA region, which for example, cannot distinguish between closely related L. danicus and L. hargravesii. This lower resolution can be an issue in recently diversified diatom genera. Finally, the lower performance of V9 versus V4 in producing reliable distance trees can also constitute a hindrance to the detection of new taxa for which no reference sequences are available yet. In the V4 dataset, which includes both the rDNA and cDNA templates, leptocylindracean sequences are generally more prominent among those generated from cDNA than from rDNA, suggesting that cells were physiologically active during sampling.
However, in the case Figure  2. RAxML tree inferred from the alignment of 165 representative V9 sequences of leptocylindracean OTUs from the BioMarKs data, six leptocylindracean sequences from GenBank, and 96 reference sequences of bolidomonads, leptocylindraceans and other diatoms, utilizing the GTRGAMMA base substitution model and Hill Climbing algorithm. Bolidomonas pacifica and B. mediterranea were designated as outgroups. All non-leptocylindracean sequences were pruned from the tree following tree construction (see Figure  S2  for tree with outgroups included). Bootstrap values were inferred from 100 distinct alternative runs and values ,50 are deleted. OTU labels follow same principle as in Figure  1. doi:10. 1371/journal. pone. 0103810. g002 of Leptocylindraceae, sequences were recovered in each of the size fractions (0.
Geographical distribution Members of the centric diatom family Leptocylindraceae are recognizable in light microscopy. Yet, individual species therein have not been well defined until recently  [7] , and therefore, information about their distribution patterns is missing or unreliable. In several cases electron microscopy and even cultivation and strain sequencing are required to confirm species identity in the pseudo-cryptic species of the family. In this study, the identification of OTUs containing reference sequences of Leptocylindrus and Tenuicylindrus species in environmental DNA sequence datasets provided new information on the distribution of these species at several European and extra-European locations. However, the different markers and the different sequencing methods do have their shortcomings as has been illustrated above.
Based on our BioMarKs results, most species described in Nanjappa et al. [7]  are widely distributed in European Seas, and the exploration of GenBank data has demonstrated their occurrence also in places outside Europe. The most widespread seems to be L. aporus, which was found at all BioMarKs sites as well as -according to GenBank data -along the French Mediterranean coast, in the North Pacific and on the North West Atlantic coast. It was actually in Narragansett Bay (North West Atlantic coasts) that the species was first described (as L. danicus var. apora)  [7, 40]. Leptocylindrus danicus and/or L. hargravesii are also present at all BioMarKs sites, although distribution patterns of these two individual species cannot be explored in the V9 dataset. Based on V4 GenBank sequences, L. danicus might be more widespread, as it was detected along the French Mediterranean coast, the East Pacific and the Gulf of Mexico, while sequences of L.