dataesr/scientific-paragraphs-categorization huggingface dataset:
Metabarcoding is offering new insights in diatom diversity as well, shedding light on distribution and biogeographic patterns of species difficult to identify with optical methods  10, 11  and extending the exploration of their diversity over much larger and so far scarcely investigated oceanic areas  12. High throughput sequencing (HTS) of rDNA barcodes is producing a large number of sequence tags that can be used for diatom identification at the genus or even the species level, depending on the marker, thus allowing for an unprecedented detailed appraisal of the species present in a sample or an area, compared with optical methods. At the same time, the objective identification offered by the molecular signature reflects the actual diversity of diatom species far more precisely. Finally, the use of datasets collected and analysed with the same methodology across different geographic areas allows diversity assessment over wide regions and makes comparisons sounder  [13] [14] [15].
In this study we analysed diatom distribution and diversity around European coasts using a complex environmental metabarcode dataset collected within the EU BIODIVERSA project BioMarKs. Barcodes of the hyper-variable V4 region of the 18S rRNA locus were obtained from samples from different size fractions, water column depths and sediments on eight dates at six European coastal sites in the Mediterranean (Blanes and Naples), Atlantic (Gijón and Roscoff), Skagerrak (Oslo) and Black Sea (Varna). Previous studies on the same dataset analysed the overall composition of coastal protist communities  16  , the pattern of distribution of rare species  1  , the diversity of protists in the sediments  17  as well as individual protistan groups or species (e. g. 10, 18 ,). In the present study, we focused on diatoms and addressed a number of questions: What is the overall composition and geographic distribution of diatom communities at the sampling sites? Are there differences between the performances of rDNA and rRNA templates? What diatom species are present in the different size fractions? Which is the relationship between diatom communities in the water column and sediments? The general aim of this effort was to attempt a first definition of the coastal European diatom communities and identify the specific biological features of diatoms emerging from HTS-metabarcoding analyses in comparison with those recognized for the whole protistan community analysed so far.
Results The diatom V4 dataset used for the analyses consisted of 89 samples collected at the six sampling sites, two of which, Oslo and Naples, were sampled in two different seasons. Water column samples (subsurface and DCM) with the respective size fractions (pico, nano and micro-meso plankton) consisted of 73 samples (37 rDNA and 36 rRNA). Sediment samples consisted of eight rDNA and eight rRNA samples. The whole dataset contained 143,036 total diatom reads (25,606 distinct metabarcodes, or unique ribotypes) split in 53,878 rDNA and 89,158 rRNA reads. Details of samples and diatom reads used in our analyses are shown in Supplementary Tables  S1-S3. Overall, diatoms in coastal European waters accounted for about 19% of total protist reads, with their percentage varying among sites and seasons between 0.
Taxonomic analyses. The taxonomic assignment of ribotypes revealed a total of 121 genera (all habitats, size fractions and templates). The majority of ribotypes (82%, corresponding to 91% of the reads) were assigned to reference sequences at similarity level between 98 and 100%, producing a list of 236 species. The remaining ribotypes were assigned to known genera and in 4 cases ribotypes could be assigned only as "araphid pennate" and in 2 cases only to "uncultured diatom". The final assignment produced a list of 340 different taxa (Supplementary Table  S4 ). The most abundant genera in the whole dataset were Leptocylindrus, Chaetoceros and Thalassiosira, which represented 23. 8, 24. 9 and 19. 9% of the total diatom reads, respectively, followed by Skeletonema (14. 6%) and Pseudo-nitzschia (3.
3 ). For each sampling point, the three size fractions were generally dominated by the same genera and even the same species in several cases. Smaller species (e. g. small Chaetoceros in the Oslo sample) were at times more abundant in the nanoplankton size-fraction, but in other cases (e. g. Skeletonema menzelii and Thalassiosira tealata in the Naples samples) they were abundant in the microplankton fraction as well. Overall, the main taxa detected in the metabarcoding dataset matched those seen in light microscopy of the corresponding samples, and the relative abundance of species in the nanoplankton size-fraction was closest to that determined from light microscopy results.
4 ). rRNA and rDNA results for the same samples were generally paired in the cluster analyses. However, the two templates showed marked differences. The 89,158 rRNA reads grouped into almost three times more OTUs at 99% than the 53,878 rDNA reads (3,984 and 1,405 OTUs, respectively). In the analysis with normalized read numbers (Supplementary Table  S5 ), the two templates shared only 17% of OTUs, but these included the large majority of the reads (95. 1%). The high diversity of the rRNA datasets involved a high number of exclusive OTUs (69% of the total OTUs, 4. 2% of the total read number), compared with 14% (0. 7% of the total read number) of OTUs only found in rDNA datasets (Supplementary Table  S5 ). In a comparison based on the taxonomic assignment, more than one third of the named species or genera (122) were only found in the rRNA dataset, while only 9 were exclusively found in the rDNA one.
Discussion This high-throughput sequencing investigation, based on the V4 region of 18S, on plankton and sediment from six European sites provides a first overview of diatom distribution across different coastal areas, revealing shared features but also peculiarities with respect to the whole protistan community. The diatom species detected in large number of reads at European sites in this study are already known to be important in European coastal waters based on classical diatom surveys  [19] [20] [21] [22]. This is not surprising, considering the good correspondence found for the relative abundance of diatom taxa between metabarcoding and light microscopy data  12, 23. The dominance of Leptocylindrus in the whole dataset mainly reflects a bloom of species of this genus, which reached the density of up to 9 × 10 5 cells l -1 during the autumn sampling in the Gulf of Naples.
In fact the autumn 2009 samples from Naples and Oslo (dominated by Chaetoceros) were the richest in reads, covering about 75% of the total diatom dataset. Genera such as Chaetoceros, Thalassiosira and Skeletonema, which were the other most abundant taxa across coastal sites, are also found among the most abundant in offshore areas  12. Interestingly, in the present coastal dataset Chaetoceros consists mostly of the Hyalochaetae subgenus (i. e. , Chaetoceros with thin setae lacking plastids), whereas the Phaeoceros group (species with the plastids also in the thick setae) is much more represented in the prevalently offshore Tara Ocean dataset  12. The dominance of Hyalochaetae versus Phaeoceros in coastal areas is consistent with previous reports  24  and with the distribution of the two subgenera depicted in the OBIS maps (Ocean Biogeographic Information System (OBIS).
The relative rarity of Leptocylindrus in sediment samples compared to surface waters may also look unexpected since some species in this genus do form resting spores  25, 30. However, the most abundant species in the sampling period (and in the whole dataset) was L. aporus 10 , which does not form resting stages. These results highlight the need for regular comparison of benthic and planktonic samples in combination with sediment traps in order to assess the benthic-pelagic coupling along the seasons and the fate of the resting stages raining down on the sediments. Altogether, the close relationship of all samples within individual sites, including different size fractions, habitats and even sampling dates, highlights a marked individuality of each site in terms of diatom communities. This situation is consistent with that reported for freshwater diatom communities  31  , in which a strong prevalence of microevolutionary processes generates distinct regional species pools, similar to what happens for larger organisms.
This situation may derive from the low coverage of reference sequences for the numerous taxa described in this lineage based on morphology, but also from the existence of still undescribed taxa. In either case, this points at many remaining knowledge gaps for diversity even in this group of well-known organisms. Overall, diatoms largely support the view based on the whole protist assemblages that most of the unknown marine diversity is in the benthic realm  17. The high level of assignment of ribotypes to known taxa allows for some considerations on the use of OTUs as a proxy for the number of species. In the whole diatom dataset, the number of OTUs is much higher than that of distinct taxa to which reads were assigned, the two numbers drawing nearer only at an OTU cutoff of 93% sequence similarity (Supplementary Table  S7 ), which is a far too low threshold for diversity analysis.
However, both cell-size distribution and clogging rates may vary across different samples and lead to different outcomes of size fractionation which, in any case, is not informative and hence not advisable in the study of diatoms. The analysis of the BioMarKs diatom dataset provides some new insights in the characterization of microbial assemblages with rDNA and rRNA. The two templates are rarely used in parallel in eukaryotic surveys, although interest in comparing the use of either of the two molecules is growing  40, 41. In our study, the pairing of the two templates for each sample confirms the picture that emerged from the whole protistan community  16  : the main compositional patterns of diatom communities are consistently reflected by the two templates and the community structures obtained with either template in different sites are comparable.
However, while both templates are able to describe in the same way the 95% of the dataset, remarkable differences are evident in the rare component of diversity, which is captured more effectively by rRNA than by rDNA. In particular, high fractions of OTUs are exclusively found in the rRNA whereas a few are exclusively found in the rDNA. The higher diversity exclusive for the rRNA (in terms of OTUs or taxa) is in agreement with previous bacterial and protistan studies that suggest the rRNA-based approach be more effective in the description of the rare but active component of diversity, which is often lost with the rDNA  1, [42] [43] [44] [45]. This is however not true for other studies on bacteria  46  or metazoa from marine sediments  47, 48. Nevertheless, these contrasting results from different surveys are expected and congruent with the well-known complex relationship between rRNA concentrations and differences in life strategies, life histories or metabolic activity among the different taxa  49  , calling for dedicated studies that elucidate variations in rRNA-rDNA relationships at intra-group, intra-specific and also intra-individual level.
In addition, taking into account the considerable amount of time and expertise required for identification, diatom metabarcoding can effectively represent a valid complementary or alternative approach to classical methods for water quality assessment  [51] [52] [53]  , and certainly highly adequate to be translated into automated in situ technologies. Nonetheless, an effort is still required towards taxonomic studies that improve the reference datasets for the annotation of environmental tag sequences. The use of more variable marker sequences, e. g. LSU or even ITS, might allow for a sounder attribution of closely related species in metabarcoding analyses. However, in the case of diatoms, 18S fragments can already resolve taxa at the species level in many genera  10, 38  , which makes more detailed analyses of existing HTS-metabarcoding datasets worthwhile and able to produce interesting insights into the ecology and distribution of diatoms in the world's seas.
Methods Marine samples were collected within the course of the BioMarKs project (http://biomarks. eu/) at 6 coastal sites along the European coast near Blanes (BBMO station, Balearic Sea, Spain), Gijón (Gulf of Biscay, Spain), Naples (LTER-MC station, Gulf of Naples, Tyrrhenian Sea, Italy), Oslo (Skagerrak, Norway), Roscoff (SOMLIT station, Western English Channel, France) and Varna (Black Sea, Bulgaria) (Table  1 ). In Naples and Oslo the sampling was conducted on two dates. For details on sampling protocols, nucleic acid extraction, 454 pyrosequencing of the V4 18S rDNA region and data pre-processing see Massana et al. 16. Briefly, seawater samples were collected along with environmental parameters at two depths of the water column, named Subsurface and DCM in the original dataset and corresponding to ca.
The complete V4 BioMarKs sequence dataset is available at the European Nucleotide Archive under the study accession number PRJEB9133. From the V4 total cleaned BioMarKs protist datasets for two templates, rDNA and rRNA, the dissolved rDNA samples, duplicates and samples from anoxic sediment from Varna were removed and the metabarcodes (or unique ribotypes) assigned to Bacillariophyta were extracted. The initial taxonomic assignation of reads was confirmed and refined performing a local blastn against a custom version of the PR 2 database (https://doi. org/10. 6084/m9. figshare. 5913181) containing also unpublished diatom sequences. A total of 491 reads, corresponding to 0. 24% of the total Bacillariophyceae dataset, had similarity ≤90% and query coverage ≤70% to reference sequences and therefore were removed.
For the cluster analysis, the samples with less than 100 reads were removed and the remaining 61 samples (Supplementary Table  S1 ) were subsampled to 415 reads using the tool 'rrarefy' in the VEGAN R package  57  , and then log transformed, to diminish the effect of the most abundant OTUs. A distance matrix was computed with the Bray-Curtis index, and a dendrogram was constructed using the 'average' method.
Acknowledgements Financial support to the project BioMarKs was provided by the  EU  ( 2008-6530 ,  ERA-net BIODIVERSA, EU ) and, for the Italian participation, by  Stazione Zoologica Anton Dohrn  and the projects FIRB  Biodiversitalia-Approccio multitaxa allo studio delle risposte della biodiversità italiana al cambiamento climatico  ( RBAP10A2T4 ), funded by the  Italian Ministry of Education, University and Research (MIUR) , which also supported  Roberta Piredda 's fellowship. We are grateful to  Genoscope/CEA  for sequencing and to all other participants to BioMarKs for collaboration in sampling at the different sites.
Arhuaco  Adams & Bernard (1977)  is one of the least known genera of Neotropical Satyrinae. It comprises two species and presents an unusual disjunct distribution, with A. ica  Adams & Bernard (1977) , endemic to the isolated Colombian Sierra Nevada de Santa Marta, and A. dryadina  (Schaus 1913)  found in the mountains of Costa Rica and Panama. Here, the female of A. dryadina is described, and a new generic diagnosis is presented. Affinities with other genera of the subtribe Pronophilina, in particular the potential closest relatives, such as Pronophila Doubleday (1849), are investigated based on morphological, molecular, ecological, and behavioral data. Results from molecular and morphological sources are incongruent. Molecular data indicate that Arhuaco is paraphyletic, with A. dryadina segregating within the Pronophila clade.
It contains, as currently recognized, two species: A. ica  Adams & Bernard 1977, endemic  to the isolated northern Colombian Sierra Nevada de Santa Marta, and A. dryadina  (Schaus 1913 ) confined to the Mesoamerican ranges of Talamanca and Meseta Central in Costa Rica and western Panama. The latter species was described originally in the catch-all genus Catargynnis Röber, 1892, later synonymized as a junior synonym of  Pseudomaniola Röber, [1889]   (Adams 1986). The available information on Arhuaco is scanty. Adams and Bernard (1977)  described the genus for the new species Arhuaco ica Adams & Bernard, based on male adult morphology, with reference to head parts, wing shape, androconia, color pattern, and male genitalia. Pyrcz (1999)  examined the collection of Edwin Krüger and noticed that this German collector found both sexes of A.
ica some 50 years before Adams and Bernard, but for some reason did not mention it in any of his papers dedicated to Colombian Satyrinae  (Krüger 1924 (Krüger , 1925)). Pyrcz (op. cit. )  also described the female of A. ica. Catargynnis dryadina was described by  Schaus (1913) , and was later listed by  Gaede (1931)  and illustrated by D  'Abrera (1988). The only comment on the behavior and ecology of this species was by De Vries  (1987). Pyrcz (2004)  transferred C. dryadina to the genus Arhuaco based on similarities in the male adult morphology in comparison with A. ica. Both A. ica and A. dryadina are considered very rare in the field  (Adams & Bernard 1977 , De Vries 1987) , which is reflected in the scarcity of available material in scientific collections. Arhuaco ica is known only from a couple of specimens collected by Edwin  Krüger in 1919 and 1925 , by Michael Adams and George Bernard in 1972 and 1975 , and by C.
Gibson in 1974 (Adams & Bernard 1977) , and recently from two males collected by Carlos Prieto in 2011 and 2013. The only known female is that collected by Krüger. The meagerness of data on A. ica is perhaps not surprising since the Sierra Nevada de Santa Marta, although easily reachable from the coast, has been for many years nearly inaccessible due to political unrest. The scarcity of A. dryadina in scientific collections is, however, less understandable. This species occurs in central Costa Rica in the vicinity of large towns, and in areas reachable by asphalt roads. Moreover, the butterfly fauna of Costa Rica is considered as arguably the best investigated among all Central and South American countries  (De Vries 1987 , 1997 , Chacón & Montero 2007). To date, only the external morphology of the male of A.
dryadina, male and female A. ica, and male genitalia of the latter species has been examined. The female of A. dryadina was unknown prior to this study. No molecular data have been published for either species. Thus, opportunities for more rigorous systematic studies have been extremely limited, and the validity of Arhuaco is based exclusively on a comparison of wing shape and color pattern, in particular the configuration of ventral groundplan elements sensu  Nijhout (1991). In order to more reliably evaluate the monophyly of Arhuaco, therefore, both male and female genitalia were examined and compared with other genera of Pronophilina, and genetic distances were estimated in order to better establish the systematic position of the two species within the subtribe. Arhuaco is also particularly intriguing because of its disjunct distribution, which is unique among the cloud forest members of the Neotropical subtribes Pronophilina and Euptychiina.
Morphology Male and female abdomens of A. ica, A. dryadina, and several other species of Pronophilina, as listed in the "Material examined" section, were removed and soaked in 10% KOH solution for 5-10 min. Subsequently, the abdomens were preliminarily cleaned of soft tissue in water in order to expose genital parts. Female abdomens were stained in chlorazole black to better visualize weakly sclerotized structures. Dissected genitalia were subsequently cleaned in ethanol 90% and 95% solutions. A Nikon digital camera DS-Fi1 and an Olympus SZX9 stereomicroscope were used to take pictures of dissections, and images were then processed in Combine ZP and Corel PHOTO-PAINT X3 programs to enhance focus and improve quality. Genital dissections were kept in glycerol vials pinned under corresponding specimens.
Genital terminology follows largely  Klots (1956)   (Westwood 1851 ) as an outgroup. DNA was isolated using a Sherlock AX (A&A Biotechnology) extraction kit. Amplification of part of the mitochondrial gene COI and the nuclear gene RpS5 was done using the following pairs of primers, respectively: LCO1490 and HCO2198  (Folmer et al 1994) , HybrpS5degF and HybrpS5degR  (Wahlberg and Wheat 2008) , with standard PCR protocol. The PCR fragments of 569 bp (COI) and 526 bp (RpS5) were sequenced using a BigDye Terminator v. 3. 1. Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) and ran on an ABI 3100 Automated Capillary DNA Sequencer. All newly obtained sequences were deposited in GenBank (accession numbers are provided in Table  1 ). Sequences for each marker were aligned in MAFFT version 7  (Katoh & Toh 2008)  using the default settings, and manually checked against non-conservative alignments in BioEdit 5.
0. 0. (Hall 1999). Uncorrected pairwise distances were calculated in MEGA version 7  (Kumar et al 2016). A maximum likelihood (ML) topology for COI, RpS5, and concatenated sequences was constructed using RAxML v8. 0. 19  (Stamatakis 2014). The strength of support for internal nodes of the ML tree was measured using 1000 rapid bootstrap replicates. All trees were visualized in FigTree v. 1. 4. 3 (http://tree. bio. ed. ac. uk/software/figtree).
Molecular data The genetic pairwise distance between analyzed COI sequences ranged from 3. 3 to 13. 9%, and from 1. 2 to 15. 3% between RpS5 gene sequences. The genetic distance between Arhuaco and Pronophila was below 7% (Tables  1, 2 ). Analysis of COI resulted in two sister clades, with one grouping three species of  Pronophila, A. ica, A. dryadina, and Junea dorinda (Felder & Felder, 1862) , and the second clade consisting of other four genera of Pronophilina (  Fig.
In Arhuaco  (Fig 5) , in contrast to Pronophila, the forewing has an acute apex and straight outer margin, the hindwing is subtriangular with a scalloped outer margin with sharp tips at vein ends (in this respect, the wing shape of Arhuaco strongly resembles that of Pseudomaniola, as a result of which A. dryadina has been previously associated with this genus). Importantly, on the hindwing venter, the median band is discontinuous in the discal cell along cross vein Cu A 1-Cu A 2, with its basal edge connected to the postbasal line (Fig  6 ). Additionally, on the forewing underside, there is a row of black submarginal ocelli roughly parallel to the outer margin (in A. ica, the ocelli do have small violet pupils as in Pronophila, whereas in A. dryadina the pupils are white and very large; in A.
Female of Arhuaco dryadina Sexual dimorphism of A. dryadina is slight (Fig  5 ). The female is marginally larger (~2 mm in wingspan) than the male. On the upperside, its brown ground color is a shade lighter, and the white submarginal ocellar elements on the fore and hindwing are larger, bordered with a light brown halo, and on the hindwing additionally ringed with red. The underside ground color is lighter, predominantly sandy yellow, except for the basal half on the forewing which is brown with a light yellowish suffusion. The female genitalia (Fig  8 ) are characterized by an oval bursa with prominent signa, a large membranous pocket enclosing the antrum, and a single slat-like sclerotized postvaginal process.