Jazmin Blaz

Javier Luis

Aaron A Galindo

Harpreet Heiss

Guifré Kaur

Ashley Torruella

L Alexa Yang

Alexander Thompson

Sally Filbert

Apurva Warring

Narechania

Luis Javier Galindo

Aaron A Heiss

Harpreet Kaur

Guifré Torruella

Ashley Yang

L Alexa Thompson

Filbert

Sally Warring

Shiratori

Ken-Ichiro Ishid

Joel B Dacks

Purificación López-García

David Moreira

Eunsoo Kim
email: eunsookim@ewha.ac.kr

Laura Eme
email: laura.eme@universite-paris-saclay.fr

High quality genome and transcriptome data for two new

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Background & Summary

Free-living heterotrophic flagellates play important roles in the nutrient cycling of marine and freshwater ecosystems. However, the extent of their genomic diversity is still dramatically uncharacterized. Amongst the lesser-known of these is Mantamonas, a genus of marine gliding flagellates initially described as very divergent from all other known eukaryotes [START_REF] Glücksman | The novel marine gliding zooflagellate genus Mantamonas (Mantamonadida ord. n.: Apusozoa)[END_REF] . Although Mantamonas was originally thought to be related to the poorly-known lineages Apusomonadida and Ancyromonadida, based on ribosomal RNA gene phylogenies and some of their morphological characteristics [START_REF] Glücksman | The novel marine gliding zooflagellate genus Mantamonas (Mantamonadida ord. n.: Apusozoa)[END_REF] , recent transcriptome-based phylogenomic analyses instead robustly placed Mantamonas plastica as sister to a clade comprising Collodictyonidae (also known as diphylleids) and Rigifilidae, altogether forming the "CRuMs" supergroup [START_REF] Brown | Phylogenomics Places Orphan Protistan Lineages in a Novel Eukaryotic Super-Group[END_REF][START_REF] Lax | Hemimastigophora is a novel supra-kingdom-level lineage of eukaryotes[END_REF] . This clade presents diverse cell morphologies and branches at the base of Amorphea [START_REF] Brown | Phylogenomics Places Orphan Protistan Lineages in a Novel Eukaryotic Super-Group[END_REF][START_REF] Burki | The New Tree of Eukaryotes[END_REF] (Amoebozoa plus Obazoa, the latter including animals and fungi, among others). The genomic exploration of members of this supergroup therefore represents an important resource for uncovering the characteristics of this deep-branching clade, and may help us better understand evolutionary transitions within the eukaryotic tree of life, such as the acquisition of complex multicellularity in several lineages of the Obazoa. However, to date, only partial transcriptomic data is available for a handful of CRuMs taxa, including M. plastica [START_REF] Brown | Phylogenomics Places Orphan Protistan Lineages in a Novel Eukaryotic Super-Group[END_REF][START_REF] Lax | Hemimastigophora is a novel supra-kingdom-level lineage of eukaryotes[END_REF] . Here, we isolated and described two new species of mantamonads, Mantamonas sphyraenae sp. nov. and Mantamonas vickermani sp. nov., and generated a high-quality nuclear genomic assembly for the former and transcriptomic assemblies for both species.

Overall, the cell morphology and behavior under light microscopy of these two new species (Fig. 1, Movie 1 and Movie 2) are comparable to what was reported in the original description of the genus Mantamonas 1 and to our own observations of the type strain of M. plastica. Nonetheless, our strains appear to be slightly smaller than the 5 × 5 µm dimensions of M. plastica. Cells of this genus have one anterior and one posterior flagellum. They are flattened and somewhat plastic, with shapes ranging from wide, with more or less pointed lateral "wings" resembling the fins of a manta ray, to kite-shaped, to oval, to spherical. The left side of the cell body often displays a characteristic blunt projection, which we sometimes observed in our new strains, although less conspicuously (Fig. 1; see the detailed morphological description of each of the new species in Methods and formal species description in Data Usage Notes).

All previously known mantamonad strains were isolated from marine sediments [START_REF] Glücksman | The novel marine gliding zooflagellate genus Mantamonas (Mantamonadida ord. n.: Apusozoa)[END_REF] , which was also the case for our strain M. vickermani sp. nov., isolated from marine lagoon sediment. However, we isolated the other strain (M. sphyraenae sp. nov.) from the skin surface of a barracuda, which could suggest that either this species is epizootic (normally inhabiting the skin of the fish) or that the cells that we isolated were dislodged from their normal habitat and adhered to the fish skin by chance. Additional sampling and culturing efforts should help resolve this matter.

The assembled nuclear genome sequence of M. sphyraenae is highly contiguous (Table 1). This genome sequence was generated using long (PacBio) and short (Illumina) reads (see Methods). The average sequencing coverage was 112x for PacBio and 115x for Illumina. Three different genome assembly strategies, using Canu 5 , FALCON [START_REF] Chin | Phased diploid genome assembly with single-molecule real-time sequencing[END_REF] , and MaSuRCA 7 , yielded comparable results (see Methods, Table 2), with >90% representation of the 255 Benchmarking Universal Single Copy Orthologs (BUSCO 8 ) of the eukaryota_odb10 dataset (Fig. 2), indicating high completeness. For downstream analyses, we opted to use the FALCON assembly because it was the most contiguous of the three, with the majority of the contigs (59 out of 78 primary contigs) bearing TTAGGG telomeric repeats at both ends. In addition, 14 of the remaining contigs had telomeric repeats at one end. While the presence of such conserved motives towards the end of the contigs suggests the complete assembly of most of the chromosomes and leads to an estimation of ~66 pairs of chromosomes in the M. sphyraenae nucleus, experimental evidence is needed to confirm the chromosome number in this species. Biallelic single nucleotide polymorphism (SNP) frequencies cluster around a ratio of 0.5/0.5 for each major/minor allele (Fig. 3a). This is indicative of a diploid genome, which was also supported by the statistical model of SNP frequency distribution (Table 3).

The M. sphyraenae genome contains 9,416 predicted protein coding sequences. Genes have an average length of 2,282 bp and are mostly mono-exonic (Fig. 3b). De novo characterization of repetitive elements indicates that around 12% of the genome is represented by transposable elements and other repeats. While some of these were classified into different known families of DNA transposons and long terminal repeat (LTR) retroelements, the vast majority comprises unclassified types (Fig. 3c). In comparison, the transcriptome assembly of Mantamonas sphyraenae contains 9,256 contigs from which we predicted 8,885 non-redundant proteins and the presence of 85.5% of BUSCO eukaryota_odb10 gene set (Fig. 2). 96% of these proteins are also found in the genome-based predicted proteome, suggesting that the genome assembly represents nearly the protein repertoire represented in the transcriptome (see details in the Technical validation section, Completeness analysis). The de novo assembled transcriptome of M. vickermani had an average sequencing coverage of 80x and led to the inference of 9,561 non-redundant proteins. As for the genome and transcriptome of M. sphyraenae, the proteome inferred from this transcriptome resulted in a high BUSCO score, indicating a high completeness of the predicted gene complement for this species (Fig. 2). Some BUSCO genes are consistently missing in all the four Mantamonas predicted proteomes, suggesting a true absence of these genes in the genus.

We inferred the phylogenetic relationships of our species within the CRuMs clade using publicly available data to reconstruct a dataset of 182 conserved protein markers and recovered the monophyly of the Mantamonas genus and the placement of M. sphyraenae as sister to a clade containing M. vickermanii and M. plastica (details in Methods, Phylogenomics analyses).

To explore the gene content diversity of our new mantamonad species, we annotated the predicted proteomes genes with EggNOG mapper [START_REF] Cantalapiedra | eggNOG-mapper v2: Functional Annotation, Orthology Assignments, and Domain Prediction at the Metagenomic Scale[END_REF] and reconstructed the minimal core proteome for the genus Mantamonas and the CRuMs lineage (see details in Methods CRuMs orthologue analyses).

Finally, as an additional way of assessing the completeness of the M. sphyraenae and M. vickermanii sequence data and capturing a sense of the complexity of the cellular systems in these organisms, we interrogated the complement of one well-studied set of proteins, the membrane-trafficking system. This complex protein machinery underpins normal cellular function and is critical for feeding, cell growth, and interaction with the extracellular environment [START_REF] More | Evolution and Natural History of Membrane Trafficking in Eukaryotes[END_REF] . While some proteins are highly conserved across eukaryotic lineages, others have rarely been retained during evolution but were nonetheless present in the Last Eukaryotic Common Ancestor (LECA) [START_REF] More | Evolution and Natural History of Membrane Trafficking in Eukaryotes[END_REF] . Among them, the so-called "jotnarlogs" represent LECA proteins present in diverse extant eukaryotes but not in the major opisthokont model organisms.

The identification of homologs of the majority of the protein complement associated with the membrane trafficking system as well as some jortnalogs in the proteomes of the new Mantamonas species (details in Methods, Analysis of the conservation of the membrane-trafficking system complement) corroborated the high completeness of our genomic and transcriptomic datasets, and suggests that these datasets may provide interesting insights in the evolution of anciently originated protein machineries. Overall, our new Mantamonas nuclear genome and transcriptome sequences provide high quality data for a major, yet poorly known, eukaryotic supergroup. They will allow more comprehensive comparative studies of genetic diversity in microbial eukaryotes and a better understanding of deep eukaryotic evolution. Okinawa Prefecture, Japan (24° 23′ 36.762″ N, 123° 45′ 22.572″ E). It was isolated manually from the rough sample with a micropipette, and maintained in Erd-Schreiber medium 11 fortified with 2.5% (final volume) freshwater Cerophyl medium (ATCC 802). Stock cultures were kept in 8 ml volumes in 25 ml culture flasks at 16-18 °C, and transferred at three-week intervals. Bulk cultures were grown at room temperature in 10 cm Petri plates containing ~10 ml medium. Live cells were observed on an Zeiss Axiovert 100 M inverted microscope equipped with DIC and phase contrast optics. Images were captured with an Olympus DP73 17.28-megapixel camera. Morphometric data were obtained at 1,000x magnification on 20 cells.

Morphological description of Mantamonas sphyraenae sp. nov. Mantamonas sphyraenae cells exhibited three general morphologies: 'balloons' , which were typically ~5 µm long and ~3 µm wide, with a circularly curved anterior and a posterior end tapering to a point; 'kites' , which were roughly diamond-shaped, about 3.5-4 µm long and wide; and 'mantas' , which were 4-5 µm wide and ~3 µm long, having a broadly curved anterior end, a more tightly rounded right side, a bluntly rounded projection on the left side, and a posterior comprising either straight edges culminating in a point, two shallowly concave curves, or one of each. All three morphologies were plastic to some extent, although 'mantas' were noteworthy in that the left-side projection appeared rigid, and the curved right side frequently very plastic. Intermediates between the three morphologies were sometimes observed. In general, all cells in any given culture flask exhibited the same morphology, which often changed from one observation to the next, one to three weeks later. Exceptions to the prevalent morphology were almost always intermediate forms. We did not observe active transitions from one cell type to another, including to or from intermediate forms. Cells of all morphologies glided slowly and with constant speed, although occasionally stopping; the cell body frequently deformed when changing direction or colliding with other objects.

In all cases, a flagellum, 6-10 µm long, trailed behind the cell, always in a straight line except when the cell was turning, in which case it followed the cell's path. No movement of the flagellum was seen besides this. Under extremely favourable conditions, a second flagellum could be seen projecting from the anterior-left of 'manta' cells, at about a 45° angle. This second flagellum was invariably very thin, stiff, and 1-2 µm long. Very occasionally, we observed an additional protrusion, about the full width of a flagellum and about 1-2 µm long. This was always seen projecting from the posterior of the cell, immediately to the left of, and usually parallel to, the posterior flagellum. It appeared entirely static, and never appeared to change its length or orientation. Cysts were never observed at any stage of culture. Likewise, we never observed dividing cells.

Mantamonas sphyraenae nucleic acid extraction and genome/transcriptome sequencing.

To obtain nucleic acids, initially, five plates were inoculated with 500 µl from mature stock cultures. When these had reached high density (qualitatively determined), for each plate, the supernatant was discarded, cells were collected with the use of disposable cell scrapers, and the resulting 0.3-0.5 ml of concentrated cells were inoculated into 50 ml of fresh medium, which was then distributed into five new plates. This process was repeated, for a final count of 125 plates, for DNA extraction and 14 plates used for RNA extraction. For both preparations, cells were harvested with disposable cell scrapers and resuspended in sterile medium. The resuspension was prefiltered using 5.0-µm-pore polycarbonate filters, to remove bacterial flocs, and refiltered using 0.8-µm-pore filters, to remove individual bacteria.

For DNA extraction, filters were incubated in lysis buffer (50 mM Tris, 5 mM EDTA, 50 mM NaCl, pH 8), proteinase K (~300 µg/ml final concentration) and SDS (1% final concentration) for 1 hr on a rotator at 37°. The resulting solution was divided into two aliquots. From these, DNA was extracted in parallel using phenol/ chloroform/isoamyl alcohol (25:24:1), extracted again using chloroform/isoamyl alcohol (24:1) and precipitated overnight in 95% EtOH at -20. The DNA was then pelletted in a centrifuge at 4°, washed with 80% EtOH, and resuspended in ddH 2 O. The total yield was ~90 µg.

Long-read genomic sequences were obtained by using Single Molecule Real Time (SMRT) cell technology in a PacBio RSII system at the Cold Spring Harbor Laboratory. A total of 2,304,908 reads (18.7 Gbp) were acquired from 33 SMRT cells. Additional DNA samples were used to prepare two Illumina Nextera short-insert and mate-pair libraries following the manufacturer's protocols. The sequencing was done with the HiSeq 2500 System and a PE150 run option. A total of 62,929,978 read pairs (18.9 Gbp) and 53,901,870 read pairs (16.2 Gbp) were generated for the paired-end library and the mate pair library, respectively. For RNA extraction, the cell filters were incubated in TRI Reagent (Sigma-Aldrich) and RNA was isolated according to the manufacturer's instructions, using spin columns for elution. The total RNA sample was subjected to poly-A selection followed by Illumina TruSeq RNA library preparation and a total of 24,187,884 read pairs (7.3 Gbp) were sequenced using the Illumina HiSeq 2500 platform and a PE150 run option. All the genomic and transcriptomic Nextera library preparation and sequencing were conducted at the Weill Cornell's Genome Resources Core Facility.

Mantamonas sphyraenae genome assembly, gene prediction and ploidy analysis.

As the presence of co-cultured bacterial contamination in the sequencing data was expected, b oth the PacBio and Illumina reads were screened for contamination (see details in the technical validation section) and more than 60% of the original data identified as contaminant was discarded (see technical validation section). After this initial decontamination step, a total of 5.89 Gbp of long-read data was assembled using the Canu [START_REF] Koren | Canu: scalable and accurate long-read assembly via adaptive -mer weighting and repeat separation[END_REF][START_REF] Kriventseva | OrthoDB v10: sampling the diversity of animal, plant, fungal, protist, bacterial and viral genomes for evolutionary and functional annotations of orthologs[END_REF] and FALCON [START_REF] Chin | Phased diploid genome assembly with single-molecule real-time sequencing[END_REF] pipelines.

The resulting genomic contigs from the Canu and FALCON approaches were then polished by aligning the screened PacBio reads to the draft genome using minimap2 [START_REF] Li | Minimap2: pairwise alignment for nucleotide sequences[END_REF] and generating a consensus with Racon v1.3.1 [START_REF] Vaser | Fast and accurate de novo genome assembly from long uncorrected reads[END_REF] . Subsequently, a second step of polishing was performed with the high quality Illumina reads by mapping them with bwa-0.7.15 [START_REF] Li | Fast and accurate short read alignment with Burrows-Wheeler transform[END_REF] and using Pilon v1.22 [START_REF] Walker | Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement[END_REF] to correct for single base errors.

Additionally, MaSuRCA v3.2.6 [START_REF] Zimin | Hybrid assembly of the large and highly repetitive genome of Aegilops tauschii, a progenitor of bread wheat, with the MaSuRCA mega-reads algorithm[END_REF] was used to generate a hybrid assembly using the PacBio as well as the short-insert and mate-pair Illumina data that were retained after bacterial read filtering.

After these assembly efforts, any remaining bacterial contigs were identified by using a combination of homology searches and tetramer frequency-based binning (see details in the technical validation section). From the Canu assembly, a contig corresponding to mtDNA was identified and removed. Clean assemblies were then assessed based on their contiguity and completeness (Table 2) and the FALCON assembly was chosen for further analyses. Because of the specific parameter set utilized, our FALCON analysis did not assemble mtDNA due to its much higher sequence coverage compared to that for the nuclear DNA.

A custom library of repetitive elements was generated for the polished and cleaned nuclear genomic sequence by combining the results of RepeatModeler2 17 and Transposon-PSI (http://transposonpsi.sourceforge.net/) pipelines. The gathered repeat sequences from both analyse s were merged and clustered to generate a single consensus and refined repeat library that was further compared against the Dfam database 18 to classify the repetitive elements using RepeatModeler [START_REF] Flynn | RepeatModeler2 for automated genomic discovery of transposable element families[END_REF] refiner and classifier modules. Repetitive elements identified by this procedure were then masked out of the nuclear genome using RepeatMasker 17 before the prediction of protein-coding genes. Subsequently, the RNA-seq libraries were mapped against the genome sequence with HISAT-2 [START_REF] Kim | HISAT: a fast spliced aligner with low memory requirements[END_REF] to generate spliced alignments, and BRAKER2 [START_REF] Brůna | BRAKER2: automatic eukaryotic genome annotation with GeneMark-EP+ and AUGUSTUS supported by a protein database[END_REF] was employed to predict the nuclear protein coding genes integrating the extrinsic evidence from the RNA-Seq data.

Ploidy was inferred by assessing the distribution of allele frequencies at biallelic single nucleotide polymorphisms (SNPs) visually, and with modeling 21,22 using nQuire [START_REF] Weiß | nQuire: a statistical framework for ploidy estimation using next generation sequencing[END_REF] . Briefly, the Nextera Illumina reads were mapped to the final genome assembly with Bowtie2 v2.3.5.1 [START_REF] Langmead | Fast gapped-read alignment with Bowtie 2[END_REF] and the resulting.bam file was used to calculate base frequencies for each biallelic site. These results were denoised using nQuire. The resulting frequencies were plotted in R version 3.3.3 [START_REF] Core Team | A language and environment for statistical computing[END_REF] . Finally, we ran the nQuire's Gaussian Mixture Model (GMM) command, which models the distribution of base frequencies at biallelic sites, and uses maximum likelihood to select the most plausible ploidy model (Table 3).

Isolation and microscopy of Mantamonas vickermani sp. nov. Mantamonas vickermani

CRO19MAN was isolated from a sediment sample collected in July 2014 from the shallow marine lagoon Malo jezero (42°47′05.9"N 17°21′01.3"E) on the island of Mljet (Croatia, Mediterranean Sea). The sample was taken from the upper layer of the sediments at the shore of the lagoon with a sterile 15 ml Falcon tube at a depth of 10 cm below the water surface and stored at -20 °C. In September 2019, a small amount of sediment was inoculated in a Petri dish with 5 ml of sterile seawater supplemented with 1% YT medium (100 mg yeast extract and 200 mg tryptone in 100 ml distilled water, as in the protocol from the National Institute for Environmental Studies [NIES], Japan). After observation of some mantamonad cells, serial dilution was performed in a multiwell culture plate to further enrich the culture. We transferred 250 µl of culture to a well with 1 ml of fresh 1% YT seawater medium and then retransferred the same volume to a new well, repeating the process 5 times for a total of 24 wells. Single mantamonad cells were then isolated from one of the enriched cultures with an Eppendorf PatchManNP2 micromanipulator using a 65 µm VacuTip microcapillary (Eppendorf) and a Leica Dlll3000 B inverted microscope. This cell was inoculated into 1 ml of growth medium and after 48 hr incubation we confirmed an established monoculture of M. vickermani CRO19MAN.

Optical microscopy observations were performed with a Zeiss Axioplan 2 microscope equipped with oil-immersion differential interference contrast (DIC) and phase contrast objectives. Images were acquired with an AxiocamMR camera using the Zeiss AxioVision 4.8.2 SP1 suite. Videos were recorded using a Sony α9 digital camera. Morphometric data were obtained at 1,000x final magnification on 20 cells. Images were captured at multiple focal planes in order to visualise different cell parts. Measurements of flagella pertain to the visible parts, i.e., the posterior flagellar length is measured beginning from the point at which it emerges from underneath the cell at the body's posterior end.

Morphological description of Mantamonas vickermani sp. nov. Mantamonas vickermani cells are

∼3 μm wide and ∼3.5 μm long; thus noticeably smaller than those of Mantamonas plastica (∼5 μm wide and ∼5 μm long) (Glücksman et al. [START_REF] Glücksman | The novel marine gliding zooflagellate genus Mantamonas (Mantamonadida ord. n.: Apusozoa)[END_REF] ) (Fig. 1g-l). Like M. plastica, M. vickermani also has a strongly flattened and plastic morphology. However, the characteristic blunt projection on the left-hand side of the cell observed in M. plastica is less conspicuous in M. vickermani, and not always observed in cells possessing an overall spherical to oval morphology (Fig. 1). The anterior flagellum of M. vickermani is ∼2 μm long, rigid in all of its length, and vibrates with a small amplitude; its posterior flagellum is ∼7 μm long and considerably thicker than the anterior one, having a very small acroneme that when seen is never longer than 1-2 μm. Both flagella are also shorter than those reported for M. plastica (∼3 μm anterior and ∼10 μm posterior).

Mantamonas vickermani glides in a smooth and continuous manner on the substrate with a similar speed and turning behavior to that observed for M. plastica (Glücksman et al. [START_REF] Glücksman | The novel marine gliding zooflagellate genus Mantamonas (Mantamonadida ord. n.: Apusozoa)[END_REF] ; AAH, pers. obs.) (Movie 1 and Movie 2). As with M. plastica, M. vickermani is a bacterivore with a voracious appetite, engulfing bacteria at a high rate. Interestingly, and in contrast with Glücksman et al. [START_REF] Glücksman | The novel marine gliding zooflagellate genus Mantamonas (Mantamonadida ord. n.: Apusozoa)[END_REF] , we did observe one cell possessing two posterior flagella, which strongly suggests that it was undergoing cellular division (Fig. 1).

Mantamonas vickermani RNA purification and transcriptome sequencing. This new strain was

grown for a week in 75 cm 2 cell culture flasks with ~10 ml of medium. Fully grown cultures were collected by gently scratching the bottom of the flasks with a cell scraper to resuspend the gliding flagellates and pooled in 50 ml Falcon tubes to be centrifuged at 10 °C for 15 minutes at 15,000 g. Total RNA was extracted from cell pellets with the RNeasy mini Kit (Qiagen) following the manufacturer protocol. Two cDNA Illumina libraries were constructed after polyA mRNA selection, and these were sequenced using the paired-end (2 × 125 bp) method with Illumina HiSeq 2500 Chemistry v4 (Eurofins Genomics, Germany).

transcriptomes assembly and proteome prediction. The transcriptomic sequence of M vickermani

and M. sphyraenae were assembled de novo using Spades v3.13.1 [START_REF] Bushmanova | rnaSPAdes: a de novo transcriptome assembler and its application to RNA-Seq data[END_REF] with the rna mode and default parameters specified. Transcripts were then screened to identify remaining contaminants using the Blobtools2 26 pipeline and homology searches against a custom database (see technical validation section). Predicted proteins were obtained from the clean transcripts using Transdecoder v2 (http:transdecoder.github.io) alowing for a single prediction by transcript (-single-bes-only option) and using the universat genetic code. Subsequently, CD-HIT [START_REF] Li | Clustering of highly homologous sequences to reduce the size of large protein databases[END_REF] clustering was employed (with a threshold of > = 90% of identity) to produce a non-redundant data set of proteins for each of the trancriptomes, and to eliminate falsely duplicated proteins stemming from alternatively spliced transcripts. phylogenomic analyses. The dataset of 351 conserved protein markers from Lax et al. [START_REF] Lax | Hemimastigophora is a novel supra-kingdom-level lineage of eukaryotes[END_REF] was updated by BLASTP searches [START_REF] Altschul | Basic local alignment search tool[END_REF] against the inferred proteomes of representatives of other eukaryotic lineages, including the proteomic data for our two new mantamonad strains. Each protein marker was aligned with MAFFT v.7 [START_REF] Katoh | MAFFT multiple sequence alignment software version 7: improvements in performance and usability[END_REF] and trimmed using TrimAl 30 with the -automated1 option. Alignments were manually inspected and edited with AliView 31 and Geneious v6.06 [START_REF] Kearse | Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data[END_REF] . Single-protein trees were reconstructed with IQ-TREE v1.6.11 [START_REF] Nguyen | IQ-TREE: A Fast and Effective Stochastic Algorithm for Estimating Maximum-Likelihood Phylogenies[END_REF] under the corresponding best-fitting model as defined by ModelFinder 34 implemented in IQ-TREE [START_REF] Nguyen | IQ-TREE: A Fast and Effective Stochastic Algorithm for Estimating Maximum-Likelihood Phylogenies[END_REF] . Each single-protein tree was manually inspected to discard contaminants and possible cases of horizontal gene transfer or hidden paralogy. At the end of this curation process, we kept a final taxon sampling of 14 species, including members of Ancyromonadida, Malawimonadida, Opisthokonta, and CRuMs (concatenated alignment and supplementary trees are available at Figshare [START_REF] Eme | One high-quality genome and two transcriptome datasets for two new species of Mantamonas, a deep-branching eukaryote clade, Figshare[END_REF] ), and 182 protein markers that were present in all mantamonad species (with at least 80% of markers identified in each taxon). All proteins were realigned, trimmed as previously described, and concatenated, creating a final supermatrix with 62,088 amino acids.

A Bayesian inference tree was reconstructed using PhyloBayes-MPI v1.5a [START_REF] Lartillot | PhyloBayes 3: a Bayesian software package for phylogenetic reconstruction and molecular dating[END_REF] under the CAT-GTR model [START_REF] Lartillot | A Bayesian mixture model for across-site heterogeneities in the amino-acid replacement process[END_REF] , with two MCMC chains, and run for 10,000 generations, saving one of every 10 trees. Analyses were stopped once convergence thresholds were reached (i.e. maximum discrepancy <0.1 and minimum effective size >100, calculated using bpcomp). Consensus trees were constructed after a burn-in of 25%. Maximum likelihood (ML) analyses were done with IQ-TREE v1.6.11 [START_REF] Nguyen | IQ-TREE: A Fast and Effective Stochastic Algorithm for Estimating Maximum-Likelihood Phylogenies[END_REF] , first by calculating the ML tree under the LG+F+R4 model, which was used as guide tree for the PMSF approximation [START_REF] Wang | Modeling Site Heterogeneity with Posterior Mean Site Frequency Profiles Accelerates Accurate Phylogenomic Estimation[END_REF] run under the LG+C60+F+R4 model.

Consistent with previous studies, our maximum likelihood (ML) and Bayesian inference (BI) phylogenetic trees recovered the monophyly of CRuMs with high BI posterior probability (0.99) and ML bootstrap support (95%), although it is worth noticing that the outgroup is highly reduced since resolving the position of CRuMs in the tree of eukaryotes is outside the scope of this paper. The monophyly of Mantamonas received full support from both methods. We found Mantamonas sphyraenae to be sister to a maximally-supported clade containing M. plastica and M. vickermani (Fig. 4).

CRuMs orthologue analysis and protein functional annotation.

Orthologous gene families were identified among the predicted proteomes of Mantamonas sphyraenae, Mantamonas vickermani and the publicly available proteomes of Mantamonas plastica, Diphylleia rotans and Rigifila ramosa as obtained from the EukProt Fig. 4 Phylogenomic analysis of CRuMs clade. Bayesian inference (BI) phylogeny based on 182 conserved proteins from Lax et al. [START_REF] Lax | Hemimastigophora is a novel supra-kingdom-level lineage of eukaryotes[END_REF] . The tree was obtained using 62,088 amino acid positions with the CAT-GTR model. Statistical support at branches was also estimated using maximum likelihood (ML) under the LG+C60+F+R4 model with the PMSF approximation. Numbers at branches indicate BI posterior probabilities and ML bootstrap values, respectively; bootstrap values <50% are indicated by dashes. Branches with support values higher than or equal to 0.99 BI posterior probability and 95% ML bootstrap value are indicated by black dots. The tree was rooted between CRuMs and everything else. v3 database 39 using OrthoFinder v2.5.4 [START_REF] Emms | OrthoFinder: phylogenetic orthology inference for comparative genomics[END_REF] . For this, we used DIAMOND [START_REF] Buchfink | Sensitive protein alignments at tree-of-life scale using DIAMOND[END_REF] ("ultra-sensitive" mode, and query cover > = 50%), an inflation value of 1.5, and the MCL clustering algorithm (Fig. 5a).

Then, the predicted proteomes of M. sphyraenae, M. vickermanii, M. plastica, D. rotans, and R. ramosa were functionally annotated with the EggNOG-mapper pipeline [START_REF] Cantalapiedra | eggNOG-mapper v2: Functional Annotation, Orthology Assignments, and Domain Prediction at the Metagenomic Scale[END_REF] , using DIAMOND ultra-sensitive mode and all domains of life as the target space. During this process, individual sequences composing the CRuMs orthogroups generated by OrthoFinder were assigned a COG functional category. This information was summarized at the orthogroup level by assigning to each orthogroup a single COG category corresponding to the most frequent annotation of its individual sequences, provided that it represented at least 50% of the sequences within the orthogroup.

A total of 1,718 orthogroups were found to be conserved among all CRuMs taxa (Fig. 5b), while 4,378 were identified as shared between the three Mantamonas species, representing the minimal core proteome of the genus Mantamonas as currently known, among which 2,161 orthogroups are not found in the other two CRuMs lineages. Our species also display a smaller number of unique proteins than the publically available proteomes likely due to the methodological strategy that we employed to assemble the transcriptomes and infer open reading frames that reduces the number of short and incomplete ORFs and sioforms when compared with the proteomes derived from the other CRuMs transcriptomes. However, beyond the absolute numbers of predicted coding sequences, the comparison between all these proteomes gives us an indication about the degree of the diversity of gene content in each of our two Mantamonas species.

Most of the proteins conserved among the CRuMs taxa (99.6%) were found to have an ortholog in the EggNOG database and to belong to at least one Cluster of Orthologous Groups (COG) [START_REF] Tatusov | A genomic perspective on protein families[END_REF][START_REF] Galperin | Expanded microbial genome coverage and improved protein family annotation in the COG database[END_REF] functional category, where the most highly represented were "Function unknown" and "Post-translational modificationand "Intracellular trafficking" (Fig. 5c). By contrast, a substantial amount of orthogroups conserved among mantamonads (12%), but absent in other CRuMs lineages, could not be assigned to any cluster in the EggNOG database. In addition, most orthogroups conserved in mantamonads but absent in other CRuMs that could be connected to an existing EggNOG cluster were annotated as "Function unknown" (Fig. 5c). Altogether, this large number of Mantamonas-specific genes of unknown function suggests that many genetic innovations occurred at the origin of this group. analysis of the conservation of the membrane-trafficking system complement. To assess the complement of the membrane trafficking system encoded in our Mantamonas genome and transcriptome datasets, we performed homologous searches of a selection of protein query sequences from the genomes of Homo sapiens (GCF_000001405.40), Dictyostelium discoideum (GCF_000004695.1), Arabidopsis thaliana (GCF_000001735.4) and Trypanosoma brucei (GCF_000002445.2) available at the GenBank of the NCBI (National Center for Biotechnology Information) database. These proteins included components of the machinery for vesicle formation (HTAC-derived coats, ESCRTs, and ArfGAPs) and vesicle fusion (SNAREs and SM proteins, TBC-Rab GAPs, and Multi-subunit tethering complexes) [START_REF] More | Evolution and Natural History of Membrane Trafficking in Eukaryotes[END_REF] .

BLASTP and TBLASTN were used to search the predicted proteomes and nucleotide coding sequences, respectively, of M. sphyraenae and M. vickermani. The HMMER3 package was used to find more divergent protein sequences using the hmmsearch tool [START_REF] Finn | HMMER web server: interactive sequence similarity searching[END_REF] . In cases in which only TBLASTN hits were retrieved, these were translated using Exonerate [START_REF] Slater | Automated generation of heuristics for biological sequence comparison[END_REF] . Potential orthologs (i.e., hits with an E-value below 0.05) were further analyzed by the Reciprocal Best Hit (RBH) approach, using the Mantamonas candidate orthologs as queries against the H. sapiens, D. discoideum and A. thaliana proteomes. If the best hit was the protein of interest and had an E-value two orders of magnitude lower than the next non-orthologous hit, this was considered as orthology validation. Forward and reverse searches were performed using the AMOEBAE tool [START_REF] Barlow | Comparative genomics for evolutionary cell biology using AMOEBAE: Understanding the Golgi and beyond[END_REF] .

We detected most proteins of the membrane-trafficking system in the two new Mantamonas species, making it one of the most complete known protein complements for this system. Notably, when compared to representatives of well-characterized model organisms from other supergroups (Fig. 6). Mantamonas encodes some rarely retained proteins, such as the AP5 complex [START_REF] Hirst | Correction: The Fifth Adaptor Protein Complex[END_REF] and syntaxin 17 [START_REF] Arasaki | A role for the ancient SNARE syntaxin 17 in regulating mitochondrial division[END_REF] . We also identified several jotnarlogs (Fig. 6), including a near-complete TSET complex, and the SNAREs NPSN and Syp7 [START_REF] Eme | One high-quality genome and two transcriptome datasets for two new species of Mantamonas, a deep-branching eukaryote clade, Figshare[END_REF] .

Data Records

The read data associated with the nuclear genome and transcriptomic datasets of Mantamonas sphyraenae and the transcriptome of Mantamonas vickermani have been submitted to the NCBI SRA database [START_REF] Eme | NCBI Sequence Read Archive[END_REF] (Table 4). The Transcriptome Shotgun Assemblies have been deposited at DDBJ/EMBL/GenBank under the accessions GKLA00000000 and GKKZ00000000 for M. vickermani and M. sphyraenae respectively. The final nuclear genome assembly of Mantamonas sphyraenae has been deposited at GenBank under the accession GCA_026936335.1 [START_REF] Blaz | genome assembly ASM2693633v1[END_REF] . The versions described in this paper are the first versions. The prediction of protein-coding genes from the genome and transcriptome assemblies of Mantamonas sphyraenae, as well as from the transcriptome assembly of M. vickermani are available at Figshare [START_REF] Eme | One high-quality genome and two transcriptome datasets for two new species of Mantamonas, a deep-branching eukaryote clade, Figshare[END_REF] .

Phylogenomic analysis alignments and trees, and membrane-trafficking predicted proteins table can be found on Figshare [START_REF] Eme | One high-quality genome and two transcriptome datasets for two new species of Mantamonas, a deep-branching eukaryote clade, Figshare[END_REF] .

technical Validation

Quality assessment of sequencing datasets. All Illumina paired-end raw reads used for genome polishing were quality-checked with FastQC v0.11.8 [START_REF] Fastqc | FastQC: a quality control tool for high throughput sequence data[END_REF] and trimmed using TRIMMOMATIC [START_REF] Bolger | Trimmomatic: a flexible trimmer for Illumina sequence data[END_REF] to retain only reads with maximum quality scores. PacBio reads resulted in an N50 of 11,048 bp and an average coverage of 106x after filtering out the identified contaminant sequences (see below).

Identification and filtering of contaminant sequences. Mantamonads grow in non-axenic cultures

with co-cultured prokaryotes on which they feed. Therefore, various methods were employed to ensure the correct identification and filtering of contaminant sequences in the genomic and transcriptomic datasets of M. sphyraenae and M. vickermani.

For the genomic dataset of M. sphyraenae, we first identified the main bacterial contaminants from the initial genome assemblies [START_REF] Aponte | The Bacterial Diversity Lurking in Protist Cell Cultures[END_REF] . In addition, we established a custom database consisting of contigs assembled from Illumina sequencing data from bacteria only enrichment cultures derived from the lab's several xenic protist cultures. These were used to screen PacBio reads using BLASR v5.1 [START_REF] Chaisson | Mapping single molecule sequencing reads using basic local alignment with successive refinement (BLASR): application and theory[END_REF] The Illumina reads were screened similarly using Bowtie2 v2.3.5.1 [START_REF] Langmead | Fast gapped-read alignment with Bowtie 2[END_REF] . Only Illumina reads in which neither pair aligned to the bacterial database were retained for further assembly recovering 57% and 49% of the pair-end and mate pair reads from the total libraries respectively.

After genome assembly using the filtered reads, remaining contaminant contigs were identified by using MyCC v1 [START_REF] Lin | Accurate binning of metagenomic contigs via automated clustering sequences using information of genomic signatures and marker genes[END_REF] , which bins contigs based on their tetranucleotide frequencies and coverage. Clusters were formed using the affinity propagation (AP) algorithm and visualized in a 2-dimensional Barnes-Hut-SNE plot. BLASTN searches using default parameters were conducted against the 'nt' database from the NCBI to taxonomically classify the bins. Contigs were identified as contaminants if they contained no hits other than to prokaryotes, and if they were clustered away from the main eukaryotic bin. Finally mitochondrial sequences were screened out from the short and long read libraries of M. sphyraenae by mapping them against the mitochondrial genome using bwa-0.7.15 [START_REF] Li | Fast and accurate short read alignment with Burrows-Wheeler transform[END_REF] and minimap2 [START_REF] Li | Minimap2: pairwise alignment for nucleotide sequences[END_REF] respectively.

The assembled transcriptomes of M. sphyraenae and M. vickermani were decontaminated with the Blobtools2 pipeline [START_REF] Challis | BlobToolKit-interactive quality assessment of genome assemblies. G3: Genes, Genomes[END_REF] . Briefly, this approach helps to identify contaminant sequences based on their biases in coverage and GC content, as well as on a taxonomic classification established by DIAMOND searches [START_REF] Buchfink | Sensitive protein alignments at tree-of-life scale using DIAMOND[END_REF] against the 'nt' and Uniprot databases [START_REF]UniProt: the universal protein knowledgebase in 2021[END_REF] . In addition, a second cleaning step was done by performing DIAMOND searches against a database containing all the proteins of the prokaryotic Genome Taxonomy Database (GTDB) [START_REF] Parks | GTDB: an ongoing census of bacterial and archaeal diversity through a phylogenetically consistent, rank normalized and complete genome-based taxonomy[END_REF] and the eukaryotic-representative EukProt v3 database [START_REF] Richter | EukProt: a database of genome-scale predicted proteins across the diversity of eukaryotes[END_REF] . A protein was considered as a probable contaminant and excluded from further analyses if its best hit corresponded to any protein from GTDB, with strict cutoffs of identity ≥50% and query coverage ≥50%. Finally, a blobplot was generated for the final genomic and transcriptomic contigs of M. sphyraenae and M. vickermani, respectively, to verify the absence of contaminant sequences (Fig. 7).

Completeness analysis.

To assess the completeness of the decontaminated genome and transcriptome datasets, we employed the BUSCO v5.3.2 pipeline. We identified the percentage of near-universal single copy orthologs of the eukaryote_odb10 database [START_REF] Kriventseva | OrthoDB v10: sampling the diversity of animal, plant, fungal, protist, bacterial and viral genomes for evolutionary and functional annotations of orthologs[END_REF] on the predicted proteomes of M. sphyraenae and M. vickermani, as well as those of other species belonging to the CRuMs supergroup available in the EukProt v3 [START_REF] Richter | EukProt: a database of genome-scale predicted proteins across the diversity of eukaryotes[END_REF] database for comparison purposes (Fig. 2). Moreover, the comparison of the transcriptomic dataset and the genomic dataset of Mantamonas sphyraenae revealed that 96% of the proteins predicted in the transcriptome share similarity with the proteins derived from the genome (80% of these being identical) and 271 proteins were found to be present uniquely in the transcriptome. Adittionally, the mapping coverage from the clean transcriptomic reads to the genome sequence was of 97.38%, suggesting a near complete representation of the gene space in the genome-predicted proteins.

Data usage notes. Formal species descriptions

All taxonomic descriptions in this work were approved by all authors. Eukarya: 'CRuMs' Order Mantamonadida Cavalier-Smith 2011 Family Mantamonadidae Cavalier-Smith 2011 Genus Mantamonas Cavalier-Smith and Glücksman 2011 Mantamonas sphyraenae sp. nov. Description: Cells with varying morphologies: shaped as manta rays (as for genus in Glücksman et al. [START_REF] Glücksman | The novel marine gliding zooflagellate genus Mantamonas (Mantamonadida ord. n.: Apusozoa)[END_REF] ), ~3 µm long and ~5 µm wide; diamonds, 4 ± 1 µm in both dimensions; or rounded anteriorly and tapering posteriorly, ~5 µm long and ~3 µm wide. Anterior flagellum stiff, 0.5-1.0 µm long. Other characters as for genus.

Type culture: SRT306 Type locality: Surface of barracuda caught in lagoon on Iriomote Island, Taketomi, Okinawa Prefecture, Japan (24° 23' 36.762″ N, 123° 45' 22.572″ E).

Isolator: Takashi Shiratori Etymology: From Sphyraena, the genus name for barracuda, the fish from which the type strain was obtained. Gene sequence: The nuclear genome and transcriptomic read sequencing data from Mantamonas sphyraenae (strain SRT306) were deposited in GenBank under BioProject accession number PRJNA886733.

Mantamonas vickermani sp. nov. Description: Cell size ∼3 μm (2.5-4.3 μm) long, ∼3.5 μm (3.0-4.0 μm) wide; cells almost perfectly round, although in some cases possessing a small projection to the left side of the cell; without pseudopodia; anterior flagellum usually ≤2 μm long (1.2-2.7 μm), held forwards and to left ∼40-50° to longitudinal axis, does not beat except for slight terminal vibration; posterior flagellum ∼7 μm long (6-8.9 μm), conspicuous and sometimes acronematic. Other characters as for genus.

Type culture: CRO19MAN Type locality: Specimen isolated from the sediments of the marine lake Malo jezero in the island of Mljet, Croatia.

Isolator: Luis Javier Galindo. Etymology: The name vickermani honors work on heterotrophic protists by Keith Vickerman. Gene sequence. The full transcriptome read data from Mantamonas vickermani (strain CRO19MAN) were deposited in GenBank under BioProject accession number PRJNA886733.

Fig. 1

 1 Fig. 1 General morphology of Mantamonas sphyraenae sp. nov. and Mantamonas vickermani sp. nov. (a-c) Differential interference contrast light micrographs of living M. sphyraenae cells. Note acroneme (white arrowheads), most visible in panel (a) but present in all micrographs. The extremely thin anterior flagellum is visible in panel (b). The left projection, present in all cells, is most distinct in (b). A posterior protrusion is often visible, usually parallel and immediately adjacent to the posterior flagellum (a,b), but sometimes at an angle to it (c). (d-f) Individual M. sphyraenae cell imaged over a 12-second period; numbers in lower right indicate elapsed time in seconds. Note the plastic nature of the cell and lack of movement of the posterior flagellum except to trail behind the cell body. (g-l) Phase and differential interference contrast light micrographs of living interphase M. vickermani cells. Note contrast between thick and long posterior flagellum and thin and short anterior flagellum in (g,k). (l) Laterally dividing cell of M. vickermani with two posterior flagella. Scale bars: 10 μm. AF = anterior flagellum; LP = left projection; PF = posterior flagellum; PP = posterior protrusion; arrowhead = acroneme.

Fig. 2

 2 Fig. 2 Distribution of BUSCO orthologs (eukaryota_odb10) in inferred proteomes of mantamonad assemblies of this study (in bold) in comparison with those of other members of the CRuMs supergroup. Proteomes were inferred from genome (G) and transcriptome (T) assemblies. The top panel represents the BUSCO output for each CRuMs dataset, whereas bottom panel illustrates the patterns of presence/absence of each BUSCO gene (X axis) in the four Mantamonas predicted proteomes.

Fig. 3

 3 Fig. 3 Genomic features of Mantamonas sphyraenae sp. nov. (a) Biallelic SNP frequency distribution. (b) Length distribution and intron frequency of protein-coding genes. (c) Genomic occupancy of the families of repetitive elements identified de novo.

Fig. 5

 5 Fig. 5 Orthology analysis across the CRuMs supergroup. (a) Distribution of coding sequences shared among CRuMs representatives (magenta) or that are species-specific in one or several copies (dark and light green, respectively). Note that these numbers do not represent genes but open reading frames identified in assembled transcripts, except for M. sphyraenae. (b) Number of orthogroups shared among compared CRuMs species. (c) COG functional categories associated with orthogroups shared among all CRuMs, and those associated with orthogroups shared across Mantamonas species but absent in other CRuMs taxa. COG counts were scaled by column using z-score standardization.

Fig. 6

 6 Fig. 6 Distribution of proteins associated with the membrane trafficking system in new Mantamonas species and other model organisms. (a) Selected vesicle formation machinery; (b) Selected vesicle fusion machinery. Names of proteins with jotnarlogs are in purple; those with patchy distribution are in orange.

Fig. 7

 7 Fig. 7 Blob plot of read coverage against GC proportion in genome and transcriptomic contigs. (a) M. sphyraenae genomic sequences. (b) M. sphyraenae transcripts. (c) M. vickermani transcripts. Records are coloured according to their similarity to different phyla. Circles are sized in proportion to records cumulative length. The assembly has been filtered to exclude records whose taxonomic assignment matches "Bacteria". Histograms show the distribution of record length sums along each axis.

Mantamonas sphyraenae Mantamonas sphyraenae Mantamonas vickermani

	Assembly type	genome	transcriptome transcriptome
	Assembly length (Mb)	25.06 (31.49)	20.52	19.78
	Number of contigs	78 (199)	9,255	9,796
	Contig mean length (Kb)	321.30	2.218	2.019
	Longest contig (Kb)	751.365	32.28	21.503
	Shortest contig (Kb)	17.266	0.0202	0.0201
	N50 (Kb)	375.07	3.05	2.79
	L50	26	1,917	2,083
	GC content	59.19	59.02	46.4
	Total repeat content	12.12%	-	-

Table 1 .

 1 Genomic and transcriptomic assemblies statistics for Mantamonas sphyraenae sp. nov. and Mantamonas vickermani sp. nov. Values within parentheses correspond to primary plus associate contigs produced by FALCON.

	Assembly approach	Canu	Falcon	MaSuRCa
	Total length (Mb)	27.35	25.06	26.11
	Number of contigs	172	78	136
	Mean length (bp)	159,014.56	321,299.41	191,995.24
	Longest contig (bp)	732,584	751,365	1,133,621
	Shortest contig (bp)	20,756	17,266	1,222
	N_count	0	0	4,688
	Gaps	0	0	9
	N50 (bp)	303,774	375,077	386,663
	N50n	32	26	24
	N70 (bp)	224,361	300,753	269,297
	N70n	52	41	40
	N90 (bp)	50,673	226,430	146,039
	N90n	95	60	65
	BUSCO eukaryota odb10	C:89.1%[S:82.0%,D:7.1%], F:2.0%,M:8.9%	C:91.4%[S:90.6%,D:0.8%],F:2.0%,M:6.6% C:89.8%[S:86.7%,D:3.1%], F:2.0%,M:8.2%

Table 2 .

 2 Mantamonas sphyraenae sp. nov. genome assembly statistics produced by the tested assembly strategies.

Table 3 .

 3 nQuire Gaussian Mixture Model delta log-likelihood values for the Mantamonas sphyraenae genome.

Table 4 .

 4 Summary of sequencing data records.

Scientific Data | (2023) 10:603 | https://doi.org/10.1038/s41597-023-02488-2

Code availability

All the employed software as well as their versions and parameters were described in the method section. If no parameters were specified, default settings were employed. Data visualization plots were generated using R v4.1.2 (https://cran.r-project.org/, R development core team) and https://bioinformatics.psb.ugent.be/webtools/Venn/.

acknowledgements

The authors thank Drs. J.Z. Xiang, D. Xu, and H. Shang at the Weill Cornell's Genome Resources Core Facility for their assistance with Illumina sequencing. We also thank Dr. S. Goodwin in the NGS sequencing core at Cold Spring Harbor Laboratory for her help with PacBio sequencing, Drs. J.A. Burns and A.A. Pittis for their initial data analysis efforts and K. Lukacs for her help with maintaining the M. sphyraenae cultures.

Competing interests

The authors declare no competing interests.


B R O U A R D

† , R E ´G I S C E ´R E ´g H I N O ‡

§ , B R U N O C O R B A R A * , † , C E ´L I N E L E R O Y { L A U R E N T P E L O Z U E L O ‡

A L A I N D E J E A N ‡, -A N D J E A N -F R A N C ¸o I S C A R R I A S

Understorey environments influence functional diversity in tank-bromeliad ecosystems

Keywords: food webs, French Guiana, invertebrates, microorganisms, phytotelmata, rainforest

A substantial fraction of the freshwater available in neotropical forests is impounded within the rosettes of bromeliads that form aquatic islands in a terrestrial matrix. The ecosystem functioning of bromeliads is known to be influenced by the composition of the contained community but it is not clear whether bromeliad food webs remain functionally similar against a background of variation in the understorey environment. 2. We considered a broad range of environmental conditions, including incident light and incoming litter, and quantified the distribution of a very wide range of freshwater organisms (from viruses to macroinvertebrates) to determine the factors that influence the functional structure of bromeliad food webs in samples taken from 171 tank-bromeliads. 3. We observed a gradient of detritus-based to algal-based food webs from the understorey to the overstorey. Algae, rotifers and collector and predatory invertebrates dominated bromeliad food webs in exposed areas, whereas filter-feeding insects had their highest densities in shaded forest areas. Viruses, bacteria and fungi showed no clear density patterns. Detritus decomposition is mainly due to microbial activity in understorey bromeliads where filter feeders are the main consumers of microbial and particulate organic matter (POM). Algal biomass may exceed bacterial biomass in sun-exposed bromeliads where amounts of detritus were lower but functional diversity was highest. 4. Our results provide evidence that tank-bromeliads, which grow in a broad range of ecological conditions, promote aquatic food web diversity in neotropical forests. Moreover, although bromeliad ecosystems have been categorised as detritus-based systems in the literature, we show that algal production can support a non-detrital food web in these systems.

Introduction

Bromeliaceae are flowering plants represented by 59 genera and some 2400 species native mainly to the neotropics, with a few species found in the American subtropics and one in West Africa [START_REF] Mabberley | The Plant Book[END_REF]. The leaves of tank-forming bromeliads are tightly interlocking, forming wells that collect water, leaf litter and other organic detritus. These tanks, or phytotelmata ('plant-held waters'), provide a habitat for aquatic organisms in rainforests where ponds and lakes are naturally scarce. The aquatic food web inhabiting tank-bromeliads consists of micro-and macroinvertebrates (reviewed in [START_REF] Kitching | Food Webs and Container Habitats: The Natural History and Ecology of Phytotelmata[END_REF] and microorganisms such as bacteria [START_REF] Cochran-Stafira | Integrating bacteria into food webs: studies with Sarracenia purpurea inquilines[END_REF], algae [START_REF] Laessle | A micro-limnological study of Jamaican bromeliads[END_REF][START_REF] Maguire | Phytotelmata: biota and community structure determination in plant-held waters[END_REF], fungi and protozoa (Carrias, Cussac & Corbara, Correspondence: Re ´gis Ce ´re ´ghino, INP, UPS EcoLab (Laboratoire Ecologie Fonctionnelle et Environnement), Universite ´de Toulouse, 118 Route de Narbonne, 31062 Toulouse, France. E-mail: regis.cereghino@univ-tlse3.fr 2001; [START_REF] Foissner | Endemic ciliates (Protozoa, Ciliophora) from tank bromeliads Bromeliaceae): a combined morphological, molecular, and ecological study[END_REF]. Because they can be exhaustively sampled and contain multiple trophic levels, tankbromeliads and their aquatic biota have recently proven to be ideal model systems for controlled studies of many basic ecosystem processes [START_REF] Srivastava | Habitat structure, trophic structure and ecosystem function: interactive effects in a bromeliadinsect community[END_REF], ranging from the rules by which communities are assembled [START_REF] Ce ´re ´ghino | Ants mediate the structure of phytotelm communities in an antgarden bromeliad[END_REF][START_REF] Ce ´re ´ghino | Ant-plant mutualisms promote functional diversity in phytotelm communities[END_REF] to the relationships between diversity and ecosystem function [START_REF] Leroy | Ants mediate foliar structure and nitrogen acquisition in a tankbromeliad[END_REF][START_REF] Srivastava | Reducing horizontal and vertical diversity in a food web triggers extinctions and impacts functions[END_REF].

In tank-bromeliads, detritus constitutes a source of nutrients for the aquatic food web as well as for the host plant itself. Debris-chewing invertebrates process incoming litter. Small particles of organic matter, including faeces, are then washed into the plant pools where particulate organic matter (POM) is further processed in the gut of invertebrate collectors and filterers. Dead organisms, litter and faecal particles, which collect in the leaf bases, are utilised by bacteria and other microorganisms. Observations and experimentation have revealed that, at a given site (i.e. a single location), ecosystem functions in bromeliad tanks are strongly influenced by the composition of the community inhabiting them; for instance, nitrogen uptake by the bromeliad is positively influenced by invertebrate species richness [START_REF] Leroy | Ants mediate foliar structure and nitrogen acquisition in a tankbromeliad[END_REF] while predators facilitate nutrient uptake by limiting the emergence of detritivorous insects that would otherwise represent a loss of nitrogen [START_REF] Ngai | Predators accelerate nutrient cycling in a bromeliad ecosystem[END_REF]. However, we do not know whether environmental conditions, such as available understorey light and incoming litter, determine community structure and whether this in turn determines ecosystem function. Moreover, our understanding of the relationships between food web structure and environment in bromeliads primarily comes from ecological studies on a target fraction of the food web (e.g. invertebrates, insects, microorganisms excluding components such as bacteria and viruses).

Taxonomic resolution is inevitably very heterogeneous when dealing with many phyla simultaneously, both because compromises must be made to obtain sufficient information to detect the expected biological responses in the various taxa and because of the cost of obtaining this information (e.g. bacteria versus insects, lack of taxonomic knowledge in tropical areas). Biological traits have the potential to provide additional information on the mechanisms structuring entire communities because they reflect adaptations to environmental characteristics, including spatiotemporal variability and stochasticity [START_REF] Townsend | Species traits in relation to a habitat templet for river systems[END_REF]. Functional groups (FGs), mostly based on morphological and behavioural adaptations related to food acquisition, aggregate taxa into fewer categories than do species lists [START_REF] Bonada | Taxonomic and biological trait differences of stream macroinvertebrate communities between mediterranean and temperate regions: implications for future climatic scenarios[END_REF], thus simplifying a priori predictions of the responses of communities to environmental fluctuations.

This study focussed on the tank-bromeliads found in a primary rainforest characteristic of the eastern Amazon. We analysed the functional variability of the aquatic communities extracted from 171 tank-bromeliads ranging from 80 to 420 m above sea level in relation to environmental variables (incident radiation, POM, water volume, position in the host trees). To the best of our knowledge, there is no evidence of species-specific associations between tank-bromeliad species and particular sets of aquatic animals [START_REF] Benzing | Vascular Epiphytes. General Biology and Related Biota[END_REF]. Instead, when several tank-bromeliads co-occur, the set of plant species present form a metahabitat. Earlier bromeliad studies highlighted the role of habitat characteristics (amount of water, surrounding vegetation) and biological interactions (predation, competition) in shaping species assemblages in these natural microcosms [START_REF] Armbruster | Factors influencing community structure in a South American tank bromeliad fauna[END_REF][START_REF] Srivastava | Habitat structure, trophic structure and ecosystem function: interactive effects in a bromeliadinsect community[END_REF]. Therefore, assuming that the bromeliads that grow in the forest understorey collect leaf litter and nutrients leached from the canopy, we predicted that their detritus-based food webs rely on high abundances of microbial decomposers such as bacteria and ⁄ or fungi. Conversely, we expected that bromeliads that grow in open areas would collect lower amounts of detritus, but that higher incident light would promote the growth of algae. Subsequently, we predicted that algae in open areas could support a non-detrital food web (in addition to the detrital food web), thus promoting functional diversity at higher trophic levels.

Methods

Study area

This study was conducted in a primary rainforest characteristic of the eastern Amazon near the Nouragues Tropical Forest Research Station (4°5¢N, 52°41¢W, French Guiana). The area is totally uninhabited and current anthropogenic disturbance is negligible (see [START_REF] Sarthou | Successional patterns on tropical inselbergs: a case study on the Nouragues inselberg (French Guiana)[END_REF] for a detailed description). The Station is located in the Nouragues Nature Reserve (Fig. 1), 100 km as the crow flies from Cayenne, and 40 km from the nearest village (Regina). The area is delineated by hills (elevation <120 m a.s.l.) and by the Balenfois Mountains (maximum elevation: 460 m a.s.l.). The geology of the area is dominated by two types of substrate: Carribean granite and metavolcanic rocks from the Paramaca series [START_REF] Bongers | Nouragues. Dynamics and Plant-Animal Interactions in a Neotropical Rainforest[END_REF]. A granitic inselberg dominates the Station (maximum elevation: 420 m a.s.l.). The vegetation is composed of primary rainforest with small, naturally occurring stands of palm forest on poorly drained terrain ('pino swamps'), liana forests (probably indicating past Amerindian slash-and-burn agriculture) and bamboo thickets. On the inselberg, patches of savannah ('rocksavannahs') can be found, intermingled with patches of shrubby trees belonging to the Clusiaceae, Myrtaceae and Bombacaceae families [START_REF] Sarthou | Shrub vegetation on tropical granitic inselbergs (French Guiana)[END_REF]. The climate is tropical moist with 3000 mm of yearly precipitation distributed over 280 days. There is a major reduction in rainfall between September and November (the dry season) and another shorter and more irregular dry period in March. The maximum monthly temperature averages 33.5 °C (32.1-35.8 °C) and the monthly minimum, 20.3 °C (19.7-21 °C).

Field data and techniques

Sampling was carried out over 12 days, from 23 March to 3 April 2006. We selected five sampling sites located along a gradient of elevation above sea level. We chose the six most common bromeliad species among the 24 that are present in the area around the Nouragues Tropical Forest Research Station (see [START_REF] Bongers | Nouragues. Dynamics and Plant-Animal Interactions in a Neotropical Rainforest[END_REF] for a species list). Guzmania lingulata (L.) Mez was the only tank-bromeliad at site 1 (80 m a.s.l., Nouragues Creek, rainforest). At site 2 (130 m a.s.l., transition forest), G. lingulata co-existed with two other common species, namely Aechmea melinonii Hooker and Vriesea pleiosticha (Grisebach) Gouda. At site 3 (160 m a.s.l.), Pitcairnia geyskesii (L. B. Smith) Varadajaran & Gilmartin, was abundant on a plateau situated at the base of the inselberg. Site 4 (390 m a.s.l.) was a forested area where Vriesea splendens (Brongniart) Lemaire was the only tank-bromeliad. Finally, Catopsis berteroniana (Schultes f.) Mez was restricted to the summit of the granite inselberg (site 5; 420 m a.s.l.), where it grew as an epiphyte on Clusia minor shrubs. Overall, 171 bromeliad individuals (i.e. 171 aquatic communities) were sampled. The number of plants sampled per bromeliad species and their main characteristics are provided in Table 1. To characterise the percentage of total incident radiation above the bromeliads, we used hemispherical photography. Photographs were taken near dusk (to avoid direct sunlight) above every bromeliad. We used a height adjustable tripod and a digital camera (Nikon Coolpix 4500, Tokyo, Japan) equipped with a Nikon Fisheye converter lens (FC-E8 0.21X) that provides a 180°canopy view. We analysed the images using the Gap Light Analyzer (GLA, Burnaby, BC, Canada and Millbrook, NY, USA) 2.0 image processing software to calculate the percentage of total incident radiation [START_REF] Frazer | Gap Light Analyzer (GLA) 2.0: Imaging Software to Extract Canopy Structure and Gap Light Transmission Indices from True-Colour Fisheye Photographs: Users Manual and Program Documentation[END_REF]. The percentages of transmitted light are provided in Table 1.

For legal reasons (the Station is located in a protected area), the bromeliads could not be removed from their host trees. Therefore, we used flexible plastic tubes (length 10-30 cm, diameter 2-5 mm) connected to 50-mL syringes and micropipettes (with the end trimmed to widen the orifice) to sample the water retained in the tanks. The elevation above ground (m) was measured for each plant. We then carefully emptied the wells in each plant by sucking the water out using tubes and pipettes of appropriate dimensions. This technique, although less efficient than plant dissection, has already been successfully used by us and others [START_REF] Ce ´re ´ghino | Ants mediate the structure of phytotelm communities in an antgarden bromeliad[END_REF][START_REF] Ce ´re ´ghino | Ant-plant mutualisms promote functional diversity in phytotelm communities[END_REF][START_REF] Jocque | How effective are non-destructive sampling methods to assess aquatic invertebrate diversity in bromeliads?[END_REF]. It was used for all of the samples, and most of the water (>95%) was collected. The water volume extracted (mL) was recorded for each plant. A subsample of 20% of the collected volume was fixed with 4% (final concentration) formaldehyde for enumeration of microorganisms and detrital particles (see below). The remaining water (and its contents) was preserved in 70% ethanol and used to sort and count the metazoans.

Laboratory analyses

Subsamples for microbial analyses were gently mixed by inversion and the water removed from the supernatant after a 2-min sedimentation period for the larger debris. This procedure enabled us to conduct a thorough microscopic inspection of the samples and prevented size-fractionation and the subsequent loss of fragile microorganisms. For the enumeration of viruses and bacteria, 1-mL subsamples were filtered through 0.02 lm pore size Anodisc filters (Whatman, Whatman International Ltd, Maidstone, UK) using cellulose acetate backing filters (1.2 lm pore size) and were stained with SYBR Green I fluorochrome according to the method developed by [START_REF] Noble | Use of SYBR Green I for rapid epifluorescence counts of marine viruses and bacteria[END_REF]. Slides were prepared using the medium Citifluor (Citifluor, London, UK) amended with c. 20% (v ⁄ v) of Vecta Shield (Vector Laboratories, Peterborough, UK), resulting in a highly stable fluorescence of the fluorochrome. The slides were stored at )20 °C before counting. Bacteria were distinguished from virus-like particles on the basis of their relative size and brightness through 40-60 fields of view using an epifluorescence microscope (Leica DC 300F model, Leica microsystems, Wetzlar, Germany) at 1250• magnification. The abundances of algae, fungi, ciliates and rotifers were estimated from settled samples [START_REF] Utermo | Zurvervolkommungder quantitativen phytoplankton Methodik[END_REF]) using an inverted Leitz Fluovert FU microscope at 500• magnification by scanning the entire chamber area. Samples with a high detrital particle content were diluted, and total counts were pooled from triplicate subsamples. The abundance of fungi must be considered a density index rather than a real estimate because only the spore stages (conidia) were considered. POM (i.e. detrital particles <300 lm) were also counted from settled samples using the inverted microscope through 30-50 microscopic view settings and separated into three size classes (<30, 30-150 and 150-300 lm). We used particle counts instead of mass because size-fractionation by filtration was not possible with the smallest samples where most of the water permeates the detritus (e.g. P. geyskesii, G. lingulata).

Macroinverbrates were keyed to genus or morphospecies and counted under a stereomicroscope. They were then partitioned into FGs inspired by the work of [START_REF] Merritt | An Introduction to The Aquatic Insects of North America, 3rd edn[END_REF]. We also relied on the information available in [START_REF] Kitching | Food Webs and Container Habitats: The Natural History and Ecology of Phytotelmata[END_REF] for phytotelm organisms. These FGs were filterers (sift fine particulates, including living microorganisms, from the column of water); collectors (gather fine particulates of organic matter from the accumulated debris); and predators (feed on other animals). The macroinvertebrates were largely dominated by aquatic insects. Other taxa included oligochaetes that were assigned to the collectors. After counting all organisms, density was expressed as the number of individuals per unit volume of water (ind mL )1 ). It is worth noting that Dendrobates ventrimaculatus (Shreve) adults and tadpoles (amphibians) were found in A. melinonii, V. splendens and V. pleiosticha. However, because of their rarity, these organisms were not added to the analyses in this study.

Data analysis

The relationships between all of the environmental variables, bromeliads and abundance of FGs were examined using multivariate ordination. The environmental variables were elevation a.s.l. (m), incident radiation (%), elevation above ground (on the host tree, m), water volume (mL), POM<30 lm (%), POM 30-150 lm (%), POM 150-300 lm (%) and total POM present in the water (number of particulates per mL). Host tree was not included as a factor in our analysis because it was generally confounded by species of bromeliad (e.g. all C. berteroniana on Clusia minor, A. melinonii, V. splendens and P. geyskesii rooted on the soil). Organism abundance data were log (n + 1) transformed prior to analysis. An initial detrended correspondence analysis (DCA) in CANOCO v4.5 showed that a linear model was the most applicable because of low species turnover (gradient = 0.548) along Axis 1 (Leps ˇ& S ˇmilauer, 2003); thereafter, a redundancy analysis (RDA) was used to examine functional group relationships with bromeliads and with the eight environmental variables. Forward selection was employed to test which of the eight environmental variables explained significant (P < 0.05) proportions of the species variance. The significance of explanatory variables was tested against 500 Monte Carlo permutations.

Results

Axes 1 and 2 of the RDA accounted for 28.7% of the total species variance and 98% of the Functional group (FG)environment relationship (Fig. 2). Eigenvalues for Axes 1 and 2 were 0.23 and 0.05, respectively. Functional groupenvironment correlations were 0.667 for Axis 1 and 0.426 for Axis 2. Forward selection identified three variables as explaining a significant amount of the FG variance (bold arrows in Fig. 2a): incident radiation (P = 0.002), percentage of 150-300 lm POM (P = 0.006) and elevation above ground (P = 0.04). Incident radiation accounted for the greatest proportion of total canonical eigenvalues (22%; F = 42.38; P = 0.002). The scatterplot of the RDA segregated two subsets of bromeliads along Axis 1 according to their environmental context, namely forest sites (understorey, low incident radiation, right part of the scatterplot), and the inselberg (overstorey, high incident radiation, left part of the scatterplot) (Fig. 2a). Axis 1 thus displayed a gradient of habitat openness with incident radiation ranging from 15 to 24% and from 62 to 72% in closed and open areas, respectively (Table 1). Axis 2 represented a gradient of POM size [from small (bottom) to large (top)], and, to a lesser extent, a gradient of elevation above the ground [from low (bottom) to high (top)]. In other words, regardless of their location and species, all of the bromeliads were likely to bear small to large amounts of large POM (150-300 lm), but bromeliads at higher elevations in the host trees tended to have larger proportions of large POM. Guzmania lingulata, V. pleiosticha and C. berteroniana contributed to this pattern because they are epiphytes, whereas other bromeliads were rooted in the soil (Table 1).

Algae, rotifers, collectors and predators dominated the bromeliad food web in exposed areas of the inselberg, whereas filterers had their highest densities at forest sites (Fig. 2b, Table 1). More specifically, algal densities reached 88.4 • 10 2 ± 22.1 • 10 2 and 25.3 • 10 2 ± 4.7 • 10 2 ind mL )1 in P. geyskesii and C. berteroniana, respectively. Bumilleriopsis (Xanthophyceae) dominated the algal assemblage in this area. Algae were apparently abundant in understorey A. melinonii bromeliads (forest), but this was due to very high numbers of mixotrophic Euglenophyceae (Phacus sp. combining autotrophic and heterotrophic nutrition, mean = 72.9 • 10 2 ± 14.0 • 10 2 ind mL )1 ). Rotifer density was 5-20 times higher in the exposed than in the shaded bromeliads. A small bdelloid from the genus Habrotrocha (60 lm in length) was the numerically dominant species. Predatory insects (e.g. Veliidae heteropterans, Coenagrionidae odonates, Toxorhynchites culicids) and invertebrate collectors (e.g. Chironominae and Tanytarsus chironomids, and Aulophorus superterrenus Michaelsen Oligochaetes) were three times and 1.5-2 times more abundant in the exposed than in the shaded bromeliads, respectively (Table 1). Conversely, filter-feeding insects (Wyeomyia and Culex mosquito larvae) were on average 2-3 times more abundant in understorey bromeliads. Microorganisms such as viruses, bacteria and fungi did not show any clear density patterns among the bromeliads (Table 1); however, their densities clearly increased along a gradient of a decreasing percentage of large POM (150-300 lm) in the tanks (Fig. 2b). Ciliates (Colpoda sp.) were found in a few plants at low densities, at both open and closed sites.

Discussion

Up to 90% of all primary productivity may enter food webs as dead organic matter or detritus [START_REF] Moore | Detritus, trophic dynamics and biodiversity[END_REF]. In aquatic ecosystems in general, detrital inputs form a strong trophic link between plant production, decomposer microorganisms and larger metazoan detritivores and their predators. The nature and extent of the vegetation that surrounds these systems therefore has a strong influence on food web structure through food quality and availability.

In neotropical forests, a substantial fraction of the freshwater available is impounded within the rosettes of bromeliads that form freshwater islands (i.e. a metahabitat) in a terrestrial matrix. The 171 freshwater communities extracted from six tank-bromeliad species were subject to a wide range of environmental conditions. The first two axes of our RDA explained 98% of the functional groups-environment relationship, using a limited number of variables. This allows us to provide quantitative data on the distribution of a very wide range of freshwater organisms in rainforests and to determine the major factors that influence the functional structure of phytotelm food webs.

A key result of our study is that incident radiation, vertical position on the host trees, and POM size (a series of indicators of habitat openness) explain significant differences in the functional structure of bromeliad food webs in forest environments and, to a lesser extent, between individual bromeliads located at the same site (e.g. epiphytic versus soil bromeliads). Previous studies concluded that water volume (an indicator of habitat size) and the total amount of POM (an indicator of available food at the base of the food web) play key roles in shaping community composition in water-filled bromeliads [START_REF] Armbruster | Factors influencing community structure in a South American tank bromeliad fauna[END_REF][START_REF] Jabiol | Structure of aquatic insect communities in tank-bromeliads in a East-Amazonian rainforest in French Guiana[END_REF]. Overall, we note that tank-bromeliads show a gradient of detritusbased to algal-based food webs from understorey to overstorey. Our results were used to draw diagrammatic representations of these food webs (Fig. 3). This conceptual framework highlights the dominant taxa and energy pathways and is discussed below.

Tank-bromeliads growing in the forest understorey had higher amounts of detritus in their tanks and displayed higher abundances of filter-feeding insects, while densities of bacteria and fungi increased along a gradient of decreasing POM size. This suggests that leaf-litter breakdown by fungi promotes bacterial growth by increasing the surface area for colonisation [START_REF] Allan | Stream Ecology[END_REF][START_REF] Sigee | Freshwater Microbiology -Biodiversity and dynamic interactions of Microorganisms in the aquatic environments[END_REF]. Microbial activities are also known to enhance leaf palatability for shredding invertebrates, while microbial decomposition may be facilitated by the fragmentation of detritus by insects [START_REF] Gessner | A perspective on leaf litter breakdown in streams[END_REF]. In our study, the metazoan consumers were largely consumers of bacteria and fungi rather than direct shredders of leaf material. This suggests that POM decomposition is mainly the result of microbial activity in understorey bromeliads, and that microbial and POM consumers are mainly represented by filter feeders in these plants.

Abundances of algae, rotifers, collector and predatory invertebrates increased with greater exposure of the plants to sunlight and lower amounts of detritus on the inselberg. Algae were observed in the earliest limnological studies of tank-bromeliads, especially in plants in the canopy that are exposed to direct sunlight [START_REF] Maguire | Phytotelmata: biota and community structure determination in plant-held waters[END_REF]. [START_REF] Laessle | A micro-limnological study of Jamaican bromeliads[END_REF] only found algae in bromeliads on open, rocky outcrops in Jamaica. More recent research on bromeliad phytotelmata has frequently ignored algae (but see [START_REF] Brouard | Are algae relevant to the detritusbased food web in tank-bromeliads?[END_REF][START_REF] Marino | Habitat size determine algae biomass in tankbromeliads[END_REF]. The current paradigm is that these freshwater microcosms sequester and recycle nutrients to the bromeliad, and therefore, if algae are present and abundant, they should be competing with the bromeliad for dissolved inorganic nutrients. Nitrogen, rather than phosphorus, limits the productivity of bromeliads [START_REF] Ngai | Predators accelerate nutrient cycling in a bromeliad ecosystem[END_REF]. Ammonium (NH 4 + ) is the primary source of nitrogen for the bromeliad [START_REF] Inselsbacher | Microbial activities and foliar uptake of nitrogen in the epiphytic bromeliad Vriesea gigantea[END_REF], as well as for algae [START_REF] Sigee | Freshwater Microbiology -Biodiversity and dynamic interactions of Microorganisms in the aquatic environments[END_REF]. Ultimately, both algae and collector and filterfeeding invertebrates could form a nitrogen sink in tank-bromeliads. Thus, the high algal density and biomass in exposed bromeliads suggests a trade-off between carbon (light) and nutrient availability. The interactions between bromeliads and algae remain poorly understood [START_REF] Marino | Habitat size determine algae biomass in tankbromeliads[END_REF], and further investigations are needed to accurately determine the implications of algae as nutrient competitors of tank-bromeliads. Information on nutrient uptake by freshwater algae comes from studies of taxa isolated from rivers, lakes and other large ecosystems [START_REF] Sigee | Freshwater Microbiology -Biodiversity and dynamic interactions of Microorganisms in the aquatic environments[END_REF], while the metabolism of bromeliad-dwelling algae is largely unknown. Moreover, mixotrophic Euglenophyceae that are frequent in bromeliads are able to absorb organic rather than inorganic nitrogen.

On the basis of cell volume, we estimate that algal biomass might exceed bacterial biomass in some of the exposed bromeliads: algae represented 22.1% (range: 1.6-104.4%) and 8.5% (0-114.1%) of the bacterial biomass in P. geyskesii and C. berteroniana, respectively. Their role in nutrient cycling as primary producers and prey for metazoan grazers is therefore potentially important, especially in C. berteroniana where amounts of POM are low compared to understorey bromeliads, but where functional diversity is the highest. The same is true of some understorey bromeliads (A. melinonii in our study) that support large numbers of mixotrophic Euglenophyceae. Pitcairnia geyskesii was located in sun-exposed areas but hosted the highest algal densities and did not contain fungi, filterers or predators. This particular situation is certainly linked to the vegetative traits of this bromeliad which has very narrow leaves and impounds very small volumes of water (8 mL per plant on average), although exposed to a high level of incident light.

Given the small size of the tanks, it is likely that congruent patterns in functional group distributions were also generated through biotic interactions. Most viruses, for instance, are bacteriophages [START_REF] Fuhrman | Virus in marine planktonic systems[END_REF][START_REF] Weinbauer | Distribution and life strategies of two bacterial populations in a eutrophic lake[END_REF] and have patterns that are congruent with bacteria [START_REF] Weinbauer | Ecology of prokaryotic viruses[END_REF]. This is also true for tank-bromeliads (this study). The ubiquity and abundance of viruses (10 4 to 10 8 particulates mL )1 in lakes according to [START_REF] Bettarel | Viral lysis, flagellate grazing potential, and bacterial production in Lake Pavin[END_REF] 1.1 • 10 7 to 2.1 • 10 7 particulates mL )1 in the bromeliads that we studied) suggest that they are heavily involved in the dynamics and functioning of bromeliad food webs. Finally, we did not find clear density patterns for viruses, bacteria or fungi. It should be noted that there are certainly several taxa present within each of these groups, but taxonomic limitations prevented us from obtaining more precise data that would be useful to bring out more specific patterns for the microbial components of the bromeliad food web.

In conclusion, our study provides evidence that tankbromeliads, which span a broad range of ecological conditions, promote the diversity of aquatic food webs in neotropical forests. The taxonomic composition of the bromeliad food web primarily depends on the plants' vegetative traits (i.e. size, leaf display) [START_REF] Montero | The phytotelmata and foliage macrofauna assemblages of a bromeliad species in different habitats and seasons[END_REF][START_REF] Ce ´re ´ghino | Ant-plant mutualisms promote functional diversity in phytotelm communities[END_REF], but its functional structure is mainly driven by the surrounding environment (this study). Although detritus is a main source of energy in closed forests [START_REF] Benzing | Bromeliaceae: Profile of an Adaptive Radiation[END_REF], recent bromeliad research has shown that the role of algae as a potential energy source in open habitats is also a topic of interest [START_REF] Brouard | Are algae relevant to the detritusbased food web in tank-bromeliads?[END_REF][START_REF] Marino | Habitat size determine algae biomass in tankbromeliads[END_REF]. Our results therefore shed new light on the ecology of bromeliad ecosystems, which are usually categorised as detritusbased systems, but where algal production can certainly form the basis of a non-detrital food web.

Fig. 1

 1 Fig. 1 Map of the study area and location of the five sampling sites (1-5). The grey area delineates the inselberg (maximum elevation 430 m a.s.l.).

Fig. 2

 2 Fig. 2 Redundancy analysis (RDA) biplots. (a) Bromeliads and environmental variables. Environmental variables are represented as vectors; directions show the gradients, arrow length represents the strengths of the variables on the ordination space. Abbreviations are used to identify the bromeliad species (GL = Guzmania lingulata; PG = Pitcairnia geyskesii; VP = Vriesea pleiosticha; AM = Aechmea melinonii; CB = Catopsis berteroniana; VS = Vriesea splendens). (b) distribution of microbial and metazoan functional groups in ordination space. Abbreviations for environmental variables in panels (a) and (b) are as follows: EG = elevation above ground (m); EL = elevation above sea level (m); IR = percentage of total incident radiation above the bromeliads; WV = water volume (mL); Total POM = amount of particulate organic matter (number of particles mL); POM<30, POM 30-150 and POM 150-300 = percentage of particles <30, 30-150 and 150-300 lm in size. Variables explaining a significant (P < 0.05) proportion of the functional group variance are represented by bold arrows.

Fig. 3

 3 Fig. 3 Diagrammatic representations of the detrital food web (understorey bromeliads, forest sites) and the detrital-algal-based food web (overstorey bromeliads, sun-exposed areas). The numerically dominant organisms are grouped by functional groups (1-6), and arrows show the proposed energy pathways. Bold characters ⁄ boxes indicate higher density, and arrow thickness indicates differences in the strength of the relationships. 1 = Heterotrophic microorganisms, 2 = mixotrophic microorganisms, 3 = autotrophic microorganisms, 4 = filter-feeding insects, 5 = macroinvertebrate collectors, 6 = predatory insects.

Table 1

 1 Main characteristics of the 171 plants (6 bromeliad species) that were sampled during the study near the Nouragues station (French Guiana) in 2006, and mean densities (individuals mL )1 ) for the various functional groups

		Guzmania	Aechmea	Vriesea	Vriesea	Pitcairnia	Catopsis
		lingulata	melinonii	pleiosticha	splendens	geyskesii	berteroniana
	Number of plants sampled per site
	Site 1: rain forest	38	-	-	-	-	-
	Site 2: transitional forest	17	30	32	-	-	-
	Site 3: inselberg, open area	-	-	-	-	37	-
	Site 4: inselberg, forest area	-	-	-	31	-	-
	Site 5: summit inselberg, open area	-	-	-	-	-	32
	Environmental variables
	Elevation a.s.l. (m)	80-130	130	130	390	160	420
	Incident radiation (%, mean ± SE)	15.8 ± 2.3	24.2 ± 3.0	24.2 ± 3.0	16.3 ± 3.0	62.3 ± 7.2	72.5 ± 5.8
	Elevation above ground (m)	1.5 ± 0.2	0	0.4 ± 0.1	0	0	0.7 ± 0.1
	Water volume (mL, mean ± SE)	16.1 ± 9.6	134.0 ± 105.2	71.7 ± 49.8	50.8 ± 27.8	8.3 ± 1.6	40.6 ± 20.2
	Total POM (10 4 particulates mL )1 )	27.2 ± 6.2	13.5 ± 2.6	14.0 ± 2.5	15.6 ± 3.0	8.2 ± 0.02	3.0 ± 0.05
	POM<30 lm (% ± SE)	75.0 ± 2.2	63.4 ± 13.2	69.1 ± 12.4	69.7 ± 13.4	80.3 ± 20.1	57.4 ± 10.6
	POM 30-150 lm (% ± SE)	17.8 ± 1.3	20.3 ± 1.9	23.3 ± 4.0	23.1 ± 4.5	15.3 ± 3.8	25.8 ± 4.8
	POM 150-300 lm (% ± SE)	7.2 ± 1.1	11.3 ± 2.2	8.6 ± 1.5	7.2 ± 1.4	4.4 ± 1.1	16.9 ± 3.1
	Functional groups (ind mL )1 ± SE)
	Viruses • 10 7	2.1 ± 0.2	1.2 ± 0.2	1.9 ± 0.3	1.9 ± 0.6	1.8 ± 0.4	1.1 ± 0.2
	Bacteria • 10 6	8.6 ± 1.1	6.3 ± 1.2	9.0 ± 1.6	7.5 ± 1.4	9.6 ± 2.4	5.6 ± 1.0
	Fungi • 10 2	13.4 ± 6.7	3.1 ± 0.6	7.9 ± 1.4	7.1 ± 1.4	<1.0	7.3 ± 1.3
	Algae • 10 2	10.0 ± 8.1	73.0 ± 14.0	0.4 ± 0.08	7.8 ± 1.5	88.4 ± 22.1	25.3 ± 4.7
	Ciliates	<1.0	<1.0	8.1 ± 8.0	1.0 ± 1.0	<1.0	2.5 ± 0.5
	Rotifers	10 ± 3	26 ± 5	21 ± 4	13 ± 3	221 ± 55	147 ± 27
	Insects, filterers	0.15 ± 0.03	0.1 ± 0.01	0.07 ± 0.01	0.08 ± 0.01	-	0.04 ± 0.001
	Invertebrates, collectors	0.04 ± 0.01	0.04 ± 0.001	0.03 ± 0.005	0.02 ± 0.01	0.06 ± 0.005	0.08 ± 0.01
	Insects, predators	0.05 ± 0.03	0.07 ± 0.01	0.05 ± 0.01	0.1 ± 0.02	-	0.36 ± 0.06

Acknowledgments

Financial support for this study was provided by the Programme Amazonie II of the French Centre National de la Recherche Scientifique (Project 2ID) and the Programme Convergence 2007-2013 (Re ´gion Guyane) from the European Community (Project DEGA). The English text was proofread by Andrea Yockey-Dejean. Two anonymous reviewers provided insightful comments on an earlier version of this manuscript.

Literature cited