Synechocystis sp. PCC 6803. We could identify over 300 phosphorylation events in cultures grown on nitrate as exclusive nitrogen source. Chemical dimethylation labeling was applied to investigate proteome and phosphoproteome dynamics during nitrogen starvation. Our dataset describes the most comprehensive (phospho)proteome of Synechocystis to date, identifying 2382 proteins and 183 phosphorylation events and quantifying 2111 proteins and 148 phosphorylation events during nitrogen starvation. Global protein phosphorylation levels were increased in response to nitrogen depletion after 24 h. Among the proteins with increased phosphorylation, the PII signaling protein showed the highest fold-change, serving as positive control. Other proteins with increased phosphorylation levels comprised functions in photosynthesis and in carbon and nitrogen metabolism. This study reveals dynamics of Synechocystis phosphoproteome in response to environmental stimuli and suggests an important role of protein Ser/Thr/Tyr phosphorylation in fundamental mechanisms of homeostatic control in cyanobacteria.Cyanobacteria have shaped the earth's biosphere as the first oxygenic photoautotrophs and still play an important role in many ecosystems. The ability to adapt to changing environmental conditions is an essential characteristic in order to ensure survival. To this end, numerous studies have shown that bacteria use protein post-translational modifications such as Ser/Thr/Tyr phosphorylation in cell signaling, adaptation, and regulation. Nevertheless, our knowledge of cyanobacterial phosphoproteomes and their dynamic response to environmental stimuli is relatively limited. In this study, we applied gel-free methods and high accuracy mass spectrometry toward the detection of Ser/Thr/Tyr phosphorylation events in the model cyanobacterium  Among these, the highest demand is for nitrogen, whose cellular abundance amounts to a carbon to nitrogen ratio of 5:1.Cyanobacteria constitute one of the most widely distributed groups of prokaryotes in the biosphere, where they inhabit almost all illuminated environments. They are oxygenic photoautotrophs, employing photosynthetic machinery that resembles that of plants, due to the endosymbiotic origin of chloroplasts. The substrate diversity of cyanobacteria is limited since photoautotrophs only need HCyanobacteria encounter many environmental fluctuations, such as changing light conditions, nutrient availability and the ambient physico–chemical properties . During evolution, these microbes have developed sophisticated regulator systems to adapt cellular processes and maintain metabolic homeostasis in response to these challenges. Nitrogen starvation induces in non-diazotrophic cyanobacteria a highly dynamic response, which can be classified in three phases. Their temporal occurrence depends on many environmental and intrinsic conditions  has received the most thorough investigations , referred to as S/T/Y phosphorylation or O-phosphorylation, was initially thought to occur exclusively in eukaryotic signal transduction. However, primarily due to advances in mass spectrometry proteomics technologies, it is now clear that this type of modification frequently occurs in bacteria serving an important role in prokaryotic signal transduction  in response to the cellular nitrogen supply. The effector molecule 2-oxoglutarate (2-OG) whose abundance reflects the cellular state of nitrogen assimilation   chromatography coupled to high accuracy mass spectrometry. The development of chemical stable isotope labeling by dimethylation of tryptic peptides enables the application of a cost efficient and reliable quantification strategy also to autotrophic organisms, without the need of profound genetic manipulation of amino acid metabolic pathways . However, 52.7% of the total protein coding genes was thus far not identified on the proteome level, indicating a need for improvement in protein extraction methods, particularly with respect to hydrophobic (membrane)proteins proteins, we compared three common, in gel-free approaches frequently used extraction methods in a qualitative experiment. Here, in total 301 phosphorylation events could be identified, of which 262 could be localized to a specific Ser, Thr, or Tyr residue. This dataset represents the so far most comprehensive qualitative phosphoproteome dataset of Synechocystis. It encouraged us to investigate phosphoproteome dynamics in response to nitrogen starvation in a quantitative manner employing for the first time in a bacterial system the method of chemical dimethylation labeling. Extracts from Synechocystis cultures grown in the presence of nitrate or ammonia and cultures subjected to nitrogen starvation were dimethyl labeled. Thereby, we could identify altogether 2382 proteins and 183 phosphorylation events. Of those, the dynamics of 2111 proteins and 148 phosphorylation events from two independent experiments could be quantified. The PII signaling protein, which served as a positive control to validate the experiments, showed the highest increase in phosphorylation level upon nitrogen starvation. Other proteins with increased phosphorylation levels comprised functions in photosynthesis and in carbon and nitrogen metabolism, suggesting an important role of protein S/T/Y phosphorylation in such a fundamental acclimation response.In the present work, we applied state of the art mass spectrometric analysis to define the phosphoproteome of Synechocystis sp. PCC 6803 substrain Kazusa was grown photoautotrophically at a constant photon flux density of 40 μmol photons m−2 s−1 in 5 mM NaHCO3 and 20 mM HEPES supplemented BG11 medium  or 5 mM ammonium chloride (NH4Cl) as exclusive nitrogen source, at 26°C. A stationary culture of Synechocystis in BG11 nitrate medium served as a stock for the final inoculation of experimental 500 mL batch cultures in the qualitative experiments. For the quantitative experiment 1, pre-cultures were grown from the stock in fresh nitrate or ammonia medium to optical density (OD)750 = 0.6–0.8 and were directly used as inoculum for the experimental 500 mL batch cultures. In experiment 2, the pre-cultures were repeatedly cultivated to OD750 = 0.6–0.8 and after five cycles used as inoculum for the experimental 500 mL batch cultures. All experimental cultures were inoculated to an initial OD750 = 0.2 and grown to OD750 = 0.6 with ambient air bubbling and magnetic stirring. Cultures were harvested by addition of 100 g ice for rapid cooling and centrifugation at 7477 × g for 10 min. The supernatant was removed and cell pellets were resuspended in 50 mL ice cold nitrogen-free BG11 medium and centrifuged again. Cell pellets were quick-frozen in liquid nitrogen. For nitrogen starvation conditions, nitrate grown cells (at OD750 = 0.6) were shifted to nitrogen-free BG11 medium by centrifugation with 3500 × g for 8 min at room temperature (RT). The supernatant was decanted, the cell pellet was gently overlaid with nitrogen-free BG11 medium, swiveled and decanted again and finally, the cell pellet was resuspended in the same volume as before centrifugation. Cells were kept for 24 h under previous incubation conditions and harvested as described.Wild-type  Rippka, , with ei4HCO3 buffer, subsequently. For protein digestion, 50 μg trypsin  in 500 μL NH4HCO3 buffer, pH 8.0, were added onto the filter and incubated overnight (o.n.) at 37°C. Peptides were collected in a fresh tube by centrifugation as before for 10 min and additional with 500 μL NH4HCO3 buffer.Protein extraction method A: Filter Aided Sample Preparation was performed as described previously , supplemented with 10 mM ethylenediaminetetraacetic acid (EDTA). Cell lysates were sonified on ice as described in method A and subsequently reduced with a final concentration of 10 mM DTT for 1 h at RT under agitation at 650 rpm on a shaker. Alkylation was performed with a final concentration of 5.5 mM IAA for 1 h at RT under agitation in the dark, followed by centrifugation at 13,000 × g for 5 min. Proteins in the supernatant were precipitated by mixing with eight volumes ice cold acetone and one volume ice cold methanol, followed by incubation at −20°C o.n. The precipitate was washed five times with 5 mL ice cold 80% (v/v) acetone in water and centrifugation at 1000 × g. The protein pellets were air dried and dissolved in urea buffer.Protein extraction method C: Protein extraction with Y-PER  and acetone/methanol precipitation: A frozen cell pellet was resuspended in 2 mL Y-PER, supplemented with 50 μg/mL lysozyme , c0mplete protease inhibitors (Roche) and phosphatase inhibitors, as described in method A. The suspension was incubated at 37°C for 20 min under agitation at 650 rpm. Cell lysates were sonified on ice and centrifuged as described before. Proteins in the supernatant were acetone/methanol precipitated, washed and dissolved in urea buffer as described in method B. Reduction and alkylation of cysteines was subsequently performed as described in method B.Protein concentration of samples derived from methods B and C were measured by Bradford assay (Bio-Rad) and subsequently pre-digested with endoproteinase Lys-C (Waco) for 3 h and digested further o.n. with trypsin  at RT. The protease/protein ratio was for both enzymes 1:100 (w/w). The resulting peptide mixtures from methods A, B, and C were acidified to pH 2.5 with trifluoroacetic acid (TFA) and desalted by solid phase extraction on SepPak C18 columns (Waters) according to the manufacturer's instructions.2O (Sigma-Aldrich) and NaBH3CN (Fluka) for light-, CD2O (Sigma-Aldrich) and NaBH3CN for medium-heavy and 13CD2O (Sigma-Aldrich) and NaBD3CN (Sigma-Aldrich) for heavy labeling were flushed with 15 min contact time through the column. Labeled peptides were washed with 5 mL HPLC Solvent A (0.5% acetic acid) on the column and eluted with HPLC Solvent B (80% acetonitrile in 0.5% acetic acid). For validation of labeling efficiency and correct mixing of the labeled peptides, two times 5 μg of each labeled sample (based on Bradford measurements) were used for separate measurements or mixed 1:1:1 and subjected after purification by C18 stage tips  dimethylation labeled as described previously . For quantitative proteome analysis, isoelectric focusing (OffGel fractionation strategy) was performed: For SAX, 100 μg peptides were loaded onto in-house packed SAX column (Empore™) and subsequently eluted stepwise in five pH fractions with 20 mM of acetic-, phosphoric- and boric acid at pH 3, 4, 5, 6 and 8, (pH adjusted with sodium hydroxide), respectively. Each fraction was subsequently purified by C18 stage tips. For OffGel fractionation, 100 μg of the labeled peptide mixture was separated on a linear 13 cm pH 3–10 IEF strip  and focused on a OffGel Fractionator 3100 (Aglient), according to the manufacturer's protocol, with 20 kVh at maximum 50 μA. Peptides focused in 12 fractions were separately purified by C18 stage tips.2 chromatography , pre-incubated with 2,5-dihydrobenzoic acid  for five consecutive rounds of 30 min. TiO2 spheres were washed and eluted as described previously and purified by C18 stage tips. The sample volume was reduced by vacuum centrifugation at RT and subjected to nano-LC-MS/MS analysis.Phosphopeptide enrichment for qualitative analyses was performed from the digested samples of protein extraction methods A, B and C by TiO2 chromatography from each 15 mg peptide mixture. For TiO2 chromatography, a modified protocol was applied: peptides were eluted from SepPak columns with 80% acetonitrile in 6% TFA and enriched for eight consecutive rounds with each 5 mg TiO2 spheres (5 μm Sachtopore NP) for 10 min. TiO2 spheres were washed twice with each 1 mL 80% acetonitrile in 6% TFA for 1 min at 1000 rpm and loaded onto C8 (Empore™) stage tips. The spheres were washed once with 200 μL HPLC Solvent B and phosphopeptides were eluted with 30 μL 5% ammonium hydroxide solution in 60% acetonitrile, pH 11, for 30 min at 4°C into 30 μL 20% TFA in a fresh tube. Eluates were purified by C18 stage tips. The sample volume was reduced by vacuum centrifugation at RT and subjected to nano-LC-MS/MS analysis.For the quantitative study, labeled phosphopeptides were enriched by TiO2-enriched samples) segmented linear gradients with 5–90% HPLC solvent B. Eluted peptides were directly ionized and measured on a LTQ Orbitrap XL , or LTQ Orbitrap Elite mass spectrometer (quantitative study). Mass spectrometers were operated in the positive ion mode.For nanoLC-MS/MS analyses of proteome and phosphoproteome samples, peptides were loaded onto an in-house packed 15 cm reverse-phase C18  nanoHPLC column on an EasyLC nano-HPLC (Proxeon Biosystems). Separation was performed by 90 min (for OffGel-separated samples) or 130 min  scan in the Orbitrap mass analyzer was acquired at resolution 60,000 and scan range of m/z 300–2000, followed by collision induced dissociation (CID) of the 5 (phosphoenrichment) or 15 (proteome) most intense multiply charged ions in the linear ion trap mass analyzer (LTQ). For phosphoproteome analysis, multi stage activation (MSA) in all MS/MS events with neutral losses of phosphoric acid on singly (−97.97 Th), doubly (−48.99 Th) or triply (−33.66 Th) charged precursor ions, was activated.The LTQ Orbitrap Elite was conducting higher energy collision dissociation (HCD) of the 20 most intense multiply charged ions at the same scan range at resolution 120,000.Dynamic exclusion of sequenced precursor ions for 90 s and the lock mass option  via the PRIDE partner repository  and Uniprot (http://www.uniprot.org), containing 3672 and 3507 protein sequences and 245 common contaminants, with the following database search criteria: Trypsin was defined as cleaving enzyme and up to two missed cleavages were allowed. Carbamido-methylation of cysteines was set as a fixed and methionine oxidation, protein N-termini acetylation and S/T/Y phosphorylation were set as variable modifications. Light-, medium-heavy- and heavy- dimethylation labeling on peptide N-termini and lysine residues was defined for samples from the quantitative experiments. The initial mass tolerance of precursor ions was limited to 6 ppm and 0.5 ppm for fragment ions. False discovery rates (FDRs) of peptides and proteins were set to 1%, respectively. Quantification of dimethylation labeled peptides required at least two ratio counts. Peptides were only allowed with a posterior error probability (PEP) <1% at the peptide- and <5% at the phosphopeptide level. Phosphopeptide MS/MS spectra were further manually filtered and validated with stringent acceptance criteria: Phosphorylation site localization with ≥ 75% localization probability was manually validated as well as comprehensive coverage of b- and y-ion series for CID and HCD spectra as well as a low noise to signal ratio (Supplementary MS Spectra). To exclude quantitation bias of phosphopeptide ratios due to fluctuations of corresponding proteins, phosphopeptide ratios were normalized by division with corresponding protein ratios. Significance analysis of regulated phosphorylation events was performed with Perseus software (version 1.5.0.31), normalized phosphopeptide ratios were log2 transformed and plotted against the respective log10 transformed phosphopeptide intensities. Significantly regulated phosphorylation events were identified by significance A analysis with a p-value of 0.05.All raw MS spectra were processed with MaxQuant software suite (version 1.5.1.0) , respectively. Pre-cast Tris-glycine 4–20% gradient SDS polyacrylamide gels (NuSep) or 4–16% gradient native polyacrylamide gels (Serva) were used according to the manufacturer's protocols. Protein transfer was performed on a semi-dry blotting system (peqlab) onto polyvinylidene fluoride  membranes. Membranes were blocked o.n. in TBS and 1% Tween20 at 4°C. Polyclonal primary antibodies against the PII protein  or the AtpB protein  were incubated for 2 h at RT in a 1:5000 dilution in TBS-T after washing of the membrane with TBS-T buffer. Membranes were washed again and peroxidase coupled secondary antibody (Sigma A6154) was added in a 1:2000 dilution for 1 h at RT. Proteins were visualized by Lumi-Light Plus detection reagent (Roche).Proteins from 2 mL cell culture aliquots were extracted in 50 mM Tris/HCl, 5 mM EDTA buffer, pH 7.4, with a RiboLyser, conducting five cycles with a speed of 6.5 m sSynechocystis PCC 6803, we first performed a conventional qualitative gel-free phosphoproteome analysis, based on TiO2 enrichment of phosphopeptides from a tryptic digest of the crude protein extract, followed by mass spectrometry (MS) analysis. In order to achieve the most comprehensive coverage of the phosphoproteome, we compared three common sample preparation workflows for MS identification of phosphorylation events as well as for total proteome analysis (see below). Based on the obtained results, we applied the most successful protein extraction strategy for a quantitative study to analyze phosphoproteome dynamics in response to nitrogen starvation.As an initial step to systematically approach the global phosphoproteome of Synechocystis culture at mid exponential growth phase (OD750 = 0.6) and split into three equal parts that were subjected to the following workflows: (A) Filter Aided Sample Preparation (FASP), (B) protein extraction as in the FASP protocol in combination with acetone/methanol protein precipitation, and (C) extraction with Y-PER in combination with acetone/methanol protein precipitation . For total proteome analyses, one portion of the sample was fractionated into five fractions by strong anion exchange chromatography. The remaining sample was used for five consecutive rounds of phosphopeptide enrichment by TiO2 chromatography. All samples were analyzed by nanoLC-MS/MS on an LTQ Orbitrap XL mass spectrometer.Cells were harvested from a nitrate-grown 2 chromatography. Method A recovered 1835 proteins, method B 2014 proteins and method C 1983 proteins in total  proteins detected from the TiOWe concluded that all three methods had a similar performance on the protein level; however, striking differences in the performance were observed on the phosphoproteome level, with method B obviously being the method of choice. These results clearly demonstrate the importance of a potent and suited protein extraction protocol for the analysis of the phosphoproteome in organisms with a high content of membranous compartments such as cyanobacteria. As evidenced here, the overall number of identified proteins did not automatically lead to high numbers of identified phosphorylation events. Since phosphorylation events have in many cases a low occupancy in prokaryotic organisms proteome in response to nitrogen starvation. Since autotrophically growing cyanobacteria are not suitable for metabolic stable isotope labeling, we used a protocol in which samples are labeled after protein digestion, using stable isotope dimethylation of peptides on N-termini and lysine side chains , ammonium , and nitrogen starved  cells were then mixed in a ratio of 1:1:1 based on the underlying protein amounts. Correct mixing of labeled peptides was validated by a dedicated MS measurement. The mixed sample was used for proteome and phosphoproteome analyses. For proteome analysis, isoelectric focusing (IEF) of the peptide mixtures was performed (OffGel strategy) and phosphopeptide enrichment was conducted by TiO2 chromatography in two technical replicates. All samples were analyzed by nanoLC-MS/MS on an LTQ Orbitrap Elite mass spectrometer. The experimental workflow is shown in Supplementary Information Proteins were extracted from cell pellets by “method B” as described in Materials and Methods, precipitated by acetone-methanol mixture and subjected to in-solution digestion by trypsin. Obtained peptide solutions were loaded onto C18 reverse phase columns and subsequently chemically labeled by dimethylation. Different isotopoloques of formaldehyde and cyanoborohydride were used: Samples from nitrate grown cultures were light labeled with CH−3 ratios and 0.937 for –N/NH+4 ratios for the first experiment and 0.948 for –N/NO−3 ratios and 0.913 for –N/NH+4 ratios for the second experiment, revealing a good technical reproducibility and high overlap of phosphorylation events. Correlation plots from technical replicates of phosphopeptide ratios are shown in the supplement  was 0.18% at the peptide level and 1.04% at the protein level. Of the identified 2382 proteins, 2111 proteins fulfilled the requirement of having at least two quantification events (ratio counts) in at least one of both experiments and were considered as quantified.Synechocystis sp. PCC 6803 proteome by combining previously published proteome studies. Thereby, they reported a total of 1738 experimentally identified proteins, which amounts to 47.3% of the possible 3672 protein coding genes listed on Cyanobase , with a quantification rate of 57.5% of the total proteome (2111 of 3672). These numbers are presumably close to the completeness of the expressed proteome under the actual experimental conditions, since of the previously identified 1738 proteins, 90.0% (1565) were also identified in the present study. Of the 1935 previously unidentified proteins, 44.3% (857) were identified and 32.0% (620) were even quantified in our combined dataset. In a global transcriptome study, analyzing ten different growth and stress conditions, Kopf and coworkers could recently identify a total of 3101 expressed genes in Synechocystis. Under nitrogen starvation and nitrate growth conditions, approximately 75% of these expressed genes were present (approx. 2300)  from the quantitative study are shown in the supplement  and on the phosphorylation level by non-denaturing PAGE , showing up-regulation under nitrogen starvation proteome data, we analyzed the expression profile of the nitrogen regulatory signal protein Pn Figure . This isSynechocystis phosphoproteome to nitrogen starvation, we classified the quantified –N/NO−3 and –N/NH+4 phosphorylation event ratios into up- or down-regulated and static patterns, separately for both experiments . In experiment 1, characterized by a short pre-adaptation to the respective nitrogen sources, the majority of phosphorylation events revealed a static pattern, whereas, in experiment 2, characterized by a long pre-adaptation, the majority revealed up-regulation. In both experiments, down-regulated phosphorylation events revealed the lowest occurrence. The occurrence and frequency of InterPro terms was analyzed in the three classes  between –N/NO−3 and –N/NH+4 ratios for each experiment  and RuBisCO, as well as P-loop NTPase and N-regulatory PII and PII-like proteins. However, in experiment 1, these phosphoprotein classes did not fulfill the requirement of having at least two up-regulated evidences. The respective classified proteins are indicated in Supplementary Information To analyze the response of the Synechocystis. In order to obtain a more detailed insight about specific phosphorylation events with strong dynamics, and their abundance under the studied conditions, we plotted the quantified phosphorylation event –N/NO−3 and –N/NH+4 ratios against the respective phosphopeptide intensities, separately for each experiment . Phosphorylation events revealing significant up- or down-regulation are depicted in the scatter plots as stars (in red), and the respective phosphoprotein and modification site  is indicated. All significant phosphorylation events (between N/NO−3 and –N/NH+4 ratios) from both experiments are listed in Table This classification provides a comprehensive overview about general protein functions, which are frequently regulated under nitrogen starvation by S/T/Y phosphorylation in II (Ssl0707) Ser49 phosphorylation exhibited the highest level of increase in phosphorylation and intensity throughout all experiments in response to nitrogen starvation. Interestingly, two Ser/Thr protein kinases with contrary phosphorylation dynamics were identified: The Ser/Thr protein kinase C (Slr0599) revealed multiple significantly down-regulated phosphorylation events in experiment 1 and for one of them in experiment 2. Four out of five events could be localized on a specific Thr residues , and one event could be quantified on the phosphopeptide 324VYTQEFTGYTETQEGSPLMK343. In contrast, the putative Ser/Thr protein kinase F (Slr1225) revealed one significantly up-regulated phosphorylation event, which was quantified on the phosphopeptide 324APPGATVSTPQGTNTQIQPTPASSASPLTAPK355 for –N/NO−3 ratios in experiment 1.As expected from previous studies, P28TDVGPITTPNPQK40 and 187LVAAGVILPLSERSASR203 of the hypothetical proteins Slr0483 and Sll0505, respectively. The phosphopeptide 100LQSELSTLEQELAQQDSSPTGPGEEQKSSR129, Ser4 and Ser49 of the hypothetical proteins Slr1686 and Sll0822 and the phycocyanin beta subunit (Sll1577), respectively, revealed down-regulated phosphorylation events in experiment 2. Interestingly, down-regulated phosphorylation on Ser65 of the 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (Slr1945) was detected in both experiments for only –N/NH+4 ratios. Among other proteins with significantly up-regulated phosphorylation events in our study, Ser64 phosphorylation of the unknown protein Sll0981 was detected in experiment 1, whereas, phosphorylations on Thr4 of the hypothetical protein Slr0397 and Thr349 and Thr361 of the hypothetical protein Sll0103 were detected in both experiments. Remarkably, the sodium-dependent bicarbonate transporter SbtA (Slr1512) revealed in our study down-regulation of Thr189 phosphorylation in experiment 1, whereas, the neighboring gene product, the PII-like membrane-associated protein SbtB (Slr1513), revealed significant up-regulation on Thr53 in experiment 2 in response to nitrogen starvation. For a complete overview of the detected phosphorylation events revealing significant regulation in response to nitrogen starvation, see Table Other significantly down-regulated phosphorylation events were detected in experiment 1 on Ser144 of the large-conductance mechanosensitive channel (Slr0875) and on the phosphopeptides Synechocystis proteome and shed first light into the phosphoproteome. However, most of these studies were exclusively focused on specific cellular compartments such as different membrane fractions or the cytoplasm or on specific proteins. Therefore, most previous studies mapped only “snapshots” of either delimited cellular compartments or only one particular metabolic condition of the proteome or the phosphoproteome, mainly performed in a qualitative manner, based on 2D gel strategies. The present study aimed to establish a method to analyze the global dynamic changes of the Synechocystis phosphoproteome in a site specific and quantitative manner between distinct metabolic conditions. In the present study, this was exemplified using nitrate and ammonia grown cells in comparison to 24 h nitrogen starved cells.Numerous previous studies revealed insights with regards to the Synechocystis. The efficient extraction of phosphoproteins contributed in combination with a gel-free MS strategy to a high percentage of detections in the qualitative phosphoproteome dataset. Although similar identification numbers were detected on the proteome level, when comparing method B with the detergent buffer based method C (Y-PER), significantly more localized phosphorylation events (235 vs. 67) could be obtained exclusively with method B. This may be due to the fact that the overall extraction efficiency of hydrophobic proteins was higher with method B compared the other methods. In bacteria, phosphorylation events are predominantly present in low occupancy compared to eukaryotes. Therefore, an overall small percentage of phosphoproteins are actually phosphorylated compared to the respective unmodified protein . The correlation between technical replicates of the phosphoproteome revealed high reproducibility and the overlap of quantified phosphorylation events between technical replicates , with the PII Ser49 phosphorylation preventing this interaction to ensure efficient ammonia uptake under N-limited conditions. Interestingly, we identified Amt1 as a phosphoprotein, which is increasingly phosphorylated under N-limitation. This mechanism could reveal an additional and more sensitive regulative control mechanism to ensure maximum ammonia uptake. Remarkably, the PII-like membrane-associated protein SbtB (Slr1513) revealed significant increasing Thr53 phosphorylation under N-limitation, like the GlnB PII protein. Interestingly, the sbtB gene is neighboring the sodium-dependent bicarbonate transporter Slr1512 (sbtA) gene on the chromosome, which revealed in our analysis significant decreasing Thr189 phosphorylation under the same conditions. It is thus tempting to speculate that SbtB may be involved in regulation of SbtA activity, in agreement with a recent analysis of the sbtAB operon  on Ser17 increased strongly upon N-starvation. In addition, two phosphorylation events were quantified on the small and large subunits of the ribulose bisphosphate carboxylase/oxygenase RuBisCO (Slr0012 and Slr0009), both showing a three- to fourfold increase in response to nitrogen starvation. Both phosphorylation events are localized on seryl-residues, Ser78 on the small and supposedly Ser323 on the large subunit. Interestingly, similar phosphorylation sites were identified in a phosphoproteome study of Arabidopsis thaliana on the respective subunits. Here, Ser71 and Ser321 were phosphorylated on the small and large subunits, respectively, besides several other phosphorylation events  proteins M and N (Sll1031 and Sll1032), multiple phosphorylation events were detected, of which three could be quantified and localized on Sll1031  and one on Sll1032 (pThr121). These phosphorylation events generally showed either static or slightly increasing phosphorylation under N-depletion, with the most prominent regulation on pThr609, showing up to threefold increase for –N/NHSeveral enzymes involved in the pentose phosphate pathway and in glycolysis were detected as phosphoproteins, generally with static or increasing phosphorylation dynamics toward N-starvation, such as the fructose-bisphosphate aldolase (Sll0018) and the 6-phosphogluconate dehydrogenase (Sll0329). Interestingly, the 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (Slr1945) elicited an opposite response, with the level of Ser65 phosphorylation significantly decreasing under N-depletion.Synechocystis, and dephosphorylation was driven upon long-term exposure to high light intensities and under nitrogen limitation, initiating proteolytic cleavage and degradation  revealed only slight decreasing or static levels under nitrogen starvation. This result could indicate that either the process of chlorosis has not occurred to a level that is required in order to significantly reduce the level of phycobiliproteins, or phycobiliproteins were only partially degraded and peptide fragments were still present in the cells, which were then detected by MS analysis. To solve this issue, further experiments containing earlier and later time points are required.324APPGATVSTPQGTNTQIQPTPASSASPLTAPK355. Contrary, we identified four phosphothreonines to be significantly decreasing under the same conditions on the Ser/Thr protein kinase C (Slr0599) and one unlocalized phosphorylation event  and phosphorylation events (301 under nitrogen growth conditions) described thus far. We showed quantitative dynamics of the total proteome in response to nitrogen starvation. This is not only of broad relevance for Synechocystis molecular biology, but sets the ground for a comprehensive analysis of the phosphoproteome, with quantified dynamics of 148 phosphorylation events. Using nitrogen starvation acclimation as a test case, this study reveals that protein S/T/Y phosphorylation may play an outstanding regulatory role for metabolic adaptation in the model cyanobacterium Synechocystis. This analysis paves the way for an in depth analysis of the role of protein phosphorylation in cyanobacteria, to unravel its contribution to homeostatic control.In conclusion, this study reports the first global phosphoproteome analysis of The Guest Associate Editor Ivan Mijakovic declares that, despite having coauthored an article with the author Boris Macek in 2014, the review process was handled objectively. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Ardisia crispa roots, it was shown that Ardisia crispa root inhibited inflammation-induced angiogenesis in vivo. The present study was conducted to identify whether the anti-angiogenic properties of Ardisia crispa roots was partly due to either cyclooxygenase (COX) or/and lipoxygenase (LOX) activity inhibition in separate in vitro studies.In our previous studies conducted on Ardisia crispa ethanolic rich fraction (ACRH), quinone-rich fraction (QRF) and BQ were screened for COX assay to evaluate their selectivity towards two isoforms (COX-1 and COX-2), The experiment on soy lipoxygenase (LOX) inhibitory assay was also performed to determine the inhibitory effect of ACRH, QRF and BQ on soy LOX.Benzoquinonoid fraction (BQ) was isolated from hexane extract by column chromatography, and later analyzed by using gas chromatography–mass spectrometry (GC-MS). Anti-angiogenic effect was studied on mouse sponge implantation assay. BQ was confirmed to consist of 2-methoxy-6-undecyl-1,4-benzoquinone, when compared with previous data. Antiangiogenesis study exhibited a reduction of mean vascular density (MVD) in both ACRH and QRF, compared to control. In vitro study showed that both ACRH and QRF inhibited both COX-1 and COX-2, despite COX-2 inhibition being slightly higher than COX-1 in BQ. On the other hand, both ACRH and QRF were shown to have poor LOX inhibitory activity, but not BQ.In conclusion, ACRH and QRF might possibly exhibit its anti-angiogenic effect by inhibiting cyclooxygenase. However, both of them were shown to possess poor LOX inhibitory activity. On the other hand, BQ displayed selectivity to COX-2 inhibitory property as well as LOX inhibitory effect. Angiogenesis is a fundamental process of new capillary formation which is physiologically important in wound healing and reproduction . Under n2 (PGH2), which eventually leads to the biosynthesis of other prostanoids  , with mass break down also present at m/z 193.1, 179.1, 154, 124, and 69  was determined in the present study. There was no significant difference in MVD of VEGF control  and vehicle control (VEGF and DMSO), which also indicated DMSO as a vehicle did not possess any significant blood vessel- reducing activity in this study. However, treatment with licofelone 100 ng), ACRH (100 ng), and QRF (100 ng) resulted in a significant reduction in mean vascular density (MVD), with the MVD value being 3.67 ± 0.843 (P < 0.01), 5.00 ± 0.894 (P < 0.05), 2.83 ± 0.833 (P < 0.01), respectively, compared to VEGF control (7.76 ± 2.34). In addition, there was no significant difference observed in ACRH and QRF treatment group, compared to licofelone. The MVD of the treatment of ACRH and QRF at similar dose (100 ng), was also observed not to significantly differ as well. Interestingly, QRF is shown to be more potent than licofelone , followed by celecoxib (29.10 ± 1.878 μg/mL), ACRH (37.79 ± 1.843 μg/mL) and QRF (35.24 ± 1.958) respectively. Aspirin displayed non-selectivity towards COX by inhibiting both isoforms competitively.ACRH and QRF were assessed in vitro to investigate their possible effects in COX activity inhibition. Percentage of inhibition of both isoforms of COX (COX-1 and COX-2) was found to be dose-dependent. Aspirin was shown to be the most effective COX-2 inhibitor with the lowest IC50 of both COX-1 and COX-2 following treatments were shown in Table Meanwhile, celecoxib displayed more selectivity towards COX-2 than COX-1 with approximately 2-fold percentage of inhibition increase, notably in the concentration of 25 μg/mL and above. ACRH and QRF showed approximate result, as both ACRH and QRF did not display specific selectivity towards COX-2 inhibition. Both COX-1 and COX-2 were inhibited competitively, though COX-2 inhibition was more prominent, especially in high concentration (100 μg/mL and above). The pattern of COX-1 and COX-2 inhibition in ACRH and QRF was comparable to aspirin. However, BQ showed preference to COX-2 inhibition, with a 2.1-fold inhibition of COX-2 compared to inhibition of COX-1. The result was comparable to that of celecoxib. ICArdisia crispa roots shown in the present study, there are also several other 1, 4- benzoquinones isolated from other Ardisia species possessing potent 5-lipoxygenase inhibitory activity which were reported in previous studies, such as Ardisiaquinone A, isolated from Ardisia sieboldii[Ardisia japonica[The use of soybean lipoxygenase in the in vitro inhibition study served as an appropriate model for the screening of plants with anti-inflammatory properties , as liposieboldii, and Ard japonica. Ardisia japonica.50: 44.37 ± 2.65 μg/mL). ACRH showed minimum LOX inhibition, with IC50 of 304.5 ± 0.32 μg/mL. Apart from that, QRF was also shown to be a weak inhibitor of LOX, with IC50 being 95.86 ± 2.17 μg/mL. BQ was found to be a moderate LOX inhibitor, with IC50 was found to be 66.65 ± 2.71 μg/mL. The IC50 of each treatment was depicted in Table LOX-suppressive effect of ACRH, rich fraction and BQ were assessed in vitro. The inhibitory effect of soy lipoxygenase was found to be prominent in rutin  major compound in BQ at Rt=39.537, and (b) the reference, 2-methoxy-6-undecyl-1,4-benzoqunone [.oqunone .Click here for file


To evaluate total and specific E immunoglobulin (IgE) antibody concentrations in underage subjects with respiratory allergic diseases.Aspergillus, Penicillium, Fusarium and Neurospora were selected for the preparation of sensitizing antigens from ELISA dishes. IgE total concentrations were estimated using the same method.This study was a transversal-type study in 100 underage subjects between 4 and 14 years old, with asthma and/or allergic rhinitis. Total and specific IgE were quantified for airborne fungi in the city of São Luís, Maranhão, Brazil. Five distinct regions—North, South, Center, East and West—were selected so fungi could be collected monthly for 1 year. Twenty genera were identified. Aspergillus concentrations, 87 % presented IgE anti-Penicillium, 45 % presented IgE anti-Fusarium, and 46 % presented IgE anti-Neurospora.IgE total serum concentration was increased in 97 % of the atopic subjects: 75 % of the subjects presented increased IgE anti-Atopic subjects presented simultaneous IgE total and specific elevations for the tested fungi, possibly due to polysensitization caused by the presence of fungi in all of the areas all year. However, determining the clinical significance of the results was not yet possible because most of the data were isolated variables. Respiratory disorders are higher prevalent in children. The different manifestations of allergic diseases involve the upper and lower respiratory airways.Fungi are important pathogens associated with airway diseases. They interact with epithelial cells, induce increases in inflammatory cytokines, which enhance IgE production, and increase toll-like receptor (TLR) expression , 2. ExpoAlternaria, Aspergillus, Cladosporium, Fusarium, Penicillium, Stachybotrys are producers of mycotoxins and allergens that cause respiratory diseases [Fungi and fungal-like organisms are the cause of a lot of diseases in humans and animals, but are also economically important . Alternadiseases .Alternaria, Aspergillus, Bipolaris, Cladosporium, Curvularia and Penicillium being the most prominent [Allergic rhinitis and allergic asthma are characterized as type 1 allergy. Type 1 allergy is caused by a large number of fungi . Allergirominent .Alternaria, Aspergillus, Cladosporium, Helminthosporium, Epicoccum, Aureobasidium and Penicillium have frequently been implicated in allergic asthma [Allergic asthma is a respiratory disease characterized by exposure to environmental agents, which provoke allergic inflammation and transitory bronchiolar obstruction, resulting in typical symptoms of coughing and dyspnea . In chilc asthma –13.Before the outcomes of experimental models, asthma was considered to be a disease caused by IgE because it only occurred due to antigen–antibody complexes formed in mastocyte membranes . Most rePenicillium and Aspergillus species propagules have been shown to increase the risk of asthma development in children. [Indoor dampness increases the risk of indoor fungal growth. The indoor fungal concentrations and species diversity could improve the risk of having asthma, exacerbation of asthma symptoms, or both. hildren. , 19.Alternaria, Aspergillus, Aureobasidium, Cladosporium, Epicoccum, Helminthosporium and Penicillium, are most frequently implicated in fungi-related allergic asthma exacerbations amongst adults in indoor and outdoor settings [In general, outdoor fungi are generally more abundant than indoor fungi, but the association between outdoor fungi and child asthma exacerbations has not been clearly established . The funsettings .Cladosporium (17.86 %) and Aspergillus/Penicillium (15.03 %) in the atmospheric air of Porto Alegre, similar to other countries, such as France, Chile, USA and Cuba. Mendes et al. [Aspergillus, Botryodiplodia and Curvularia in Aracaju.In Brazil, Mezarri et al.  found a s et al.  mainly iThe current research aimed to isolate and extract the most common airborne antigens to detect specific IgE concentrations in the serum of atopic subjects, and the expectation was that these concentrations would be correlated according to the patients’ sex, age and place of residence. By doing so, the findings could actually contribute to better control of the disease.The island of São Luís, which is the capital of Maranhão State, Brazil, is in the center of the Maranhão seashore, at 2°20′00′′S to 2°45′00′′S and longitude 44°01′21′′W to 44°24′54′′W. It is located in the north of the state, bordering the Atlantic Ocean; it stretches south to Estreito dos Mosquitos, which separates it from the continent, east to São José Bay and west to São Marcos Bay. It features hot and humid weather and a steady temperature around 27–33 °C .To calculate the sample size, Epi Info 3.4.2  statistical software was used. The calculation was based on the prevalence of asthma in the reference service at 5 % significance.The study sample consisted of 100 patients (64 % boys and 36 % girls), between 4 and 14 years old enrolled in the Pediatric Pneumology Program, University Hospital, Federal University of Maranhão (HUUFMA). Through-chip protocol, clinical data were obtained from patients in the period from 2007 to 2008.From these subjects, 10 ml of total blood was collected by intravenous puncture after the parent’s/tutors signed the instrument of free consent. The serum was separated and aliquoted for storage in a freezer at 20 °C until the moment of performing the techniques.Airborne fungi were monthly collected by Bezerra , in fiveFungal spores were collected by passive gravitational settling on Petri dishes containing Sabouraud agar, 1.5 m height, for 15 min , 25. A tAspergillus, Penicillium, Fusarium and Neurospora showed detectable amounts of protein and were used as antigens to sensitize ELISA plates to investigate the concentration of specific IgE by ELISA index (EI) [Only the genera dex (EI) .The protocol for the extraction of antigens from filamentous fungi was performed as follows, based on the literature , 28 withThe sera from 100 patients with clinically characterized respiratory diseases were subjected to ELISA to detect the concentrations of total and specific IgE against fungi.Polystyrene plates with 96 wells containing immobilized monoclonal antibodies of the anti-IgE type formed specific complexes with serum IgE through high-affinity bonds. The plates were then washed to remove proteins that had not interacted. A secondary antibody conjugated to peroxidase was added to form a ternary complex with the immobilized.IgE, followed by washing. Next, 3-30,5-50-tetramethylbenzidine and hydrogen peroxide were added to the solution, which under the action of the immobilized peroxidase, produced a blue color. A strong acid was added to the mixture to halt the reaction, producing a yellow color. A reading was performed at a wavelength of 450 nm. The absorbed intensity was proportional to the concentration of IgE in the sample.Optical densities were determined spectrophotometrically at 450 nm. The rates of antibodies were expressed as the EI, according to Alves et al. . The EI Aspergillus spp.; 0.466 (EI ≥ 0.466) for IgE anti-Penicillium spp.; 0.298 (EI ≥ 0.298) for IgE anti-Neurospora spp.; and 0.565 (EI ≥ 0.565) for IgE anti-Fusarium spp.EI values equal to or higher than 0.102 (EI ≥ 0.102) were considered to be positive for total IgE; 0.603 (EI ≥ 0.603) for IgE anti-The cut-off was calculated by averaging the increased negative samples three times to the standard deviation of these samples, where the number of standard deviations used in the formula ensures the confidence level of the result. Cut-off = μ DO + 3 × s .This research was approved by the ethics committee of University Hospital under number 406/06. The Informed Consent Term (ICF) was signed by the parents or guardians of minors who participated in this research.Statistical analysis was performed using the software Stata/SE 9.0 for Windows . The study was cross-sectional and observational, and descriptive statistical techniques were used to assess all of the variables, with the aid of graphs and tables of frequencies. The Pearson correlation was used to study the relationship between total IgE levels and age (years). The correlation coefficient Phi was used to verify the correlation between positivity for two fungi concomitantly. We considered a p value <0.05 as the significance criterion.We studied 100 patients (67 % boys and 33 % girls), between 4 and 14 years old enrolled in the Pediatric Pneumology Program, University Hospital, Federal University of Maranhão (HUUFMA).A total of 100 patients with a diagnosis of asthma were studied. In total, 45 (45.0 %) of these patients had rhinitis, and 35 (35.0 %) had sinusitis.Table 3 and the dry season (July–December) of 35.50 mm3. The median number of CFUs/dish in the rainy season (January–June) was 20 (maximum = 279 and minimum = 0), and the corresponding figure for the dry season was 14 (maximum = 227 and minimum = 0) using the Mann–Whitney test p = 0.96.Observing Table Aspergillus (33.5 %), Penicillium (18.8 %), Cladosporium (14.2 %), Curvularia (10.6 %) and Fusarium (7.6 %). A comparison of the average number of CFUs for the five most frequent fungi using Tukey’s test, showed that the number for Aspergillus differed significantly (p < 0.05) from the numbers for the genera Cladosporium, Curvularia and Fusarium.Twenty genera of fungi were isolated in this study. The main genera found in all of the regions were Aspergillus and Penicillium were frequently isolated in all of the areas studied, regardless of the month of the year. It was possible to see that, in the Central area, the lowest CFU number (115) was noted; in contrast, in the East area, the highest figures were measured (228) (Table 8) Table .Table 3AAspergillus, Penicillium, Fusarium and Neurospora as antigens.In this study, we used the genders Neurospora, anti-Fusarium, anti-Aspergillus and anti-Penicillium IgE) were screened in the afore mentioned 100 blood samples. Among the 100 patients enrolled in the Pediatric Pneumology Program, 97 (97 %) demonstrated that IgE total concentrations were detected , age (p = 0.47) and local of residence (p = 0.13).Sex (p = 0.86), local of residence (p = 0.90) and age (p = 0.19) were not statistically significant with anti-Neurospora; anti-Fusarium; anti-Aspergillus and anti-Penicillium concentration IgE, in children´s serum. A prevalence of seropositivity to fungi was noted, particularly for the genera Aspergillus  demonstrated that IgE total concentrations were detected. 75 % were sensitized to Rhizopus, Aspergillus, Cladosporium and Candida. Total IgE was found to be high in 77.2 % of patients, and specific IgE was found to be high in 31.4 % [A Mexican study involving pediatric patients with respiratory allergy showed that the most frequent allergens were n 31.4 % .Penicillium. The result is similar to that of the patients in our study (87 %), can be explained by the use of ELISA in both studies and the high frequency of Penicillium in Brazil and Malaysia. According to Burge et al. [Penicillium is common in outdoor and indoor environments. Patients with asthma usually present positive responses for spores and other isolated Penicillium antigens.In Malaysia, Goh et al.  found the et al. , PenicilPenicillium and Aspergillus, in 12 of 13 asthmatic schoolchildren in their study in the city of Recife. In a study conducted in Taiwan, Candida (72.1 %) was the most common sensitizing agent, followed by Aspergillus (51.2 %) and Penicillium (41.9 %) [Osorio et al.  found hy(41.9 %) .Aspergillus and Penicillium in this study. Considering that Aspergillus was the most frequently isolated antigen in this research, and it was also present in all areas of the city all year, it is reasonable to suppose that these fungus spores are naturally sensitizing. However, most of the subjects of this research lived in the central area of the city, which was where the fewest fungi were detected, possibly due to a larger concentration of buildings and homes.There was not statistic correlation with sex and local of residence and IgE total antibody concentrations or specific IgE to Aspergillus and anti-Penicillium. Chang et al. [Regarding age, there was statistical significant correlation between age and total IgE, but not between age and IgE anti-g et al.  found thAspergillus IgE in the current research; however, this finding led us to suppose that elevated anti-Aspergillus IgE concentrations might have contributed to the elevation of serum total IgE.There was no correlation between total IgE and anti-Aspergillus and Penicillium, or less frequently to Fusarium and Neurospora spp., shows the importance of these sensitisations.This study with a large number of patients sensitized to However, this study has some limitations. First, we have not accounted or discounted the possibility that the findings could be a result of indoor exposures on elevated IgE. In addition, the sample included only 100 children. The technique used to assess the levels of IgE ELISA was the index and not by Phamacia CAP unit system. Limitations in the statistical analysis. This is a cross-sectional study and there was no assessment of the levels of exposure to fungi and the development of allergic diseases. There was not a control group. Besides, the prick test was not conducted to confirm allergy to fungi of the study.The genera of fungi identified in the present study were correlated with natural systems and could be useful in assessing the impact of environmental changes on the region studied.In short, based on the data obtained in this study, the following aspects of the causal relationship between exposure and the symptoms of respiratory allergy may be noted. This study shows the importance of airborne fungi sensitizations in children with respiratory allergy. Combined diagnosis of total IgE test, specific IgE and clinical aspects is very useful for deciding specific therapies.Finally, it was possible to conclude that the presence of specific IgE antibodies for the studied airborne fungi might be due to polysensitization because the fungi were present in all of the areas over the whole year in the city of São Luis-MA, Brazil, so the topic requires more study to understand fully airborne fungus respiratory allergies.


Surales landscapes, comprised of densely packed, regularly spaced mounds, cover large areas of the Orinoco Llanos. Although descriptions of surales date back to the 1940’s, their ecology is virtually unknown. From data on soil physical and chemical properties, soil macrofauna, vegetation and aerial imagery, we provide evidence of the spatial extent of surales and how they form and develop. Mounds are largely comprised of earthworm casts. Recognizable, recently produced casts account for up to one-half of total soil mass. Locally, mounds are relatively constant in size, but vary greatly across sites in diameter (0.5–5 m) and height (from 0.3 m to over 2 m). This variation appears to reflect a chronosequence of surales formation and growth. Mound shape (round to labyrinth) varies across elevational gradients. Mounds are initiated when large earthworms feed in shallowly flooded soils, depositing casts that form ‘towers’ above water level. Using permanent galleries, each earthworm returns repeatedly to the same spot to deposit casts and to respire. Over time, the tower becomes a mound. Because each earthworm has a restricted foraging radius, there is net movement of soil to the mound from the surrounding area. As the mound grows, its basin thus becomes deeper, making initiation of a new mound nearby more difficult. When mounds already initiated are situated close together, the basin between them is filled and mounds coalesce to form larger composite mounds. Over time, this process produces mounds up to 5 m in diameter and 2 m tall. Our results suggest that one earthworm species drives self-organizing processes that produce keystone structures determining ecosystem functioning and development.The formation, functioning and emergent properties of patterned landscapes have recently drawn increased attention, notably in semi-arid ecosystems. We describe and analyze a set of similarly spectacular landforms in seasonal tropical wetlands.  They are found in the Orinoco Llanos of Colombia and Venezuela, mostly in the seasonally shallowly flooded waters of the alluvial overflow plain )ow plain . Bates  and size. Here, we explore this variation as a result of environmental variation and as a process of development over time that leads to increasing topographic heterogeneity, which in turn affects the distribution of plants and other organisms in surales landscapes.Although  example ). Given  example ,10), it Patterned landscapes in a great range of environments, from estuarine marshes to boreal peatlands and arid and semi-arid shrublands, are studied using the concept of spatial self-organization in ecosystems ,11. Thistacurú landscapes built by Camponotus punctulatus ants in Argentina  not only to the soil moisture but also to the flooded conditions, and during the flood the earthworm produces an earth mound in order to search for aeration” ) using the open source software QGIS 2.6.1-Brighton [2) was exhaustively surveyed for the presence (score of 1) or absence (score of 0) of surales landforms. Using this methodology, we surveyed 9,817 km2 of the 376,547 km2 covered by the Orinoco Llanos. We interpolated the binary data obtained on the regular grid to estimate the probability of surales presence at the scale of the Orinoco Llanos. To do this, we used a thin plate spline regression method, suited for data that do not respect the hypothesis of stationarity required for statistical methods such as kriging. Our analysis did not take into account surveyed points scored “NA”, i.e., points for which satellite imagery of sufficient resolution was not available. To calculate the estimated area of the Orinoco Llanos that features patches of surales, we counted the number of pixels (pixel size: 443.67 x 443.67 m) of the Orinoco Llanos that were ascribed an interpolated value > 0.5. In order to show a sample of some of the most spectacular sites discovered doing the satellite imagery survey, we created a commented tour using Google Earth Pro™ and its movie maker module https://mycore.core-cloud.net/public.php?service=files&t=e01a65c5769726d35c4f005a13c46502.We estimated the spatial extent of Brighton . Each noOur study was conducted in the seasonally flooded savannas of the municipality of San Luis de Palenque, Casanare, Colombia, which is included in the alluvial overflow plain. Field studies were carried out on privately owned land with the permission of landowners, and did not involve endangered or protected species. Climate is seasonal humid tropical; mean annual temperature is 28°C, mean annual rainfall is 2000 mm. An intense rainy season occurs from May to July, followed in most years by a short dry period, of varying duration and intensity, in August and September. A less intense rainy season occurs from October to mid-November, followed by an intense dry season from December to April.surales landscapes, characterized by differences in mound sizes, in the depth of the inter-mound basin, in duration of the flooding period and in vegetation cover  was characterized by the smallest mounds, with an average (± SD) height of 37.5 ± 7.3 cm (distance from the base of the inter-mound basin to the top of the mound) and an average diameter of 112 ± 47.1 cm. In Site 2  the size and shape of surales mounds varied among locations within the site. In the area within this site that we studied, mounds averaged 61.7 ± 10.8 cm in height and 160.6 ± 32.5 cm in diameter. Site 3 , in contrast to the others, was covered by trees and mounds were very large. In the part of this site we studied, the average height of mounds was 114.7 ± 17.6 cm and the average diameter was 373 ± 109 cm. Elsewhere in the site, some mounds reached 2 m in height and up to 5 m in breadth. Site 4  was considered a control or reference site. It was located close to Site 2 on a banco, i.e., a bank on higher ground that is not seasonally flooded and where surales mounds are absent. The sites investigated are all semi-natural grasslands, mainly used for extensive cattle grazing.During a first field mission in July 2012, we distinguished three main categories of round- mound on cover . Consequ2, each separated from the others by a distance of 30 meters representing an equilateral triangle. No large termite nests were present in any of the sites we studied. Samples for soil properties and earthworm species composition and abundance were collected in five sampling locations within each plot. In surales habitat (Sites 1–3), three of the five sampling locations in each plot were located on mounds and two in the inter-mound basin, giving a total of 27 sampling locations for mounds (nine in each site) and 18 sampling locations for inter-mounds (six per site). Sampling locations in each plot were separated by varying distances, as a function of differences between sites in size and spacing of mounds. In Site 4, three plots (and 15 sampling locations) were located in apparently homogeneous banco habitat. We surveyed earthworm abundances and spatial distribution in mound and inter-mound habitats during two field missions at the end of the rainy season, in July 2012 and July 2013. At these times, soils were still wet but the inter-mound basin was no longer completely flooded. In these conditions, earthworms were active and close to the soil surface in the mounds, facilitating sampling. A sampling campaign was also conducted during the dry season in December 2012 to study phytolith composition, soil properties and morphology of soil macroaggregates in the different study sites. At this period the inter-mound basin was not flooded, except for Site 3, in which the basin is flooded all year round.In each of Sites 1–4, we collected samples to obtain data on soil properties, earthworm species composition and abundance and plant microfossils (phytoliths) in soil, in three plots of 25 msurales mounds and in inter-mound basins for Sites 1 to 3, as well as in a control (reference) site in a banco. For logistical reasons, banco vegetation (never flooded) was sampled only during the rainy season (July 2013) and not in Site 4, but in a nearby site, Site 5 , a natural levee located close to Sites 2 and 4. Vegetation was sampled using the point-intercept method [surales (Sites 1–3), habitat type (mound or inter-mound) was also recorded for each sampling point. In Site 5, the banco habitat was apparently homogeneous.Plant communities were sampled during the dry season in December 2012 and again, using the same sites and methods, during the rainy season in July 2013, when the inter-mound basin was flooded. Plant species composition and distribution were recorded on t method , which p2 comprising both mound and inter-mound habitats.Whereas vegetation in Sites 1, 2 and 5 was strongly dominated by graminoids, along with some herbaceous and small woody dicots, Site 3 was dominated by trees averaging about five meters in height. In this site, in addition to the point-intercept method, all trees with a diameter at breast height (DBH) greater than 10 cm were sampled using a line transect method  along a http://www.biovirtual.unal.edu.co/ICN/) and of the Herbario Forestal Gilberto Emilio Mahecha Vega . Sterile specimens that could not be identified were assigned to morphospecies.We collected voucher specimens of each plant species encountered and identified plants using published keys –53 and bbanco), in at least one of the four sites. For analysis, we grouped all successive points lying within the same habitat type into a single unit (referred to as a ‘segment’). To do this, we summed the number of times each plant species was contacted on each mound and inter-mound segment and divided the sum by the total number of contacts recorded in the segments of each type in the site. We analyzed a total of 42 mound and 37 inter-mound segments for the dry season and 44 mound and 41 inter-mound segments for the rainy season. This method allowed controlling for the difference in sampling efforts due to the differing representation of mound and inter-mound areas in the landscape.We performed statistical analyses of plant community structure using only species that accounted for 5% or more of the vegetation cover (as estimated by the line-intercept method) in at least one of the three habitats . To study the distribution of different plant life forms between mounds and inter-mounds and among sites, plant species records were grouped as herbaceous plants, including “graminoids” , “aquatic herbs” and “other herbaceous species” (including one herbaceous climber); as suffrutescent plants ; and as woody plants, including “trees” and “other woody plants”, the latter consisting of shrubs as well as three woody vines. Cover abundance was scored for each species and grouped for each life form. All the statistical analyses conducted during this study were performed using the software R version 3.1.1 [We used a non-metric multidimensional distance scaling analysis (NMDS) based on a Bray-Curtis dissimilarity matrix to assess differences in species composition and assemblage structure between habitats (mounds and inter-mounds) and among sites . We performed separate NMDS analyses for the dry and the rainy seasons. Site 5 was included only in the analysis of the rainy season. Site 3 was not included in the analysis because it shared no plant species (in either habitat) with the other sites in the dry season and only two species, both at low abundance in Site 3, in the rainy season. To examine the contributions of site and of habitat type (nested factors) in explaining variation in the vegetation composition, we used a non-parametric multivariate analysis of variance (MANOVA) based on the distance matrix we performed for the NMDS. This test was followed by a permutation test . In each trench we took samples from profiles at two points: (i) at the highest central point of the mound and (ii) in the lowest central point of the basin. At Sites 1 and 2, we took samples at 5 cm intervals from the surface to 40 cm b.s. (below surface) (see Phytoliths offer important advantages for studying vegetation histories in soil-depth profiles because (i) being made of silica, they are preserved in the hot-humid conditions and acid soils of the tropics where other types of plant remains are not  and (ii)ace) see . At SiteAristida sp.) was based on a morphological classification first proposed by Twiss et al. [Heliconia sp. and Trichomanes sp., followed Piperno [Soil samples were processed, and phytoliths identified and counted, at the University of Exeter Archaeobotany Laboratory following standard procedures . A minims et al. , and lats et al. ,62–65).  Piperno . The sum Piperno . Globula Piperno .ISO 23611–5:2011 standard) applied to soil monoliths (25 x 25 x 30 cm depth) collected from the 60 sampling locations described in section 3 . Earthworms were extracted from the soil blocks by hand sorting. They were identified based on their external and internal morphological characteristics to the species level by A. Feijoo . Fresh biomass of all juvenile and adult earthworms was calculated for each of the two ecological groups represented  according to criteria described by Bouché [Andiorrhinus (Turedrilus) sp. (from here forward referred to simply as Andiorrhinus sp.), was measured separately, because of the high proportion of the total biomass of earthworms accounted for by this species in the surales ecosystem and because it does not fit neatly into either of the two ecological groups mentioned above. A two-way ANOVA was used to test the effects of the factors “site” (field sites) and “earthworm ecological group”  on variation in earthworm fresh biomass. Prior to this analysis, normality and homoscedasticity of data were confirmed using Shapiro-Wilk and Bartlett tests, respectively. A Wilcoxon rank-sum test was used to test the difference in earthworm biomass between mounds and inter-mounds, separately for each field site (α = 0.05).Earthworms were sampled and counted in July 2012  and July 2013 (at Site 3) using the standard Tropical Soil Biology and Fertility (TSBF) method , giving a total of 135 samples in surales habitat . In Site 4, 45 samples were collected for determination of bulk density. Organic matter content (OM) was measured by the Loss On Ignition (LOI) method, with two replicates for each sampling location, giving a total number of 90 samples in surales habitat . In Site 4 we collected 30 soil samples for analysis of OM content.Sand, silt and clay contents (%) were measured on soil samples taken from 0–10 cm depth in each of the 60 sampling locations described in section 3. Analyses of soil texture were conducted following the method of Bouyoucos , the samSoil profiles were described on two plots in each of the first two field sites along a trench cut one meter deep (from the soil surface of the inter-mound basin to the bottom) and four meters long. Each included a vertical profile of three mounds and the intervening basin. Mounds cut by the trench had an average (± SD) height of 15 ± 4.2 cm in Site 1 and of 37 ± 9.3 cm in Site 2.surales habitat (4 samples per field site).X-ray analysis of the clay fraction was performed on two soil samples per soil profile in Sites 1–3, taken at 10 cm below the surface in the basin and from a mound, giving a total of 12 samples from To characterize the contribution of biogenic macroaggregates to the composition of the uppermost 10 cm of the soil matrix, we adapted to the needs of our study the method proposed by Velasquez et al. , itself surales sites . Surales were absent in 18% of the circles and satellite imagery of sufficient resolution was not available in 30.4% of the circles, mainly located in Venezuela . The NMDS  in at least one of the two sites. The habitat type  and the site respectively accounted for 14% and 22% of the variation in plant assemblage structure . In Site 2, mound and inter-mound habitats shared 45% of the number of species recorded . In contrast to our results in the dry season, the NMDS  and Site 5. Regarding plant life forms, herbaceous aquatic plants were present in the inter-mound areas of both Sites 1 and 2, but were more abundant in Site 2, as were other herbaceous species (excluding graminoids) and suffrutescent plants. Vegetation of the Site 5 featured very low diversity of plant life forms, composed almost only of graminoids.Of the total of 67 species recorded in Sites 1, 2 and 5, 37 attained 5% or more of the vegetation cover in at least one of these three sites. Results of the non-parametric MANOVA revealed that habitat type  accounted for 34%, and site for 12%, of the variation in the plant assemblage structure (Davila nitida) and one with Site 2 (Miconia sp.).In Site 3, during the rainy season eight tree species were recorded on mounds, ranging in height from two to nine meters. Using the point-intercept method, seven plant species were recorded, of which only one species was shared with Site 1  was rarely recorded in the deep basins of this site.Among the taxa abundantly represented in our vegetation samples, the subfamily Panicoideae of the grass family (Poaceae) is the only one to possess phytoliths that are quasi-diagnostic , therebZea mays), manioc (Manihot esculenta) or arrowroot (Maranta arundinacea) were recovered in the samples analyzed. There is thus no indication that these mounds of natural origin were ever used as agricultural raised fields.Overall, the phytolith record showed no clearly discernible patterns. Not surprisingly, the proportion of arboreal phytolith morphotypes tended to be greatest in Site 3, the only site where trees were present. However, few other patterns were found. Surprisingly, there was little relation between composition of current vegetation and that of phytoliths in surface soil horizons. The great variation between mound and inter-mound habitats in each site, and in each of these habitat types across sites, was by and large not mirrored in the phytolith composition of surface soil. Phytolith morphotypes characteristic of panicoid grasses illustrate this point particularly clearly. In Sites 1 and 2, although grasses of this taxon were 2–15 times more abundant on mounds than in inter-mound basins, depending on site and season, no marked difference was apparent in the representation of phytolith morphotypes characteristic of panicoids in surface soils between these two habitats in either site . VariatiThe composition of phytoliths found in earthworm casts was similar, in terms of diversity and relative abundances, to that found in soils of the site (Site 1) from which they were collected. Phytoliths characteristic of panicoid grasses accounted for 27–59%, followed by globular psilate phytoliths (9–26%) and silicified anticlinal polyhedral epidermal cells (9–27%). As mentioned above, these last two morphotypes have very low taxonomic resolution, being produced by both herbaceous and woody dicots, as well as by herbaceous monocots . Phytolisurales field sites. Site 1 had the lowest species richness, with four species, all of which were also encountered in the two other sites: Andiorrhinus sp., which does not fit neatly into ecological groups, and three endogeic species . Site 2 harbored, in addition to these four, three additional endogeic species . Site 3 also harbored seven species: the four species found in both other sites, one of the two undetermined morphospecies found in Site 2 and two epigeic species (two undetermined species of Martiodrilus). Epigeic species were thus found only in the surales site with the largest mounds and best-drained mound-top soils. In all three sites, earthworm species richness was higher on mounds than in inter-mound habitat, and Site 3 was characterized by a complete absence of earthworms in inter-mound habitat , gray in color with brown mottling and containing abundant earthworm burrows and roots. The horizon at the surface of the inter-mound basin (and underlying the mounds) was noted A2. In both sites, this horizon was 15–20 cm deep, light gray in color and characterized by the presence of very coarse subangular blocky structures (aggregates > 80 mm) according to the FAO Guidelines for Soil Description [operties . This Bgf Site 2 . All soind basin .-3 (-3), as was OM content (4.5 ± 0.2%) (gleyic properties), as observed in the Bg horizon of Sites 1 and 2. The walls of the mounds in this site were not vertical: beneath the mound’s rounded dome, walls were concave. Mounds were thus “mushroom-shaped” ; average bulk density was 1.5 ± 4.6 g x cm-3 . During  ± 0.2%) . The Bg ped” see . Large asurales sites (Sites 1–3), allowed us to classify the soil as Fluvic Gleysols , according to the World Reference Base for Soil Resources [Description of soil profiles and analyses of soils of the three esources .banco) showed a single horizon with a sandy texture. Soils had relatively high OM contents (3.8 ± 0.6%) and were characterized by an average bulk density of 1.3 ± 0.01 g x cm-3  and quartz (15–30%). It is noteworthy that quantities of 2:1 clay minerals (< 15% of the clay fraction) were insufficient to generate gilgai microtopography by shrinking/swelling dynamics typical of Vertisols. Furthermore, we observed neither a Vertic horizon nor shrink-swell cracks at the soil surface of Sites 1–3, excluding any scenario for genesis of these landscapes based on purely physical factors.surales field sites showed large differences in the proportions of macroaggregates of different origins between the two kinds of habitats, mounds and inter-mound basin  origin, and this dominance increased from Site 1 to Site 3. Physicogenic aggregates accounted for 48.4 ± 7.2% (mean ± SD) of total soil mass in Site 1, 83.7 ± 11.2% in Site 2 and 100 ± 0.0% in Site 3. In contrast, mound soils were dominated by various types of macroaggregates formed by organisms. Biostructures produced by earthworms comprised most of the mass of macroaggregates in mound soils in Sites 1 and 2, accounting for 46.7 ± 8.1% and 44.0 ± 1.5% of total soil mass, respectively. These two proportions were not significantly different from each other . In Site 3, biostructures identifiable as having been recently produced by earthworms comprised a smaller proportion of the total mass of mound soils, 25.1 ± 14.9%. In all three sites, earthworm-produced biostructures accounted for smaller proportions of total soil mass in the inter-mound basin compared to mounds, with means of 35.9 ± 3.9% in Site 1, 13.5 ± 8.1% in Site 2 and 0% in Site 3. The proportional difference between mound and inter-mound in the contribution of earthworm-produced biostructures to soil structure was greater in Site 2 than in site 1, and greatest (infinity) in Site 3. This difference was not significant in Site 1  but was highly significant in Sites 2 and 3 . Macroaggregates (including nest structures) produced by social insects were present in mound soils in Sites 1 and 2, but completely absent from inter-mound soils in all three sites. As the proportion of soil mass constituted by biostructures produced by earthworms and by social insects decreased in mound soils from Site 1 to Site 2 to Site 3, the proportion constituted by root-associated macroaggregates simultaneously increased, from 15.1 ± 11.3% of total soil mass in Site 1, to 32.4 ± 7.0% in Site 2 and 53.2 ± 14.4% in Site 3.Morphological assessment of macroaggregates collected from the upper 10 cm of soil in nd basin . In all banco) was dominated by root-associated macroaggregates, which accounted for a mean of 73.3 ± 13.7% of total soil mass. Earthworm-produced aggregates accounted for 5.4 ± 2.9%, and 21.3 ± 3.6% of soil was non-aggregated. Physicogenic macroaggregates were not observed in these sandy, relatively well-drained soils.Soil of Site 4 . As satellite images of sufficient resolution for Venezuela (and of improved resolution for Colombia) become publicly available, our estimate could be revised. An important corpus of literature acknowledges the effects of such earth-mound landscapes on species diversity and spatial distribution and on ecosystem processes. This first tangible evidence of the great spatial extent of these landscapes presented here reveals that surales can no longer be ignored in work on the ecology of this ecoregion. But these ecosystems are under threat from industrial agriculture, and are being leveled to make way for highly intensified commercial production of rice [surales in the area and better understanding of their ecology could spur efforts to conserve these landscapes, including research on how they can be managed for the benefit of both biodiversity and human well-being. With this study, we provide the first pieces of the puzzle to help us understand the processes that create surales, determine their development and mediate their effects on biodiversity.We estimated that  of rice ,83, withsurales landscapes.In this paper, we present several lines of evidence supporting the contention that bioturbation by earthworms—and possibly other soil engineers—explains the formation of surales sites studied  and vertically (over soil profiles) by bioturbating soil engineers, most importantly earthworms. In principle, phytolith composition of soil surface layers should reflect the current vegetation cover. Because leaves and other organic matter containing phytoliths usually decompose where they fall, rather than being moved, soil phytoliths reflect vegetation composition at a very local scale (in contrast to pollen) and thus reflect the variation of vegetation cover across habitats ,88. Givees sites . Howeversurales landscapes (further discussed below)—is often marked by variation in phytolith composition with depth in soil [surales grew vertically in a flooded plain to rise above the flood water level, increasing in size from towers to mounds, creating drier platforms colonized by vegetation that cannot thrive under waterlogged conditions , in the absence of bioturbation we should expect an increase in phytoliths of these taxa near the surface and in the upper levels of surales soils, and in turn a decrease in the proportion of wetland-adapted taxa . We were anticipating a pattern similar to that we had already documented in vestiges of human-made raised fields in French Guiana, where a clear decrease in wetland taxa towards the upper levels of raised fields was apparent, with a concomitant increase in panicoid grasses [Thorough mixing of soil by bioturbating soil engineers could also explain the absence of marked vertical patterns of variation in phytolith composition with depth in soil, in a given habitat. Vegetation change over time—which we believe has been substantial with the development of  in soil ,79. With grasses , 79,90. surales sites, where phytolith distribution is virtually homogenized across two very different habitats and over soil profiles. In fact, we know of no other site where phytolith composition shows so little variation with soil depth, and where phytolith composition near the soil surface shows so little correspondence with current vegetation cover, as in the surales sites studied here.Several processes can modify the distribution of phytoliths in soil after their deposition ,91,92. Hsurales soil matrix is the result of the accumulation of aggregates that have been subjected to high rates of bioturbation, with soil being moved both vertically and horizontally.In conclusion, the relative homogeneity of soil phytolith composition across habitats, sites and depths in soil is consistent with the hypothesis that the surales mounds were unusually large, with a mean diameter of 5.3 ± 3.0 cm, indicating that most casts were produced by very large earthworms. One earthworm species, Andiorrhinus sp., the largest species in these sites , accounted for most of the total earthworm biomass in all mound soils , indicating that this species is responsible for initiating the formation of mounds. Secondly, with the exception of Ocnerodrilus occidentalis, found at very low biomass density in Site 1, Andiorrhinus sp. is virtually the only earthworm found in the waterlogged inter-mound soils, and thus the only one whose foraging activities could move soil from inter-mound areas to mounds, where all its casts are found. Third, this species appears to be restricted to the seasonally flooded soils in which surales appear, being absent from the never-flooded banco site (Site 4).Most earthworm casts found on the soil surface and in the soil matrix of nd soils . The hypsurales mounds is explained by the activity of bioturbating earthworms, in our sites Andiorrhinus sp. A combination of four features of the behavior of this species can explain how mounds are initiated and grow: (1) This earthworm feeds in flooded soils (and is absent from non-flooded sites). (2) Like some other earthworms in flooded habitats [Andiorrhinus sp. were exclusively found in large vertical burrows, located directly below their surface casts on mounds , to be rrestris . Elevatehe mound . These felongata , as an esurales earth-mound landscapes are analogous to those that lead to spatial self-organization in semi-arid shrublands [surales, the key limiting resource is well-aerated soils during the flooding season. In shallowly flooded soils, Andiorrhinus sp. builds elevated structures  to respire, creating patches of this resource. As the mound grows through movement of soil to it from the surrounding area, the basin near the mound gets deeper, decreasing the probability that a new small mound can emerge within a threshold minimal distance of an existing mound, a distance determined by the foraging radius of individual earthworms , diverse soil engineer organisms—additional earthworm species, but predominantly plant roots—contribute along with Andiorrhinus sp. to soil structure in the higher, broader mounds of Sites 2 and 3. For instance, in the largest mounds (Site 3), we showed that roots played a key role in formation of soil structure, with root-associated aggregates accounting on average for 53.2% of the macroaggregates of the soil matrix. These observations support the hypothesis that Andiorrhinus sp. initiates surales formation, creating favorable well-drained habitat that can then be colonized by other earthworms and by plants. While Andiorrhinus sp. dominates earthworm biomass in mounds of all sizes, other engineers may contribute to growth of mounds and, by reducing soil erosion [Morphological analysis of soil aggregates indicated that although biostructures in smaller mounds (Site 1) are predominantly produced by the earthworm  erosion , to theisurales sites we studied, mounds were all of similar size and shape and featured similar vegetation cover, earthworm communities and macroaggregate composition in the upper levels of the soil matrix. However, among the three sites, and among areas within them, there was great variation in all these parameters. We suggest that our results show this variation among sites to represent a gradient of mound development, marked by increasing aggradation of soils in mounds over time, with corresponding degradation (soil subsidence) in the area surrounding each mound. Five arguments support this hypothesis (1) Geomorphology. The smallest mounds are low and flat, and occur in shallowly flooded basins. These coalesce into broader, but still low and flat, mounds, which then grow even broader and taller, with narrower, but deeper, spaces between mounds. (2) Soil morphology. As mounds grow broader and taller, they contain an increasingly large proportion of biogenic macroaggregates, indicating the importance of bioturbation and aggradation in mound development. At all stages, mound soils have greater contents of biogenic aggregates than do inter-mound soils. The small, low mounds that first appear are comprised entirely of fresh earthworm casts. In the larger mounds, fresh earthworm casts are concentrated at the periphery of the mound, suggesting that mounds grow as small mounds coalesce and earthworms continue to forage in the adjacent basin, depositing casts on the growing mounds . As the inter-mound basin gets deeper, it is flooded for a longer period each year. Aquatic plants increase in frequency and diversity, then decline where trees on mounds create shady conditions in the basin. (4) Earthworm communities. Species composition of earthworm communities varies across stages of growth, and species richness increases in the initial stages. Andiorrhinus sp. dominates in contribution to biomass at all stages, and aside from scattered individuals of Ocnerodrilus occidentalis, Andiorrhinus sp. is the only species found in inter-mound as well as in mound soils. (5) Soil phytoliths. Soils of the tree-covered mounds of Site 3, which virtually completely lack herbaceous vegetation today , show phytolith composition typical of earlier stages in the hypothesized chronosequence, even in the top few centimeters of the soil profile. Landowners in Site 3 reported that around 1950 the site was covered by herbaceous vegetation, confirming this vegetational change. Succession from dominantly herbaceous vegetation to tree cover was apparently too rapid to allow a detectable accumulation of phytoliths from the recent tree vegetation cover near the surface, particularly given the high rates of bioturbation.Within the area sampled in each of the three g mounds . (3) Plasurales landscapes form only within a narrow range of flooding depths. In the absence of flooding, mounds do not form. Andiorrhinus sp. may simply not be present in such sites, and if it is, it would gain no advantage from mound construction. In shallowly flooded areas, Andiorrhinus sp. constructs mounds and the surales-forming process is initiated. However, when flooding exceeds a certain depth, mounds are either not initiated or do not persist through the high-water season. The threshold depth beyond which mounds do not form is not known with certainty, but the smallest surales mounds we observed were in basins that were flooded by less than about 15 cm during the high-water season. Surales appear to form readily in such shallowly flooded areas, even in very small depressions in otherwise unflooded areas . surales in Site 2 took forms ranging from spotted to labyrinthic, the variation corresponding to a topographical gradient from the center to the margin of the basin. Whereas process-based models can account for the range of forms observed in patterned vegetation in semi-arid shrublands [surales landscapes have not been investigated. Small differences in elevation over this gradient can affect the period and the depth of flooding, but precisely how this environmental variation affects the strength and spatial scale of feedback effects of earthworms, and thereby the shape of mounds, is a subject for further study. Modelling the bioturbation effected by Andiorrhinus sp., integrating more detailed information on the behavior and activity of this earthworm and taking into account micro-topography and eco-hydrological functioning of the seasonal floodplains within which surales form, could provide a better understanding of how self-organizing processes generate the vast and complex mosaic of surales landscapes in the Orinoco Llanos.Within the range of flooding depth in which rublands , mechanisurales mounds does reflect, at least in part, different stages of development, why then are all sites that bear surales not covered with hypothesized advanced stages, like the large tree-covered mounds of Site 3? The continued existence of putative early stages suggests that there exist processes that generate new, flat, shallowly flooded areas in which surales are initiated. What are these processes? Today, disturbances related to changes in land use by humans produce new areas in which surales can form. For example, since the creation of an airstrip for small planes ten years ago near an oil-well installation neighboring Site 2, small surales mounds have appeared in the drainage ditches parallel to the strip. Also, many areas of surales are being graded to produce rice paddies. Abandoned rice paddies could produce large areas where conditions favor initiation of surales. At longer time scales, given the restriction of surales formation to a narrow range of flooding depths, hydrological changes could reset the process. Shifting fluvial dynamics, for example, could lead to sedimentation of more deeply flooded areas, favoring the initiation of surales in areas formerly occupied by esteros, and to shallow flooding of areas that previously were not subject to flooding. Cycles of variation in rainfall at scales of decades or centuries could have similar effects.If variation in the size and shape of surales development, and if so what this ‘equilibrium state’ might resemble, are unknown. For example, surales mounds like those in Site 3 may continue to grow, producing mounds even larger than those so far observed. On the other hand, the erosion that produces ‘mushroom-shaped’ mounds in this site Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.