Combining anaerobic digestion slurry and different biochars to develop a biochar-based slow-release NPK fertilizer.

In this research, we developed a biochar-based fertilizer using biogas slurry and biochar derived from lignocellulosic agro-residues. Biogas slurry was obtained through the anaerobic digestion of the organic fraction of municipal solid waste (fresh vegetable biomass and/or prepared food), while biochars were derived from residues from quinoa, maize, rice, and sugarcane. The biochar-based fertilizers were prepared using an impregnation process, where the biogas slurry was mixed with each of the raw biochars. Subsequently, we characterized the N, P and K concentrations of the obtained biochar-based fertilizers. Additionally, we analyzed their surface properties using SEM/EDS and FTIR and conducted a slow-release test on these biochar-based fertilizers to assess their capability to gradually release nutrients. Lastly, a bioassay using cucumber plants was conducted to determine the N, P, and K bioavailability. Our findings revealed a significant correlation (r > 0.67) between the atomic O/C ratio, H/C ratio, cation exchange capacity, surface area, and the base cations concentration with N, P, and/or K adsorption on biochar. These properties, in turn, were linked to the capability of the biochar-based fertilizer to release nutrients in a controlled manner. The biochar-based fertilizer derived from corn residues showed <15 % release of N, P and K at 24 h. Utilization of these biochar-based fertilizers had a positive impact on the mineral nutrition of cucumber plants, resulting in an average increase of 61 % in N, 32 % in P, and 19 % in K concentrations. Our results underscore the potential of biochar-based fertilizers in controlled nutrient release and enhanced plant nutrition. Integration of biochar and biogas slurry offers a promising and sustainable approach for NPK recovery and fertilizer production in agriculture. This study presents an innovative and sustainable approach combining the use of biochar for NPK recovery from biogas slurry and its use as a biochar-based fertilizer in agriculture.

Phosphorus-Loaded Biochar-Assisted Phytoremediation to Immobilize Cadmium, Chromium, and Lead in Soils.

Soil contamination with heavy metals (HM) poses significant challenges to food security and public health, requiring the exploration of effective remediation strategies. This study aims to evaluate the remediation process of soils contaminated with Cd, Cr, and Pb using Lolium perenne assisted by four types of biochar: (i) activated coffee husk biochar (BAC), (ii) nonactivated biochar coffee husk (BSAC), (iii) activated sugar cane leaf biochar (BAA), and (iv) nonactivated biochar sugar cane leaf (BSAA). Biochar, loaded with phosphorus (P), was applied to soils contaminated with Cd, Cr, and Pb. L. perenne seedlings, averaging 2 cm in height, were planted. The bioavailability of P and heavy metals (HM) was monitored every 15 days until day 45, when the seedlings reached an average height of 25 cm. At day 45, plant harvesting was conducted and stems and roots were separated to determine metal concentrations in both plant parts and the soil. The study shows that the combined application of biochar and L. perenne positively influences the physicochemical properties of the soil, resulting in an elevation of pH and electrical conductivity (EC). The utilization of biochar contributes to an 11.6% enhancement in the retention of HM in plant organs. The achieved bioavailability of heavy metals in the soil was maintained at levels of less than 1 mg/kg. Notably, Pb exhibited a higher metal retention in plants, whereas Cd concentrations were comparatively lower. These findings indicate an increase in metal immobilization efficiencies when phytoremediation is assisted with P-loaded biochar. This comprehensive assessment highlights the potential of biochar-assisted phytoremediation as a promising approach for mitigating heavy metal contamination in soils.

Evaluation of different composting systems on an industrial scale as a contribution to the circular economy and its impact on human health.

Due to the production of volatile organic compounds (VOCs), large-scale composting can cause air pollution and occupational health issues. Due to this, it is necessary to determine if the amount generated poses a health risk to plant workers, which can be a starting point for those in charge of composting plant facilities. As a result, the goal of this work is to conduct a thorough analysis of both the physicochemical features and the VOC generation of three large-scale systems. For ten weeks, the three different composting plants were monitored weekly, and VOC identification and quantification were performed using GC-MS gas chromatography. It has been observed that the biggest risk related with VOC formation occurs between the fourth and fifth weeks, when microbial activity is at its peak. Similarly, it has been demonstrated that xylenes and toluene are the ones that are produced in the greatest quantity. Finally, after ten weeks of processing, it was discovered that the material obtained complies with the regulations for the sale of an amendment.Implications: The evaluation and monitoring of the composting processes at an industrial scale is very important, due to the implications they bring. VOCs are produced by the operation of composting facilities with substantial amounts of solid waste, such as the companies in this study. These may pose a health risk to those working in the plants; thus, it is critical to understand where the VOCs occur in the process in order to maintain workers' occupational health measures. This form of evaluation is rare or nonexistent in Colombia, which is why conducting this type of study is critical, as it will provide crucial input into determining when the highest levels of VOC generation occur. These are the ones that may pose a risk at some point, but with proper occupational safety planning, said risk may be avoided. This work has evaluated three composting systems, with different types of waste and mixtures. According to reports, while composting systems continue to produce VOCs and their generation is unavoidable, the potential risk exists only within the plant. These findings can pave the way for the implementation of public policies that will improve the design and operation of composting plants. There is no specific legislation in Colombia for the design and execution of this sort of technology, which allows the use of organic waste.

Changes in Lolium perenne L. rhizosphere microbiome during phytoremediation of Cd- and Hg-contaminated soils.

The contamination of soil and water by metals such as mercury (Hg) and cadmium (Cd) has been increasing in recent years, because of anthropogenic activities such as mining and agriculture, respectively. In this work, the changes in the rhizosphere microbiome of Lolium perenne L. during the phytoremediation of soils contaminated with Hg and Cd were evaluated. For this, two soil types were sampled, one inoculated with mycorrhizae and one without. The soils were contaminated with Hg and Cd, and L. perenne seeds were sown and harvested after 30 days. To assess changes in the microbiome, DNA isolation tests were performed, for which samples were subjected to two-step PCR amplification with specific 16S rDNA V3-V4 primers (337F and 805R). With mycorrhizae, changes had been found in the absorption processes of metals and a new distribution. While with respect to microorganisms, families such as the Enterobacteriaceae have been shown to have biosorption and efflux effects on metals such as Hg and Cd. Mycorrhizae then improve the efficiency of removal and allow the plant to better distribute the absorbed concentrations. Overall, L. perenne is a species with a high potential for phytoremediation of Cd- and Hg-contaminated soils in the tropics. Inoculation with mycorrhizae modifies the phytoremediation mechanisms of the plant and the composition of microorganisms in the rhizosphere. Mycorrhizal inoculation and changes in the microbiome were associated with increased plant tolerance to Cd and Hg. Microorganism-assisted phytoremediation is an appropriate alternative for L. perenne.

Remediation of Pb-contaminated soil using biochar-based slow-release P fertilizer and biomonitoring employing bioindicators.

Soil contamination by Pb can result from different anthropogenic sources such as lead-based paints, gasoline, pesticides, coal burning, mining, among others. This work aimed to evaluate the potential of P-loaded biochar (Biochar-based slow-release P fertilizer) to remediate a Pb-contaminated soil. In addition, we aim to propose a biomonitoring alternative after soil remediation. First, rice husk-derived biochar was obtained at different temperatures (450, 500, 550, and 600 °C) (raw biochars). Then, part of the resulting material was activated. Later, the raw biochars and activated biochars were immersed in a saturated KH2PO4 solution to produce P-loaded biochars. The ability of materials to immobilize Pb and increase the bioavailability of P in the soil was evaluated by an incubation test. The materials were incorporated into doses of 0.5, 1.0, and 2.0%. After 45 days, soil samples were taken to biomonitor the remediation process using two bioindicators: a phytotoxicity test and enzyme soil activity. Activated P-loaded biochar produced at 500 °C has been found to present the best conditions for soil Pb remediation. This material significantly reduced the bioavailability of Pb and increased the bioavailability of P. The phytotoxicity test and the soil enzymatic activity were significantly correlated with the decrease in bioavailable Pb but not with the increase in bioavailable P. Biomonitoring using the phytotoxicity test is a promising alternative for the evaluation of soils after remediation processes.

Application of an artificial neural networks for predicting the heat transfer in conical spouted bed using the Nusselt module.

Artificial neural networks have been used since the last decade as a satisfactory alternative for the prediction of the fluid-dynamic behavior of particles. The aim of this work has been to develop a model based on artificial neural networks (ANN) suitable for quantifying the influence of multiple factors on the heat transfer rate in a conical spouted bed reactor. The Nusselt module has been taken as an exit point and nine input factors have been evaluated, among which are the height of the bed, the diameter of the contactor, the angle of the cone, and the minimum spouting speed, among others. The model has been found to fit appropriately to the equations proposed in the literature and can be used as a suitable model to predict the behavior of heat transfer in conical spouted bed reactors operating with biomass.

Assessment of the application of two amendments (lime and biochar) on the acidification and bioavailability of Ni in a Ni-contaminated agricultural soils of northern Colombia.

Soil acidification and increased bioavailability of Ni are problems that affect agricultural soils. This study aims to compare the effects of both lime and biochar from corn stover in soil acidity correction, improving soil physicochemical properties and soil re-acidification resistance. As well as assesseing the impacts on human health risk caused by bioavailability of nickel. A greenhouse pot experiment was conducted for 30 days to determine the effect of biochar and lime on soil physicochemical properties and nickel bioavailability. Afterwards, a laboratory test was carried out to determine the repercussions of both amendments on soil resistance to re-acidification and re-mobilization of nickel. Human health risk was determined using nickle bioavailable concentration. Overall, the results of this study showed that biochar application significantly reduced soil acidity from 8.2 ± 0.8 meq 100 g-1 to 1.9 ± 0.3 meq 100 g-1, this reduction markedly influenced the bioavailability of nickel, which decreased significantly. Moreover, soil physicochemical properties and soil resistance to acidification were improved. Furthermore, biochar significantly reduced human health risk compared to lime application, even under a re-acidification scenario. It was possible to verify that Ni immobilization in the soil was increased when biochar was used. Soil Ni immobilization is associated with co-precipitation and chemisorption. Hence, it was demonstrated that biochar is more effective than lime in reducing soil acidity and remedying nickel-contaminated agricultural soils.

Use of Biochar from Rice Husk Pyrolysis: Part A: Recovery as an Adsorbent in the Removal of Emerging Compounds.

One of the main products of pyrolysis is char. For the better performance and improvement of its physicochemical properties, it is necessary to make temperature changes. In this study, different temperatures have been tested for the pyrolysis of rice husk, and the biochar obtained from the process went through an evaluation to test its yield in the removal of emerging compounds such as azithromycin (AZT) and erythromycin (ERY). For this, pyrolysis of rice husk has been carried out at temperatures of 450, 500, 550, and 600 °C, and the biochars have been characterized by ultimate analysis and proximate analysis, as well as specific surface area tests. Then, different adsorption tests have been carried out with a 200 mg L-1 drug (AZT and ERY) solution prepared in the laboratory. All biochars have been found to present removal percentages higher than 95%. Therefore, obtaining biochar from rice husk at any temperature and using it in the removal of high-molecular-weight compounds are quite suitable.

Opportunities and challenges for integrating the development of sustainable polymer materials within an international circular (bio)economy concept.

Highlights: The production and consumption of commodity polymers have been an indispensable part of the development of our modern society. Owing to their adjustable properties and variety of functions, polymer-based materials will continue playing important roles in achieving the Sustainable Development Goals (SDG)s, defined by the United Nations, in key areas such as healthcare, transport, food preservation, construction, electronics, and water management. Considering the serious environmental crisis, generated by increasing consumption of plastics, leading-edge polymers need to incorporate two types of functions: Those that directly arise from the demands of the application (e.g. selective gas and liquid permeation, actuation or charge transport) and those that enable minimization of environmental harm, e.g., through prolongation of the functional lifetime, minimization of material usage, or through predictable disintegration into non-toxic fragments. Here, we give examples of how the incorporation of a thoughtful combination of properties/functions can enhance the sustainability of plastics ranging from material design to waste management. We focus on tools to measure and reduce the negative impacts of plastics on the environment throughout their life cycle, the use of renewable sources for their synthesis, the design of biodegradable and/or recyclable materials, and the use of biotechnological strategies for enzymatic recycling of plastics that fits into a circular bioeconomy. Finally, we discuss future applications for sustainable plastics with the aim to achieve the SDGs through international cooperation. Abstract: Leading-edge polymer-based materials for consumer and advanced applications are necessary to achieve sustainable development at a global scale. It is essential to understand how sustainability can be incorporated in these materials via green chemistry, the integration of bio-based building blocks from biorefineries, circular bioeconomy strategies, and combined smart and functional capabilities.

Use of biochar from rice husk pyrolysis: assessment of reactivity in lime pastes.

Biochar has unique properties such as its porous structure, specific surface area, and stable chemical properties. The rice husk is characterized by its high content of silica, and that during the pyrolysis process it generates a considerable amount of biochar that can be used in different processes. The aim of this work is to evaluate several biochars from the pyrolysis process in the reactivity of lime pastes. For this, biochar has been obtained at four different temperatures (450, 500, 550 and 600 °C), and they have been characterized by XRF, XRD, ICP-EOS, and particle size distribution, to determine their phases and their chemical composition. Biochar has been replaced in lime pastes in different proportions (5, 10, 15, 20, 25 and 30%), and exposed to different curing times (1, 3, 7, 14, 28, 56, 90 and 180 days). It has been found that all the replacements show reactivity within the lime pastes and that the percentage of 25% in all the biochar tested could be an adequate replacement.

Assessment of the potential risk of leaching pesticides in agricultural soils: study case Tibasosa, Boyacá, Colombia.

Agricultural soils need monitoring systems to address pesticide risks for humans and the environment. The purpose of this paper was to obtain leaching risk maps of the pesticides imidacloprid, lambda-cyhalothrin, and chlorpyrifos in agricultural soil under an onion (Allium cepa L.) crop in Tibasosa, Boyacá, Colombia. This was obtained by studying the soil types in the area, analyzing the behavior of pollutants in the soil profile, using a delay factor and an attenuation factor to finally include GIS allowing visualization of the areas of greater potential risk in the study area.

Gene expression and morphological responses of Lolium perenne L. exposed to cadmium (Cd2+) and mercury (Hg2+).

Soil contamination with heavy metals is a major problem worldwide, due to the increasing impact mainly caused by anthropogenic activities. This research evaluated the phytoremediation capacity of, Lolium perenne for heavy metals such as cadmium (Cd2+) and mercury (Hg2+), and the effects of these metals on morphology, biomass production, and the changes on gene expression. Seeds of L. perenne were exposed to six concentrations of Cd2+ and Hg2+ in the range of 0 to 25 mg L-1, and two mixtures of Cd2+-Hg2. The Non-Observed Effect Level (NOEL) was established with dose response curves and the expression of specific genes was evaluated applying a commercially available quantitative reverse transcription (RT-qPCR) assay. There was no significant effect when exposing the seeds to Hg2+, for Cd2+ the maximum concentration was established in 0.1 mg L-1, and for the two concentrations of mixtures, there was a negative effect. An increase of expression of genes that regulate antioxidant activity and stress was found when the plant was exposed to heavy metals. Given the high tolerance to metals analyzed that was reflected both, the development of the plant and in its molecular response, these results highlight that L. perenne is a plant with phytoremediator potential.

Removal of Drugs in Polluted Waters with Char Obtained by Pyrolysis of Hair Waste from the Tannery Process.

The presence and final destination of pharmaceutical compounds in waters constitute one of the emerging events in current environmental chemistry. Two widely consumed compounds have been evaluated in this study, amoxicillin (AMOX) and diclofenac (DFC), at a concentration of 200 mg L-1. The presence of both in wastewater has been verified, generating problems in ecosystems and human health. Pyrolysis of hair waste from a tannery process was performed in a fixed-bed reactor. Char was obtained at different operating temperatures (300, 350, 400, and 450 °C), which underwent a characterization of heavy metals and elemental composition. An activation process was applied to the char obtained at 450 °C by means of physicochemical processes and with two chemical agents (KOH and K2CO3). For the removal of drugs, two separate tests were performed, one for 28 days and the other one for 4 h, to assess the efficiency and the percentage of removal. It was found that the char obtained at 450 °C is the one that removes most of both compounds: more than 90% of AMOX and more than 80% of DFC.

Preliminary study of the production of metabolites from in vitro cultures of C. ensiformis.

BACKGROUND: Canavalia ensiformis is a legume native to Central and South America that has historically been a source of protein. Its main proteins, urease, and lectin have been extensively studied and are examples of bioactive compounds. In this work, the effect of pH and light effects on the growth of C. ensiformis were analyzed. Also, the bioactive compounds such as phenols, carotenoids, chlorophyll a/b, and the growth of callus biomass of C. ensiformis from the effect of different types of light treatments (red, blue and mixture) were evaluated. Likewise, the antioxidative activity of C. ensiformis extracts were studied and related to the production of bioactive compounds. For this, a culture of calluses obtained from seeds were carried out. For the light experiments, polypropylene boxes with red, blue, combination (1/3, 3/1 and 1/1 R-B, respectively) lights and white LED were used as control. In each treatment, three glass containers with 25 ml of MS salts containing 0.25 g of fresh callus were seeded. RESULTS: The results have shown that the pH of the culture medium notably affects the increase in callogenic biomass. It shows that the pH of 5.5 shows better results in the callogenic growth of C. ensiformis with an average increase of 1.3051 g (198.04%), regarding the initial weight. It was found that the pH 5.5 and the 1/3 R-B LED combination had higher production of bioactive compounds and better antioxidant activity. At the same time, the red-light treatment was least effective. CONCLUSIONS: It was possible to find the ideal conditions of important growth under conditions of pH and light of C. ensiformis. Likewise, it is evaluated whether the production of compounds of interest, such as phenolic compounds and carotenoids, occurs under these conditions. The highest production of calluses occurs in the 1/3 R-B LED combined light treatment, which showed a significant increase in biomass, followed by B. From this study, it could be demonstrated that C. ensiformis produces compounds such as phenols and carotenoids in vitro culture that are essential for the antioxidant activity of the plant.

Portable and Low-Cost Respirometric Microsystem for the Static and Dynamic Respirometry Monitoring of Compost.

Composting is considered an option for the disposal of organic waste; however, the development of portable and low-cost systems for its monitoring is of high interest. Therefore, in this study, respirometric microsystems were designed and tested including two integrated oxygen sensors for the measurement of compost samples under static and dynamic conditions with high portability and ease of use. The cost of each sensor was calculated as 2 USD, while the cost of the whole respirometric microsystem was calculated as 6 USD. The electronic system for real-time monitoring was also designed and implemented. The designed systems were tested for over 6 weeks for the determination of compost quality using real samples. The respirometric microsystem was compared to a commercial respirometry system and a standard laboratory test using hierarchical analysis which included costs, portability accuracy, analysis time, and integration of new technologies. The analysis showed a global score of 6.87 for the respirometric microsystem compared to 6.70 for the standard laboratory test and 3.26 for the commercial system.

Major transitions in dinoflagellate evolution unveiled by phylotranscriptomics.

Dinoflagellates are key species in marine environments, but they remain poorly understood in part because of their large, complex genomes, unique molecular biology, and unresolved in-group relationships. We created a taxonomically representative dataset of dinoflagellate transcriptomes and used this to infer a strongly supported phylogeny to map major morphological and molecular transitions in dinoflagellate evolution. Our results show an early-branching position of Noctiluca, monophyly of thecate (plate-bearing) dinoflagellates, and paraphyly of athecate ones. This represents unambiguous phylogenetic evidence for a single origin of the group's cellulosic theca, which we show coincided with a radiation of cellulases implicated in cell division. By integrating dinoflagellate molecular, fossil, and biogeochemical evidence, we propose a revised model for the evolution of thecal tabulations and suggest that the late acquisition of dinosterol in the group is inconsistent with dinoflagellates being the source of this biomarker in pre-Mesozoic strata. Three distantly related, fundamentally nonphotosynthetic dinoflagellates, Noctiluca, Oxyrrhis, and Dinophysis, contain cryptic plastidial metabolisms and lack alternative cytosolic pathways, suggesting that all free-living dinoflagellates are metabolically dependent on plastids. This finding led us to propose general mechanisms of dependency on plastid organelles in eukaryotes that have lost photosynthesis; it also suggests that the evolutionary origin of bioluminescence in nonphotosynthetic dinoflagellates may be linked to plastidic tetrapyrrole biosynthesis. Finally, we use our phylogenetic framework to show that dinoflagellate nuclei have recruited DNA-binding proteins in three distinct evolutionary waves, which included two independent acquisitions of bacterial histone-like proteins.

Analysis of EST data of the marine protist Oxyrrhis marina, an emerging model for alveolate biology and evolution.

BACKGROUND: The alveolates include a large number of important lineages of protists and algae, among which are three major eukaryotic groups: ciliates, apicomplexans and dinoflagellates. Collectively alveolates are present in virtually every environment and include a vast diversity of cell shapes, molecular and cellular features and feeding modes including lifestyles such as phototrophy, phagotrophy/predation and intracellular parasitism, in addition to a variety of symbiotic associations. Oxyrrhis marina is a well-known model for heterotrophic protist biology, and is now emerging as a useful organism to explore the many changes that occurred during the origin and diversification of dinoflagellates by virtue of its phylogenetic position at the base of the dinoflagellate tree. RESULTS: We have generated and analysed expressed sequence tag (EST) sequences from the alveolate Oxyrrhis marina in order to shed light on the evolution of a number of dinoflagellate characteristics, especially regarding the emergence of highly unusual genomic features. We found that O. marina harbours extensive gene redundancy, indicating high rates of gene duplication and transcription from multiple genomic loci. In addition, we observed a correlation between expression level and copy number in several genes, suggesting that copy number may contribute to determining transcript levels for some genes. Finally, we analyze the genes and predicted products of the recently discovered Dinoflagellate Viral Nuclear Protein, and several cases of horizontally acquired genes. CONCLUSION: The dataset presented here has proven very valuable for studying this important group of protists. Our analysis indicates that gene redundancy is a pervasive feature of dinoflagellate genomes, thus the mechanisms involved in its generation must have arisen early in the evolution of the group.

Morphology and molecular phylogeny of Pseudotrichonympha hertwigi and Pseudotrichonympha paulistana (Trichonymphea, parabasalia) from neotropical rhinotermitids.

Pseudotrichonympha is a large hypermastigote parabasalian found in the hindgut of several species of rhinotermitid termites. The genus was discovered more than 100 years ago, and although over a dozen species have since been described, this represents only a small fraction of its likely diversity: the termite genera from which Pseudotrichonympha is known are all species rich, and in most cases their hindgut symbionts have not been examined. Even formally described species are mostly lacking in detailed microscopic data and/or sequence data. Using small subunit ribosomal RNA gene sequences and light and scanning electron microscopy we describe here the morphology and molecular phylogenetic position of two Pseudotrichonympha species: the type species for the genus, Pseudotrichonympha hertwigi from Coptotermes testaceus (described previously in line drawing only), and Pseudotrichonympha paulistana from Heterotermes tenuis (described previously based on light microscopy only).

The highly reduced and fragmented mitochondrial genome of the early-branching dinoflagellate Oxyrrhis marina shares characteristics with both apicomplexan and dinoflagellate mitochondrial genomes.

The mitochondrial genome and the expression of the genes within it have evolved to be highly unusual in several lineages. Within alveolates, apicomplexans and dinoflagellates share the most reduced mitochondrial gene content on record, but differ from one another in organisation and function. To clarify how these characteristics originated, we examined mitochondrial genome form and expression in a key lineage that arose close to the divergence of apicomplexans and dinoflagellates, Oxyrrhis marina. We show that Oxyrrhis is a basal member of the dinoflagellate lineage whose mitochondrial genome has some unique characteristics while sharing others with apicomplexans or dinoflagellates. Specifically, Oxyrrhis has the smallest gene complement known, with several rRNA fragments and only two protein coding genes, cox1 and a cob-cox3 fusion. The genome appears to be highly fragmented, like that of dinoflagellates, but genes are frequently arranged as tandem copies, reminiscent of the repeating nature of the Plasmodium genome. In dinoflagellates and Oxyrrhis, genes are found in many arrangements, but the Oxyrrhis genome appears to be more structured, since neighbouring genes or gene fragments are invariably the same: cox1 and the cob-cox3 fusion were never found on the same genomic fragment. Analysing hundreds of cDNAs for both genes and circularized mRNAs from cob-cox3 showed that neither uses canonical start or stop codons, although a UAA terminator is created in the cob-cox3 fusion mRNA by post-transcriptional oligoadenylation. mRNAs from both genes also use a novel 5' oligo(U) cap. Extensive RNA editing is characteristic of dinoflagellates, but we find no editing in Oxyrrhis. Overall, the combination of characteristics found in the Oxyrrhis genome allows us to plot the sequence of many events that led to the extreme organisation of apicomplexan and dinoflalgellate mitochondrial genomes.

Multiple protein phylogenies show that Oxyrrhis marina and Perkinsus marinus are early branches of the dinoflagellate lineage.

Oxyrrhis marina and Perkinsus marinus are two alveolate species of key taxonomic position with respect to the divergence of apicomplexans and dinoflagellates. New sequences from Oxyrrhis, Perkinsus and a number of dinoflagellates were added to datasets of small-subunit (SSU) rRNA, actin, alpha-tubulin and beta-tubulin sequences, as well as to a combined dataset of all three protein-coding genes, and phylogenetic trees were inferred. The parasitic Perkinsus marinus branches at the base of the dinoflagellate clade with high support in most of the individual gene trees and in the combined analysis, strongly confirming the position originally suggested in previous SSU rRNA and actin phylogenies. The SSU rRNA from Oxyrrhis marina is extremely divergent, and it typically branches with members of the Gonyaulacales, a dinoflagellate order where SSU rRNA sequences are also divergent. Conversely, none of the three protein-coding genes of Oxyrrhis is noticeably divergent and, in trees based on all three proteins individually and in combination, Oxyrrhis branches at the base of the dinoflagellate clade, typically with high bootstrap support. In some trees, Oxyrrhis and Perkinsus are sisters, but most analyses indicate that Perkinsus diverged prior to Oxyrrhis. Morphological characters have previously pointed to Oxyrrhis as an early branch in the dinoflagellate lineage; our data support this suggestion and significantly bolster the molecular data that support a relationship between Perkinsus and dinoflagellates. Together, these two organisms can be instrumental in reconstructing the early evolution of dinoflagellates and apicomplexans by helping to reveal aspects of the ancestors of both groups.