Benthic diatom metabarcoding: developing new approaches to research and biomonitoring in aquatic ecosystems

Les diatomees han estat àmpliament estudiades en bona part degut a la seva rellevància ecològica i per ser excel·lent bioindicadores. No obstant, la identificació de les diatomees a nivell d'espècie requereix temps i personal amb coneixements taxonòmics. Recentment, el mètode d’ADN metabarcoding s’ha postulat com una alternativa a les identificacions morfològiques basades en microscòpia òptica (MO), val a dir però, que hi ha diversos factors que poden disminuir l'eficàcia del metabarcoding. L´objectiu principal d´aquesta tesi és avaluar l´aplicabilitat de la tècnica metabarcoding per al seguiment dels sistemes aquàtics sota la Directiva Marc de l´Aigua, així com per abordar qüestions ecològiques i biogeogràfiques de les diatomees. Els nostres resultats indiquen que el metabarcoding, basat en el marcador cloroplàstic rbcL, constitueix una alternativa eficaç al MO per al seguiment de rius mediterranis, com els que hi ha a Catalunya, bàsicament gràcies a que les espècies de diatomees bentòniques més comunes d'aquests rius estan representades a la biblioteca de referència. Per contra, les espècies marines hi són molt menys representades i això comporta que el metabarcoding no pugui ser encara una alternativa a la MO en sistemes costaners, però sí una eina complementària capaç de detectar espècies difícilment identificables per MO com són espècies petites, delicades o desproveïdes de frústuls. També hem vist que l'elecció entre els dos marcadors rbcL usats actualment en metabarcoding té poques implicacions per als programes de seguiment, però sí per a estudis de biodiversitat. Aquesta tesi també mostra com les variants genètiques de diversos complexos d'espècies de diatomees difereixen a les seves preferències ecològiques i distribució espacial. A més, l'anàlisi d'una base de dades global (rbcL) de diatomees d'aigua dolça ens ha permès observar que les diatomees pennades presenten més diversitat intraespecífica que les cèntriques i alhora identificar quatre patrons filogeogràfics comuns entre les espècies analitzades.Las diatomeas han sido ampliamente estudiadas debido en parte a su gran importancia ecológica y al hecho de ser excelentes bioindicadores. No obstante, la identificación de las diatomeas a nivel de especie es una tarea que requiere de tiempo y conocimientos taxonómicos. Recientemente, el método ADN metabarcoding ha sido contemplado como una alternativa a las identificaciones morfológicas basadas en microscopía óptica (MO), si bien es cierto que existen varios factores que pueden disminuir la eficacia del metabarcoding. El objetivo principal de esta tesis es evaluar la aplicabilidad de la técnica metabarcoding para el seguimiento de sistemas acuáticos bajo la Directiva Marco del Agua, así como para abordar cuestiones ecológicas y biogeográficas. Nuestros resultados indican que el metabarcoding, basado en el marcador cloroplástico rbcL, constituye una alternativa eficaz al MO para el seguimiento de ríos mediterráneos, como los de Cataluña, debido principalmente a que las especies de diatomeas bentónicas más comunes de estos ríos están representadas en la biblioteca de referencia. Por contra, las especies marinas están en menor medida representadas lo cual conlleva que el metabarcoding no sea actualmente una alternativa a la MO en sistemas costeros, pero sí una herramienta complementaria capaz de detectar especies difícilmente identificables por MO como especies pequeñas, delicadas o desprovistas de frústulos. También hemos visto que la elección entre los dos marcadores rbcL usados actualmente en metabarcoding tiene pocas implicaciones para los programas de seguimiento, pero sí para estudios de biodiversidad. Esta tesis también muestra como las variantes genéticas de varios complejos de especies de diatomeas difieren en sus preferencias ecológicas y distribución espacial. Además, el análisis de una base de datos global (rbcL) de diatomeas de agua dulce nos ha permitido observar que las diatomeas pennadas presentan mayor diversidad intraespecífica que las céntricas e identificar cuatro patrones filogeográficos comunes entre las especies analizadas.Diatoms have been widely studied partly because of their great ecological importance and the fact that they are excellent bioindicators. However, the identification of diatoms at the species level is a time-consuming and taxonomically demanding task. Recently, DNA metabarcoding has been considered as an alternative to morphological identifications based on light microscopy (LM), although there are several factors that may decrease the effectiveness of metabarcoding. The main objective of this thesis is to evaluate the applicability of DNA metabarcoding for monitoring aquatic systems under the Water Framework Directive, as well as to address ecological and biogeographical questions. Our results indicate that metabarcoding, based on the chloroplastic marker rbcL, constitutes an effective alternative to LM for monitoring Mediterranean rivers, such as those of Catalonia, mainly because the most common benthic diatom species in these rivers are represented in the reference library. By contrast, marine species are less represented, which means that metabarcoding is not currently an alternative to LM in coastal systems, but it is a complementary tool capable of detecting species that are difficult to identify by LM, such as small, delicate species or species lacking frustules. We have also seen that the choice between the two rbcL markers currently used in metabarcoding has few implications for monitoring programmes, but does have implications for biodiversity studies. This thesis also shows how genetic variants within several diatom species complexes differ in their ecological preferences and spatial distribution. In addition, the analysis of a global database (rbcL) of freshwater diatoms has allowed us to observe that pennate diatoms show higher intraspecific diversity than centric diatoms and to identify four common phylogeographic patterns among the species analysed

Thirty-day outcomes in frail older patients discharged home from the emergency department with acute heart failure: effects of high-risk criteria identified by the DEED FRAIL-AHF trial

Objectives: To study the effect of high-risk criteria on 30-day outcomes in frail older patients with acute heart failure (AHF) discharged from an emergency department (ED) or an ED's observation and short-stay areas. Material and methods: Secondary analysis of discharge records in the Older AHF Key Data registry. We selected frail patients (aged > 70 years) discharged with AHF from EDs. Risk factors were categorized as modifiable or nonmodifiable. The outcomes were a composite endpoint for a cardiovascular event (revisits for AHF, hospitalization for AHF, or cardiovascular death) and the number of days alive out-of-hospital (DAOH) within 30 days of discharge. Results: We included 380 patients with a mean (SD) age of 86 (5.5) years (61.2% women). Modifiable risk factors were identified in 65.1%, nonmodifiable ones in 47.8%, and both types in 81.6%. The 30-day cardiovascular composite endpoint occurred in 83 patients (21.8%). The mean 30-day DAOH observed was 27.6 (6.1) days. Highrisk factors were present more often in patients who developed the cardiovascular event composite endpoint: the rates for patients with modifiable, nonmodifiable, or both types of risk were, respectively, as follows in comparison with patients not at high risk: 25.0% vs 17.2%, P = .092; 27.6% vs 16.7%, P = .010; and 24.7% vs 15.2%, P = .098). The 30-day DAOH outcome was also lower for at-risk patients, according to type of risk factor present: modifiable, 26.9 (7.0) vs 28.4 (4.4) days, P = .011; nonmodifiable, 27.1 (7.0) vs 28.0 (5.0) days, P = .127; and both, 27.1 (6.7) vs 28.8 (3.4) days, P = .005). After multivariate analysis, modifiable risk remained independently associated with fewer days alive (adjusted absolute difference in 30-day DAOH, -1.3 days (95% CI, -2.7 to -0.1 days). Nonmodifiable factors were associated with increased risk for the 30-day cardiovascular composite endpoint (adjusted absolute difference, 10.4%; 95% CI, -2.1% to 18.7%). Conclusion: Risk factors are common in frail elderly patients with AHF discharged home from hospital ED areas. Their presence is associated with a worse 30-day prognosis

Assessment of a novel microalgae-cork based technology for removing antibiotics, pesticides and nitrates from groundwater

Groundwater pollution has increased in recent years due to the intensification of agricultural and livestock activities. This results in a significant reduction in available freshwater resources. Here, we have studied the long term assessment of a green technology (1-4 L/day) based on a photobioreactor (PBR) containing immobilised microalgae-bacteria in polyurethane foam (PF) followed by a cork filter (CF) for removing nitrates, pesticides (atrazine and bromacil), and antibiotics (sulfamethoxazole and sulfacetamide) from groundwater. The prototype was moderately effective for removing nitrates (58%) at an HRT of 8 days, while its efficiency decreased at a HRT of 4 and 2 days (<20% removal). The combined use of PBR-CF enabled antibiotics and pesticides to be attenuated by up to 95% at an HRT of 8 days, but their attenuation decreased with shorter HRT, with pesticides being the compounds most affected (reducing from 97 to 98% at an HRT of 8 days to 23-45% at an HRT of 2 days). Pesticide transformation products were identified after the CF, supporting biodegradation as the main attenuation process. A gene-based metataxonomic assessment linked the attenuation of micropollutants to the presence of specific pesticide biodegradation species (e.g. genus Phenylobacterium, Sphingomonadaceae, and Caulobacteraceae). Therefore, the results highlighted the potential use of microalgae and cork to treat polluted groundwater.The authors wish to thank the financial support of the European Union through the project LIFE18 ENV/ES/000199 and the Spanish Ministry of Science, Innovation and Universities through Project CTM2017-91355-EXP. Finally, European Commission (Erasmus program) and Government of Chile for supporting Lorenzo Rambaldo and Héctor Avila Cortés's visit at IDAEA-CSIC in Barcelona, Spain.Peer reviewe

Stream diatom community assembly processes in islands and continents: a global perspective

[EN] Understanding the roles of deterministic and stochastic processes in community assembly is essential for gaining insights into the biogeographical patterns of biodiversity. However, the way community assembly processes operate is still not fully understood, especially in oceanic islands. In this study, we examine the importance of assembly processes in shaping diatom communities in islands and continents, while also investigating the influence of climate and local water chemistry variables on species distributions. Location Global. Taxon Stream benthic diatoms. Methods We used diatom datasets from five continents and 19 islands and applied beta diversity analyses with a null model approach and hierarchical joint species distribution modelling. To facilitate comparisons with continents, we used continental area equivalents (CAEs), which represent continental subsets with comparable areas and the same number of study sites as their corresponding islands counterparts. Results We found that homogeneous selection (i.e., communities being more similar than the random expectation) was the dominant assembly process within islands whereas stochastic processes tended to be more important within continents. In addition, assembly processes were influenced by study scale and island isolation. Climatic variables showed a greater influence on species distribution than local factors. However, in islands, local environmental variables had a greater impact on the distributions of unique taxa as opposed to non-unique taxa. Main Conclusions We observed that the assembly processes of diatom communities were complex and influenced by a combination of deterministic and stochastic forces, which varied across spatial scales. In islands, there was no universal pattern of assembly processes, given that their influence depends on abiotic conditions such as area, isolation, and environmental heterogeneity. In addition, the sensitivity of species occurring uniquely in islands to local environmental variables suggests that they are perhaps less vulnerable to climatic changes but may be more influenced by changes in local physicochemistrySIFor financial support, the authors thank the Academy of Finland (grant nr. 346812 to JS); the Institut Francais de Finlande; the Embassy of France to Finland; the French Ministry of Education and Higher Education; Finnish Society of Sciences and Letters. J.J. Wang was further supported by the National Natural Science Foundation of China (91851117, 41871048), CAS Key Research Program of Frontier Sciences (QYZDB-SSW-DQC043), and The National Key Research and Development Program of China (2019YFA0607100

New molecular methods to assess biodiversity. Potentials and pitfalls of DNA metabarcoding: a workshop report

This report presents the outcome of the joint work of PhD students and senior researchers working with DNA-based biodiversity assessment approaches with the goal to facilitate others the access to definitions and explanations about novel DNA-based methods. The work was performed during a PhD course (SLU PNS0169) at the Swedish University of Agricultural Sciences (SLU) in Uppsala, Sweden. The course was co-organized by the EU COST research network DNAqua-Net and the SLU Research Schools Focus on Soils and Water (FoSW) and Ecology - basics and applications. DNAqua-Net (COST Action CA15219, 2016-2020) is a network connecting researchers, water managers, politicians and other stakeholders with the aim to develop new genetic tools for bioassessment of aquatic ecosystems in Europe and beyond. The PhD course offered a comprehensive overview of the paradigm shift from traditional morphology-based species identification to novel identification approaches based on molecular markers. We covered the use of molecular tools in both basic research and applied use with a focus on aquatic ecosystem assessment, from species collection to the use of diversity in environmental legislation. The focus of the course was on DNA (meta)barcoding and aquatic organisms. The knowledge gained was shared with the general public by creating Wikipedia pages and through this collaborative Open Access publication, co-authored by all course participants

Evaluation of two short overlapping rbcL markers for diatom metabarcoding of environmental samples: Effects on biomonitoring assessment and species resolution

Two short diatom rbcL barcodes, 331-bp and 263-bp in length, have frequently been used in diatom metabarcoding studies. They overlap in a common 263-bp region but differ in the presence or absence of a 68-bp tail at the 5′ end. Though the effectiveness of both has been demonstrated in separate biomonitoring and diversity studies, the impact of the 68-bp non-shared region has not been evaluated. Here we compare the two barcodes in terms of the values of a biotic index (IPS) and the ecological status classes derived from their application to an extensive metabarcoding dataset from United Kingdom rivers; this comprised 1703 samples and was produced using the 331-bp primers. In addition, we assess the effectiveness of each barcode for discrimination of genetic variants around and below the species level. The strong correlation found in IPS values between barcodes (Pearson's R = 0.98) indicates that the choice of the barcode does not have major implications for current WFD ecological assessments, although a very few sites (55: 3.23% of those analysed) were downgraded from an acceptable WFD class (“good”) to an unacceptable one (“moderate”). Analyses of the taxonomic resolution of the two barcodes indicate that for many ASVs, the use of either marker – 263-bp and 331-bp – gives unambiguous assignations at species level though with differences in bootstrap confidence values. Such differences are caused by the stochasticity involved in the naïve Bayesian classifier used and by the fact that genetic distance, regarding closely related species, is increased when using the 331-bp barcode. However, in three cases, species differentiation fails with the shorter marker, leading to underestimates of species diversity. Finally, two ASVs from Nitzschia species evidenced that the use of the shorter marker can sometimes lead to false positives when the extent and nature of infraspecific variation are poorly known.info:eu-repo/semantics/acceptedVersio

Diatom metabarcoding reveals differences in ecological preferences among genetic variants within some key species for biomonitoring

We applied DNA metabarcoding to evaluate the ecology of genetic variants within several diatom species that are important for biomonitoring. Benthic diatoms are widely used as bioindicators for biomonitoring programmes, including those for European rivers demanded by Water Framework Directive (WFD). Morphological identification of diatoms at species level is required for assessing the ecological status in biomonitoring programmes. However, this is a time-consuming task and requires expert knowledge. In addition, closely related species, which sometimes are scarcely distinguishable on the basis of their morphology, can show different ecological preferences; these may even vary within a single diatom species. Not being able to identify the different ecological preferences shown by the genetic variants of a single species or closely related species, might have consequences for biomonitoring programmes, especially if such differences occur within common species.The key diatom species that we studied were: Fistulifera saprophila (FSAP), widely regarded as a marker for elevated nutrient levels, organic pollution and hence poor ecological status; Achnanthidium minutissimum (ADMI), which usually indicates good ecological status; and Nitzschia inconspicua (NINC) and N. soratensis (NSTS), two species that are widely separated phylogenetically but almost impossible to distinguish in the light microscope. Our dataset was based on high-throughput sequencing using a 312-bp rbcL marker. We used the denoising pipeline DADA2 to infer amplicon sequence variants (ASVs) from 554 environmental samples from river biomonitoring campaigns in Catalonia (NE Spain) and France. Ecological groupings of ASVs were distinguished according to their environmental responses given by Threshold Indicator Taxa ANalysis (TITAN); the environmental parameters that most influenced the occurrence of these groupings were tested using boosted regression trees.We could distinguish three different ecological groupings of ASVs within ADMI and three within FSAP. In each species two of the groupings were clearly separated by their opposite responses to calcium and conductivity and boosted regression trees showed that for three out of four of these groupings, these two variables were among the most important variables for explaining the ASV distributions. The third grouping in FSAP had a negative response to total organic carbon and a positive response to altitude and hence was better represented in less organically polluted waters and higher ecological status than is generally assumed for FSAP.Our analyses did not identify ecological groupings of ASVs within NINC and NSTS but confirmed earlier studies, based on more limited sampling, that indicated different preferences of these species. Conductivity and calcium were the variables that most influenced the occurrence of NINC and NSTS, NINC being better distributed in waters with higher levels of calcium and conductivity than NSTS.Our findings indicate the potential use of DNA metabarcoding for distinguishing the ecological preferences of genetic variants within a single species or closely related species. This information, coupled with the broad knowledge generated over many years using traditional microscope-based identifications, will facilitate the development of more accurate biological indexes for the biomonitoring programmes of the future

La comprensión pragmática en el niño TEA

La comunicación y el desarrollo del lenguaje es una de las áreas donde los niños que pertenecen al Trastorno del Espectro Autista (TEA) presentan más dificultades, siendo de entre los distintos componentes lingüísticos, la pragmática uno de los más afectados. Con el fin de atender a estas necesidades se han ido desarrollando diferentes sistemas de comunicación aumentativa o alternativa, considerándose ARASAAC el más empleado actualmente en nuestro país. El objeto del presente trabajo es comparar en qué medida este sistema de comunicación y otro como son láminas de sucesión temporal, favorecen la comprensión pragmática del lenguaje de estos niños. Aplicando una metodología observacional, se propone un instrumento de observación ad hoc, con la modalidad de formato de campo, a partir del cual se registrarán y analizarán las grabaciones de una serie de tareas que combinarán el empleo de ambos sistemas y que nos ofrecerán los datos que necesitamos para concluir qué tipo de sistema favorece en mayor medida la comprensión y qué aspectos debemos tener en cuenta

DNA metabarcoding reveals differences in distribution patterns and ecological preferences among genetic variants within some key freshwater diatom species

International audienceOur study evaluates differences in the distribution and ecology of genetic variants within several ecologically important diatom species that are also key for Water Framework Directive monitoring of European rivers: Fistulifera saprophila (FSAP), Achnanthidium minutissimum (ADMI), Nitzschia inconspicua (NINC) and Nitzschia soratensis (NSTS). We used DADA2 to infer amplicon sequence variants (ASVs) of a short rbcL barcode in 531 environmental samples from biomonitoring campaigns in Catalonia and France. ASVs within each species showed different distribution patterns. Threshold Indicator Taxa ANalysis revealed three ecological groupings of ASVs in both ADMI and FSAP. Two of these in each species were separated by opposite responses to calcium and conductivity. Boosted regression trees additionally showed that both variables greatly influenced the occurrence of these groupings. A third grouping in FSAP was characterized by a negative response to total organic carbon and hence was better represented in waters with higher ecological status than the other FSAP ASVs, contrasting with what is generally assumed for the species. In the two Nitzschia species, our analyses confirmed earlier studies: NINC preferred higher levels of calcium and conductivity. Our findings suggest that the broad ecological tolerance of some diatom species results from overlapping preferences among genetic variants, which individually show much more restricted preferences and distributions. This work shows the importance of studying the eco-logical preferences of genetic variants within species complexes, now possible with DNA metabarcoding. The re-sults will help reveal and understand biogeographical distributions and facilitate the development of more accurate biological indexes for biomonitoring programmes

DNA metabarcoding reveals differences in distribution patterns and ecological preferences among genetic variants within some key freshwater diatom species

Our study evaluates differences in the distribution and ecology of genetic variants within several ecologically important diatom species that are also key for Water Framework Directive monitoring of European rivers: Fistulifera saprophila (FSAP), Achnanthidium minutissimum (ADMI), Nitzschia inconspicua (NINC) and Nitzschia soratensis (NSTS). We used DADA2 to infer amplicon sequence variants (ASVs) of a short rbcL barcode in 531 environmental samples from biomonitoring campaigns in Catalonia and France. ASVs within each species showed different distribution patterns. Threshold Indicator Taxa ANalysis revealed three ecological groupings of ASVs in both ADMI and FSAP. Two of these in each species were separated by opposite responses to calcium and conductivity. Boosted regression trees additionally showed that both variables greatly influenced the occurrence of these groupings. A third grouping in FSAP was characterized by a negative response to total organic carbon and hence was better represented in waters with higher ecological status than the other FSAP ASVs, contrasting with what is generally assumed for the species. In the two Nitzschia species, our analyses confirmed earlier studies: NINC preferred higher levels of calcium and conductivity. Our findings suggest that the broad ecological tolerance of some diatom species results from overlapping preferences among genetic variants, which individually show much more restricted preferences and distributions. This work shows the importance of studying the ecological preferences of genetic variants within species complexes, now possible with DNA metabarcoding. The results will help reveal and understand biogeographical distributions and facilitate the development of more accurate biological indexes for biomonitoring programmes.info:eu-repo/semantics/acceptedVersio