General principles for assignments of communities from eDNA : Open versus closed taxonomic databases

Metabarcoding of environmental DNA (eDNA) is a powerful tool for describing biodiversity, such as finding keystone species or detecting invasive species in environmental samples. Continuous improvements in the method and the advances in sequencing platforms over the last decade have meant this approach is now widely used in biodiversity sciences and biomonitoring. For its general use, the method hinges on a correct identification of taxa. However, past studies have shown how this crucially depends on important decisions during sampling, sample processing, and subsequent handling of sequencing data. With no clear consensus as to the best practice, particularly the latter has led to varied bioinformatic approaches and recommendations for data preparation and taxonomic identification. In this study, using a large freshwater fish eDNA sequence dataset, we compared the frequently used zero-radius Operational Taxonomic Unit (zOTU) approach of our raw reads and assigned it taxonomically (i) in combination with publicly available reference sequences (open databases) or (ii) with an OSU (Operational Sequence Units) database approach, using a curated database of reference sequences generated from specimen barcoding (closed database). We show both approaches gave comparable results for common species. However, the commonalities between the approaches decreased with read abundance and were thus less reliable and not comparable for rare species. The success of the zOTU approach depended on the suitability, rather than the size, of a reference database. Contrastingly, the OSU approach used reliable DNA sequences and thus often enabled species-level identifications, yet this resolution decreased with the recent phylogenetic age of the species. We show the need to include target group coverage, outgroups and full taxonomic annotation in reference databases to avoid misleading annotations that can occur when using short amplicon sizes as commonly used in eDNA metabarcoding studies. Finally, we make general suggestions to improve the construction and use of reference databases for metabarcoding studies in the future

General principles for assignments of communities from <scp>eDNA</scp> : Open versus closed taxonomic databases

Metabarcoding of environmental DNA (eDNA) is a powerful tool for describing biodiversity, such as finding keystone species or detecting invasive species in environmental samples. Continuous improvements in the method and the advances in sequencing platforms over the last decade have meant this approach is now widely used in biodiversity sciences and biomonitoring. For its general use, the method hinges on a correct identification of taxa. However, past studies have shown how this crucially depends on important decisions during sampling, sample processing, and subsequent handling of sequencing data. With no clear consensus as to the best practice, particularly the latter has led to varied bioinformatic approaches and recommendations for data preparation and taxonomic identification. In this study, using a large freshwater fish eDNA sequence dataset, we compared the frequently used zero-radius Operational Taxonomic Unit (zOTU) approach of our raw reads and assigned it taxonomically (i) in combination with publicly available reference sequences (open databases) or (ii) with an OSU (Operational Sequence Units) database approach, using a curated database of reference sequences generated from specimen barcoding (closed database). We show both approaches gave comparable results for common species. However, the commonalities between the approaches decreased with read abundance and were thus less reliable and not comparable for rare species. The success of the zOTU approach depended on the suitability, rather than the size, of a reference database. Contrastingly, the OSU approach used reliable DNA sequences and thus often enabled species-level identifications, yet this resolution decreased with the recent phylogenetic age of the species. We show the need to include target group coverage, outgroups and full taxonomic annotation in reference databases to avoid misleading annotations that can occur when using short amplicon sizes as commonly used in eDNA metabarcoding studies. Finally, we make general suggestions to improve the construction and use of reference databases for metabarcoding studies in the future

Climate, immigration and speciation shape terrestrial and aquatic biodiversity in the European Alps

Quaternary climate fluctuations can affect speciation in regional biodiversity assembly in two non-mutually exclusive ways: a glacial species pump, where isolation in glacial refugia accelerates allopatric speciation, and adaptive radiation in underused adaptive zones during ice-free periods. We detected biogeographic and genetic signatures associated with both mechanisms in the assembly of the biota of the European Alps. Age distributions of endemic and widespread species within aquatic and terrestrial taxa (amphipods, fishes, amphibians, butterflies and flowering plants) revealed that endemic fish evolved only in lakes, are highly sympatric, and mainly of Holocene age, consistent with adaptive radiation. Endemic amphipods are ancient, suggesting preglacial radiation with limited range expansion and local Pleistocene survival, perhaps facilitated by a groundwater-dwelling lifestyle. Terrestrial endemics are mostly of Pleistocene age and are thus more consistent with the glacial species pump. The lack of evidence for Holocene adaptive radiation in the terrestrial biome is consistent with faster recolonization through range expansion of these taxa after glacial retreats. More stable and less seasonal ecological conditions in lakes during the Holocene may also have contributed to Holocene speciation in lakes. The high proportion of young, endemic species makes the Alpine biota vulnerable to climate change, but the mechanisms and consequences of species loss will likely differ between biomes because of their distinct evolutionary histories

Climate, immigration and speciation shape terrestrial and aquatic biodiversity in the European Alps.

Quaternary climate fluctuations can affect speciation in regional biodiversity assembly in two non-mutually exclusive ways: a glacial species pump, where isolation in glacial refugia accelerates allopatric speciation, and adaptive radiation in underused adaptive zones during ice-free periods. We detected biogeographic and genetic signatures associated with both mechanisms in the assembly of the biota of the European Alps. Age distributions of endemic and widespread species within aquatic and terrestrial taxa (amphipods, fishes, amphibians, butterflies and flowering plants) revealed that endemic fish evolved only in lakes, are highly sympatric, and mainly of Holocene age, consistent with adaptive radiation. Endemic amphipods are ancient, suggesting preglacial radiation with limited range expansion and local Pleistocene survival, perhaps facilitated by a groundwater-dwelling lifestyle. Terrestrial endemics are mostly of Pleistocene age and are thus more consistent with the glacial species pump. The lack of evidence for Holocene adaptive radiation in the terrestrial biome is consistent with faster recolonization through range expansion of these taxa after glacial retreats. More stable and less seasonal ecological conditions in lakes during the Holocene may also have contributed to Holocene speciation in lakes. The high proportion of young, endemic species makes the Alpine biota vulnerable to climate change, but the mechanisms and consequences of species loss will likely differ between biomes because of their distinct evolutionary histories

Pérdida del área de ocupación y del tamaño de la población de Vella lucentina (Cruciferae)

Se presenta nueva información sobre los tamaños de población de Vella lucentina, endemismo alicantino propio de matorrales esteparios calcícolas, bajo bioclima termomediterráneo semiárido. A partir de muestreos de campo durante la época de floración (abril a junio de 2008), se han delimitado en un SIG los polígonos envolventes de los tres núcleos de población conocidos. Se ha localizado y delimitado un nuevo núcleo en la Serra dels Castellans (Agost). Se han estimado los efectivos poblacionales en cada una de las subpoblaciones. La superficie total de ocupación actual es de 351,52 ha. El tamaño de población, según criterios de la UICN (individuos maduros), puede estimarse en 385.671 individuos. A partir de la realización de un diagnóstico de la situación actual y de su evolución en un período superior a 50 años, se constata que tres de las subpoblaciones se encuentran severamente fragmentadas. La subpoblación de Bec de l’Àguila, la mayor, más extensa y continua, presenta, sin embargo, un riesgo inminente de declive. A pesar de su elevado tamaño de población local, se recomienda la catalogación de V. lucentina como “en peligro” (EN) y su inclusión como “especie en peligro de extinción”en el Catálogo Valenciano de Flora Amenazada.We report new information about population size of Vella lucentina, a taxon endemic to the Thermomediterranean semiarid territories of the central areas of Alicante province (SE of Spain), which grows in calcicolous steppic bushes. We delimited polygon envelopes for the three known subpopulations in a GIS. Field work was carried out during flowering time (April to June, 2008), which resulted in the discovering of a new subpopulation in Serra dels Castellans (Agost). The current total occupation area is 351,52 ha, and the total population size is estimated in 385.671 adult individuals, according to UICN criteria. A 50-year period diagnostic indicates that three of the subpopulations are severely fragmented. The subpopulation of Bec de l’Àguila, the greatest, extensive and continuous one, showed an imminent decline risk. According to the available data, and in spite of the high local population size, we support labelling V. lucentina as “endangered” (EN), as well as its inclusion in the Valencian red list as “species threatened with extinction”.Este trabajo ha sido posible gracias al proyecto “Asistencia técnica para el conocimiento corológico y demográfico del endemismo valenciano Vella lucentina”, subvencionado por la DG de Gestión del Medio Natural de la Conselleria de Medio Ambiente, Agua, Urbanismo y Vivienda de la Generalitat Valenciana

General principles for assignments of communities from eDNA: Open versus closed taxonomic databases

Metabarcoding of environmental DNA (eDNA) is a powerful tool for describing biodiversity, such as finding keystone species or detecting invasive species in environmental samples. Continuous improvements in the method and the advances in sequencing platforms over the last decade have meant this approach is now widely used in biodiversity sciences and biomonitoring. For its general use, the method hinges on a correct identification of taxa. However, past studies have shown how this crucially depends on important decisions during sampling, sample processing, and subsequent handling of sequencing data. With no clear consensus as to the best practice, particularly the latter has led to varied bioinformatic approaches and recommendations for data preparation and taxonomic identification. In this study, using a large freshwater fish eDNA sequence dataset, we compared the frequently used zero-radius Operational Taxonomic Unit (zOTU) approach of our raw reads and assigned it taxonomically (i) in combination with publicly available reference sequences (open databases) or (ii) with an OSU (Operational Sequence Units) database approach, using a curated database of reference sequences generated from specimen barcoding (closed database). We show both approaches gave comparable results for common species. However, the commonalities between the approaches decreased with read abundance and were thus less reliable and not comparable for rare species. The success of the zOTU approach depended on the suitability, rather than the size, of a reference database. Contrastingly, the OSU approach used reliable DNA sequences and thus often enabled species-level identifications, yet this resolution decreased with the recent phylogenetic age of the species. We show the need to include target group coverage, outgroups and full taxonomic annotation in reference databases to avoid misleading annotations that can occur when using short amplicon sizes as commonly used in eDNA metabarcoding studies. Finally, we make general suggestions to improve the construction and use of reference databases for metabarcoding studies in the future.ISSN:2637-494

De las paredes del instituto al techo del mundo

La finalidad de este proyecto es trabajar el área de la Educación Física en la naturaleza, mediante la práctica de la escalada. Los objetivos más importantes son respetar la naturaleza; adoptar hábitos de vida saludables; realizar actividades en el medio natural; aprender a enfrentarse a situaciones de riesgo; y conocer los materiales necesarios para practicar la escalada. Se distinguen dos fases en el desarrollo del proyecto. En la primera los alumnos realizan actividades para adquirir destrezas útiles para los desplazamientos verticales. Estos conocimientos son después aplicados en una segunda fase, donde los alumnos practican la escalada deportiva. La evaluación del proyecto es continua y culmina con la realización de una prueba práctica y un cuestionario.Madrid (Comunidad Autónoma). Consejería de Educación. Dirección General de Ordenación AcadémicaMadridMadrid (Comunidad Autónoma). Subdirección General de Formación del Profesorado. CRIF Las Acacias; General Ricardos 179 - 28025 Madrid; Tel. + 34915250893ES

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field