Shell morphological diversification patterns and molecular systematics of the testate amoebae orders Arcellinida and Euglyphida

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología. Fecha de Lectura: 09-03-2023Para inferir los patrones generales que rigen la biodiversidad es necesario tener una buena representación de los taxones que la componen, y esto incluye también a los organismos más pequeños. Si bien se puede argumentar que el conocimiento de ciertos grupos de plantas y animales puede ser insuficiente, existe un claro vacío de conocimiento en los protistas, especialmente en el suelo y agua dulce. Para resolver esta “laguna” de conocimiento, esta tesis propone centrarse en un grupo particular de protistas que viven principalmente en ecosistemas continentales, las amebas tecadas. Pero para ello, es necesario resolver algunas faltas de conocimiento y desarrollar protocolos específicos para el estudio rápido y eficiente de la biodiversidad en estos taxones. La ausencia de tales protocolos limita enormemente su estudio, así como sus potenciales aplicaciones. Las amebas tecadas son un grupo parafilético de protistas ameboides que tienen en común un “caparazón” o teca autoconstruida. Estos organismos constituyen órdenes dentro de "supergrupos" eucariotas muy poco relacionados; Arcellinida en Amebozoa, Euglyphida y Thecofilosea en Rhizaria y Amphitremida en Stramenopiles (=Heterokonta). Dentro de cada grupo, estos organismos difieren en la composición y forma de las tecas, que constituyen la base de su taxonomía y sistemática. Intuitivamente, los investigadores han clasificado a los organismos asumiendo que morfologías de la teca similares deberían compartir un ancestro común. Esta suposición se basa en la hipótesis de que las tecas están sometidas a una selección neutral, y descarta la posibilidad de convergencias evolutivas entre especies o clados. Sin embargo, el “barcoding” molecular ha desafiado la sistemática y la taxonomía clásicas basadas en la morfología, mostrando patrones de diversificación morfológica de las tecas mucho más complejos y enmarañadas de lo que se pensaba. Estos resultados subrayan la necesidad de aplicar un enfoque molecular para caracterizar los taxones y establecer las relaciones entre ellos. Sin embargo, por el momento, casi todos los datos moleculares disponibles son de un único infraorden dentro de Arcellinida, los Hyalospheniformes. En Euglyphida, sólo el infraorden Euglyphina ha sido (relativamente) bien muestreado molecularmente. El primer objetivo de esta tesis es aumentar la base de datos molecular de las amebas tecadas, centrándose en Arcellinida y Euglyphida, recuperando las regiones genéticas 18S rRNA, COI y NADH. Dentro de estos genes que se han utilizado, el gen nuclear 18S rRNA fue el más conservado. También ha sido el más útil para la reconstrucción de relaciones más profundas, aunque demasiado conservado para discriminar entre especies. Por este motivo, nos centramos en el gen mitocondrial COI, de rápida evolución, que a su vez permite una buena resolución a nivel de especie. Siguiendo los principios de la taxonomía integrativa, también obtuvimos (además de las secuencias moleculares) datos sobre su localización, ecología y morfología de la teca. Esta tesis incluye los primeros datos moleculares para amebas tecadas de la Península Ibérica, tanto en ambientes de agua dulce, suelos, como de sedimentos marinos. También incluyen los primeros datos moleculares para géneros como Plagiopyxis o Trigonopyxis . Estas bases de datos servirán de antecedente para futuros estudios, y serán fundamentales para responder a dos preguntas que estructuran esta tesis: 1) "¿Cómo evoluciona la morfología de la teca en las amebas tecadas?": Entender los patrones de diversificación en las amebas tecadas es esencial para aclarar su taxonomía y sistemática, así como la aplicación de sus rasgos funcionales en los análisis ecológicos. Aquí nos centramos en la familia Cyphoderiidae (Euglyphida), Arcellidae (Arcellinida) y otros taxones de Arcellinida. Evaluamos las relaciones filogenéticas entre los taxones basándonos en datos moleculares y “mapeamos” las morfologías de las tecas y la ecología de los organismos en los árboles filogenéticos. Nuestros resultados muestran correlaciones entre ambientes y morfotipos, aportando varios casos de patrones convergentes. Esto sugiere que algunos rasgos de la teca pueden estar bajo selección positiva. 2) "¿Cómo generar datos moleculares de forma rápida y eficiente en Arcellinida?": La obtención de datos moleculares en amebas tecadas siempre ha sido un problema importante, debido a las dificultades de trabajar con estos organismos (en su mayoría) no cultivables. En consecuencia, la obtención de datos moleculares sobre las amebas tecadas es costosa en términos de tiempo y dinero, lo que explica en gran medida que sigan siendo relativamente poco estudiadas en comparación con otros grupos de protistas. Para resolver este problema, diseñamos un protocolo específico para obtener datos de ADN ambiental de Arcellinida, basado en los datos disponibles. Con este protocolo molecular específico de Arcellinida, se espera obtener cientos de secuencias ambientales mediante técnicas de “secuenciación de alto rendimiento”. Esto permitirá realizar experimentos ecológicos y biogeográficos de gran tamaño, así como estudios de bioindicación, todo lo cual requiere cantidades considerables de datos que eran imposibles de obtener en el pasado. Esta tesis aporta una nueva perspectiva integral de la historia evolutiva y la diversificación morfológica de las tecas de los órdenes Arcellinida y Euglyphida existentes; destacando la importancia de incorporar a los protistas, como las amebas tecadas, a la hora de sacar conclusiones generales que se apliquen a los eucariotas o a la biodiversidad en genera

A needle in a haystack: A new metabarcoding approach to survey diversity at the species level of Arcellinida (Amoebozoa: Tubulinea)

Environmental DNA-based diversity studies have increased in popularity with the development of high throughput sequencing technologies. This permits the potential simultaneous retrieval of vast amounts of molecular data from many different organisms and species, thus contributing to a wide range of biological disciplines. Environmental DNA protocols designed for protists often focused on the highly conserved small subunit of the ribosome gene, that does not permit species-level assignments. On the other hand, eDNA protocols aiming at species-level assignments allow a fine level ecological resolution and reproducible results. These protocols are currently applied to organisms living in marine and shallow lotic freshwater ecosystems, often in a bioindication purpose. Therefore, in this study, we present a species-level eDNA protocol designed to explore diversity of Arcellinida (Amoebozoa: Tubulinea) testate amoebae taxa that is based on mitochondrial cytochrome oxidase subunit I (COI). These organisms are widespread in lentic water bodies and soil ecosystems. We applied this protocol to 42 samples from peatlands, estuaries and soil environments, recovering all the infraorders in Glutinoconcha (with COI data), except for Hyalospheniformes. Our results revealed an unsuspected diversity in morphologically homogeneous groups such as Cylindrothecina, Excentrostoma or Sphaerothecina. With this protocol we expect to revolutionize the design of modern distributional Arcellinida surveys. Our approach involves a rapid and cost-effective analysis of testate amoeba diversity living in contrasted ecosystems. Therefore, the order Arcellinida has the potential to be established as a model group for a wide range of theoretical and applied studies

The voice of the little giants: Arcellinida testate amoebae in environmental DNA-based bioindication, from taxonomy free to haplotypic level

Bioindication, evaluating biological responses to environmental disturbances, is crucial for assessing the ecological status of an ecosystem. While historical bioindication relied on macroscopic organisms, the introduction of environmental DNA (eDNA) techniques allows the application of protists without the necessity of morphological identification. In this study, we propose a novel bioindication methodology utilizing Arcellinida, a group of top predators among protists, as bioindicators of freshwater ecosystems. For that purpose, we first characterized the Arcellinida diversity over 1 year at three different points of Lake Sanabria, an ancient glacier lake known to be subjected to anthropogenic disturbances. We compared this diversity with an undisturbed control site. Second, we characterized the Arcellinida diversity in other ecosystems to generate the ecological background to test the connectivity between them. Results indicate limited connectivity between the different ecosystems and an edge effect between terrestrial and aquatic ecosystems. Disturbed freshwater ecosystems exhibited reduced Arcellinida diversity at both specific and infraspecific levels, providing valuable insight into recent disturbances. Arcellinida-based bioindication provides a sensitive, accurate and easy-to-interpret protocol for monitoring disturbances in freshwater ecosystems. It represents a valuable tool for environmental assessments and conservation strategiesThis study was funded by the Spanish Government PGC2018-094660B-I00 and PID2021-128499NB-I00 10.13039/501100011033/, (MCIU/AEI/ FEDER,UE) and an ‘Atracción de Talento Investigador’ grant awarded by the Consejería de Educación, Juventud y Deporte, Comunidad de Madrid (Spain) (2017-T1/AMB-5210) to E.L. and also under the grant PID2022-136691NA-I00 to D.V; a National Geographic Society EC86022R-21 to R.G.-

Viral RNA load in plasma is associated with critical illness and a dysregulated host response in COVID‑19

Background. COVID-19 can course with respiratory and extrapulmonary disease. SARS-CoV-2 RNA is detected in respiratory samples but also in blood, stool and urine. Severe COVID-19 is characterized by a dysregulated host response to this virus. We studied whether viral RNAemia or viral RNA load in plasma is associated with severe COVID-19 and also to this dysregulated response. Methods. A total of 250 patients with COVID-19 were recruited (50 outpatients, 100 hospitalized ward patients and 100 critically ill). Viral RNA detection and quantification in plasma was performed using droplet digital PCR, targeting the N1 and N2 regions of the SARS-CoV-2 nucleoprotein gene. The association between SARS-CoV-2 RNAemia and viral RNA load in plasma with severity was evaluated by multivariate logistic regression. Correlations between viral RNA load and biomarkers evidencing dysregulation of host response were evaluated by calculating the Spearman correlation coefficients. Results. The frequency of viral RNAemia was higher in the critically ill patients (78%) compared to ward patients (27%) and outpatients (2%) (p < 0.001). Critical patients had higher viral RNA loads in plasma than non-critically ill patients, with non-survivors showing the highest values. When outpatients and ward patients were compared, viral RNAemia did not show significant associations in the multivariate analysis. In contrast, when ward patients were compared with ICU patients, both viral RNAemia and viral RNA load in plasma were associated with critical illness (OR [CI 95%], p): RNAemia (3.92 [1.183–12.968], 0.025), viral RNA load (N1) (1.962 [1.244–3.096], 0.004); viral RNA load (N2) (2.229 [1.382–3.595], 0.001). Viral RNA load in plasma correlated with higher levels of chemokines (CXCL10, CCL2), biomarkers indicative of a systemic inflammatory response (IL-6, CRP, ferritin), activation of NK cells (IL-15), endothelial dysfunction (VCAM-1, angiopoietin-2, ICAM-1), coagulation activation (D-Dimer and INR), tissue damage (LDH, GPT), neutrophil response (neutrophils counts, myeloperoxidase, GM-CSF) and immunodepression (PD-L1, IL-10, lymphopenia and monocytopenia). Conclusions. SARS-CoV-2 RNAemia and viral RNA load in plasma are associated with critical illness in COVID-19. Viral RNA load in plasma correlates with key signatures of dysregulated host responses, suggesting a major role of uncontrolled viral replication in the pathogenesis of this disease.This work was supported by awards from the Canadian Institutes of Health Research, the Canadian 2019 Novel Coronavirus (COVID-19) Rapid Research Funding initiative (CIHR OV2 – 170357), Research Nova Scotia (DJK), Atlantic Genome/Genome Canada (DJK), Li-Ka Shing Foundation (DJK), Dalhousie Medical Research Foundation (DJK), the “Subvenciones de concesión directa para proyectos y programas de investigación del virus SARS‐CoV2, causante del COVID‐19”, FONDO–COVID19, Instituto de Salud Carlos III (COV20/00110, CIBERES, 06/06/0028), (AT) and fnally by the “Convocatoria extraordinaria y urgente de la Gerencia Regional de Salud de Castilla y León, para la fnanciación de proyectos de investigación en enfermedad COVID-19” (GRS COVID 53/A/20) (CA). DJK is a recipient of the Canada Research Chair in Translational Vaccinology and Infammation. APT was funded by the Sara Borrell Research Grant CD018/0123 funded by Instituto de Salud Carlos III and co-fnanced by the European Development Regional Fund (A Way to Achieve Europe programme). The funding sources did not play any role neither in the design of the study and collection, not in the analysis, in the interpretation of data or in writing the manuscript

Arcellinida testate amoebae in environmental DNA-based bioindication, from taxonomy free to haplotypic level

&lt;p&gt;&lt;span&gt;Bioindication, evaluating biological responses to environmental disturbances, is crucial for assessing ecological status. While historical bioindication relied on macroscopic organisms, the introduction of environmental DNA (eDNA) techniques allows the application of protist without the necessity of morphological identification. In this study we propose a novel bioindication methodology utilizing Arcellinida, a group of top predators among protists, as bioindicators of freshwater ecosystems. In that purpose, we firstly characterized the Arcellinida diversity over one year at three different points of the Lake Sanabria, an ancient glacier lake known to be subjected to anthropogenic disturbances. We compared this diversity with an undisturbed control site. Secondly, we characterized the Arcellinida diversity in other ecosystems to generate the ecological background to test the connectivity between them. Results indicate limited connectivity between the different ecosystems, and an edge effect between terrestrial and aquatic ecosystems. Disturbed freshwater ecosystems exhibited reduced Arcellinida diversity at both specific and infraspecific levels, providing valuable insights into recent disturbances. Arcellinida-based bioindication provides a sensitive, accurate and easy to interpret protocol for monitoring disturbances in freshwater ecosystems. It represents a valuable tool for environmental assessments and conservation strategies.&lt;/span&gt;&lt;/p&gt

When ecological transitions are not so infrequent: independent colonizations of athalassohaline water bodies by Arcellidae (Arcellinida; Amoebozoa), with descriptions of four new species

The salinity and humidity barriers divide biodiversity and strongly influence the distribution of organisms. Crossing them opens the possibility for organisms to colonize new niches and diversify, but requires profound physiological adaptations and is supposed to happen rarely in evolutionary history. We tested the relative importance of each ecological barrier by building the phylogeny, based on mitochondrial cytochrome oxidase gene (COI) sequences, of a group of microorganisms common in freshwater and soils, the Arcellidae (Arcellinida; Amoebozoa). We explored the biodiversity of this family in the sediments of athalassohaline water bodies (i.e. of fluctuating salinity that have non-marine origins). We found three new aquatic species, which represent, to the best of our knowledge, the first reports of Arcellinida in these salt-impacted ecosystems, plus a fourth terrestrial one in bryophytes. Culturing experiments performed on Arcella euryhalina sp. nov. showed similar growth curves in pure freshwater and under 20 g/L salinity, and long-term survival at 50 g/L, displaying a halotolerant biology. Phylogenetic analyses showed that all three new athalassohaline species represent independent transition events through the salinity barrier by freshwater ancestor, in contrast to the terrestrial species, which are monophyletic and represent a unique ecological transition from freshwater to soil environments

Arcellinida testate amoebae as climate miner's canaries in Southern Spain

Southern Spain is currently under threat of desertification as a consequence of global climate change, which pressures on fragile ecosystems such as caves. The organisms living in these extremely stable environments are particularly sensitive and prone to extinction, therefore they can be used as bioindicators for climate change. Cyanobacterial mats form peculiar and vulnerable micro-ecosystems at the entrance of caves and house a diversity of protists. Amongst them, Arcellinida testate amoebae have been traditionally used as bioindicators for environmental quality, notably because their narrow ecological tolerance and their key ecological position as top predators of the microbial foodwebs. We report here two new species of Arcellinida found in the cyanobacterial mats of cave Hundidero, in Sierra de Grazalema, Malaga province, whose traits suggest a narrow tolerance for changes in humidity. We provide a formal description for Difflugia alhadiqa sp. nov. and Heleopera baetica sp. nov. based on morphometrics and 18S rRNA gene data, and propose using the presence of these species to indicate the good health of the cyanobacterial mats, like miner's canaries for local climate

Netzelia lobostoma

NETZELIA LOBOSTOMA(LEIDY, 1874)COMB. NOV. Difflugia proteiformis Lamarck, 1816: 95. Cucurbitella tricuspis – Carter, 1856: 221; Medioli et al., 1987. Difflugia tricuspis Carter, 1856: 221. Difflugia lobostoma Leidy, 1874: 79. Z o o b a n k r e g i s t r a t i o n: u r n: l s i d: z o o b a n k. org:act: 0AD69577-89AD-42E8-9C40-03A68C73EFD4.Published as part of González-Miguéns, Rubén, Soler-Zamora, Carmen, Villar-Depablo, Mar, Todorov, Milcho & Lara, Enrique, 2022, Multiple convergences in the evolutionary history of the testate amoeba family Arcellidae (Amoebozoa: Arcellinida: Sphaerothecina): when the ecology rules the morphology, pp. 1044-1071 in Zoological Journal of the Linnean Society 194 (4) on page 1061, DOI: 10.1093/zoolinnean/zlab074, http://zenodo.org/record/646126