Esta sección incluye una lista de los últimos artículos científicos del IPNA publicados en revistas incluidas en el Science Citation Index (SCI).
En DIGITAL.CSIC, repositorio institucional del CSIC, pueden encontrar el listado completo de artículos científicos desde 1962, así como otras colecciones de interés como congresos, tesis, libros, material divulgativo, etc. del centro. El objetivo de DIGITAL.CSIC es organizar, preservar y difundir en acceso abierto los resultados de nuestra investigación.
En el repositorio institucional del CSIC, pueden encontrar el listado completo de artículos científicos, así como otras colecciones de interés como congresos, tesis, libros, material divulgativo, etc.
A validated workflow for rapid taxonomic assignment and monitoring of a national fauna of bees (Apiformes) using high throughput DNA barcoding
Improved taxonomic methods are needed to quantify declining populations of insect pollinators. This study devises a high-throughput DNA barcoding protocol for a regional fauna (United Kingdom) of bees (Apiformes), consisting of reference library construction, a proof-of-concept monitoring scheme, and the deep barcoding of individuals to assess potential artefacts and organismal associations. A reference database of cytochrome oxidase c subunit 1 (cox1) sequences including 92.4% of 278 bee species known from the UK showed high congruence with morphological taxon concepts, but molecular species delimitations resulted in numerous split and (fewer) lumped entities within the Linnaean species. Double tagging permitted deep Illumina sequencing of 762 separate individuals of bees from a UK-wide survey. Extracting the target barcode from the amplicon mix required a new protocol employing read abundance and phylogenetic position, which revealed 180 molecular entities of Apiformes identifiable to species. An additional 72 entities were ascribed to nuclear pseudogenes based on patterns of read abundance and phylogenetic relatedness to the reference set. Clustering of reads revealed a range of secondary operational taxonomic units (OTUs) in almost all samples, resulting from traces of insect species caught in the same traps, organisms associated with the insects including a known mite parasite of bees, and the common detection of human DNA, besides evidence for low-level cross-contamination in pan traps and laboratory procedures. Custom scripts were generated to conduct critical steps of the bioinformatics protocol. The resources built here will greatly aid DNA-based monitoring to inform management and conservation policies for the protection of pollinators.
Creedy, Thomas J.; Norman, Hannah; Tang, Cuong Q.; Qing Chin, Kai; Andujar, Carmelo; Arribas, Paula; O'Connor, Rory S.; Carvell, Claire; Notton, David G.; Vogler, Alfred P.
Niosomes encapsulated in biohydrogels for tunable delivery of phytoalexin resveratrol
A series of biohydrogels based on mixtures of kappa-carrageenan (κ-carrageenan, κ-C) and gelatin were evaluated as potential soft delivery vehicles for the encapsulation and subsequent release of non-ionic surfactant vesicles (niosomes) loaded with resveratrol (RSV). The niosomes were prepared using a mixture of amphiphilic lipids Tween 80 and Span 80 in water. The results showed that RSV-niosomes did not significantly affect the hydrogelation properties of the biopolymer mixture. Moreover, in vitro drug release experiments from biohydrogels containing RSV-niosomes were successfully carried out under simulated gastrointestinal conditions. The RSV-niosomal liberation profiles from hydrogels were fitted using first order kinetics, Higuchi, Korsmeyer-Peppas and Weibull drug release models, showing the prevalence of diffusion mechanisms in each case. In addition, the RSV release was easily tuned by adjusting the total concentration of κ-C : gelatin. Interestingly, the niosomal-hydrogel system was also found to prevent the trans-to-cis photoisomerization of RSV.
Machado, Noelia D.; Fernández, Mariana A.; Häring, Marleen; Saldías, César; Díaz Díaz, David
First report of postmortem pH evolution and rigor mortis in guinea pigs
In the Andean region, Philippines, and central Africa most guinea pigs are raised for meat production. Guinea pig meat is of increasing interest to researchers. One of the critical questions in meat production is to know when muscles convert to meat. The objective of this study was to describe the pH evolution in guinea pig muscles and to determine the establishment and resolution time of rigor mortis. Forty-eight guinea pig carcasses were divided into four groups: 12 male and 12 female of 3 month-old fattened animals, and 12 male and 12 female of 12 month-old reproductive animals. The pH was measured using a penetration probe in Longissimus thoracis, Quadriceps femoris, Triceps brachii and Psoas major muscles for 24 h, and rigor mortis was monitored during this time. The initial and final pH ranged between 6.56–7.00 and 5.97–6.27, respectively. Rigor mortis was established at ∼5 h and resolved at ∼15 h postmortem. The Psoas major was the muscle with lower initial pH and higher final pH, while no differences were found between the other muscles. Initial, evolution and final pH were not affected by the sex. No differences were found in the productive-reproductive status between males, whereas the older female guinea pigs presented higher pH in three muscles. In conclusion, in our test conditions final pH in guinea pigs remains high enough to permit microbial growth, which could affect the shelf-life of Guinea pig meat. Moreover, it is recommended to keep guinea pig carcasses in refrigeration until 15 h postmortem, when rigor mortis is resolved, and muscle-to-meat conversion finished. Management of these carcasses should be different depending if the animals belong to a productive or reproductive animal system. It is therefore necessary to chill guinea pig carcasses at least 15 h, to achieve a correct muscle-to-meat conversion.
Sánchez-Macías, David; Cevallos-Velastegui, Lesllie; Nuñez-Valle, Diego; Morales-delaNuez, Antonio
Do mosses really exhibit so large distribution ranges? Insights from the integrative taxonomic study of the Lewinskya affinis complex (Orthotrichaceae, Bryopsida)
The strikingly lower number of bryophyte species, and in particular of endemic species, and their larger distribution ranges in comparison with angiosperms, have traditionally been interpreted in terms of their low diversification rates associated with a high long-distance dispersal capacity. This hypothesis is tested here with Lewinskya affinis (≡ Orthotrichum affine), a moss species widely spread across Europe, North and East Africa, southwestern Asia, and western North America. We tested competing taxonomic hypotheses derived from separate and combined analyses of multilocus sequence data, morphological characters, and geographical distributions. The best hypothesis, selected by a Bayes factor molecular delimitation analysis, established that L. affinis is a complex of no less than seven distinct species, including L. affinis s.str., L. fastigiata and L. leptocarpa, which were previously reduced into synonymy with L. affinis, and four new species. Discriminant analyses indicated that each of the seven species within L. affinis s.l. can be morphologically identified with a minimal error rate. None of these species exhibit a trans-oceanic range, suggesting that the broad distributions typically exhibited by moss species largely result from a taxonomic artefact. The presence of three sibling western North American species on the one hand, and four Old World sibling species on the other, suggests that there is a tendency for within-continent diversification rather than recurrent dispersal following speciation. The faster rate of diversification as compared to intercontinental migration reported here is in sharp contrast with earlier views of bryophyte species with wide ranges and low speciation rates.
Vigalondo, B.; Garilleti, R.; Vanderpoorten, A.; Patiño, Jairo; Draper, I.; Calleja, J.A.; Mazimpaka, V.; Lara, F.
A topoclimate model for Quaternary insular speciation
[Aim]: Understanding the drivers of speciation within islands is key to explain the high levels of invertebrate diversification and endemism often observed within islands. Here, we propose an insular topoclimate model for Quaternary diversification (ITQD), and test the general prediction that, within a radially eroded conical island, glacial climate conditions facilitate the divergence of populations within species across valleys. [Location]: Gran Canaria, Canary Islands. [Taxon]: The Laparocerus tessellatus beetle species complex (Coleoptera, Curculionidae). [Methods]: We characterize individual-level genomic relationships using single nucleotide polymorphisms produced by double-digest restriction site associated DNA sequencing (ddRAD-seq). A range of parameter values were explored in order to filter our data. We assess individual relatedness, species boundaries, demographic history and spatial patterns of connectivity. [Results]: The total number of ddRAD-seq loci per sample ranges from 4,576 to 512, with 11.12% and 4.84% of missing data respectively, depending on the filtering parameter combination. We consistently infer four genetically distinct ancestral populations and two presumed cases of admixture, one of which is largely restricted to high altitudes. Bayes factor delimitation support the hypothesis of four species, which is consistent with the four inferred ancestral gene pools. Landscape resistance analyses identified genomic relatedness among individuals in two out of the four inferred species to be best explained by annual precipitation during the last glacial maximum rather than geographic distance. [Main conclusions]: Our data reveal a complex speciation history involving population isolation and admixture, with broad support for the ITQD model here proposed. We suggest that further studies are needed to test the generality of our model, and enrich our understanding of the evolutionary process in island invertebrates. Our results demonstrate the power of ddRAD-seq data to provide a detailed understanding of the temporal and spatial dynamics of insular biodiversity.
García-Olivares, Victor; Patiño, Jairo; Overcast, Isaac; Salces-Castellano, Antonia; López de Heredia, Unai; Mora-Márquez, Fernando; Machado, Antonio; Hickerson, Michael J.; Emerson, Brent C.
The phylogeny of leaf beetles (Chrysomelidae) inferred from mitochondrial genomes
The high-level classification of Chrysomelidae (leaf beetles) currently recognizes 12 or 13 well-established subfamilies, but the phylogenetic relationships among them remain ambiguous. Full mitochondrial genomes were newly generated for 27 taxa and combined with existing GenBank data to provide a dataset of 108 mitochondrial genomes covering all subfamilies. Phylogenetic analysis under maximum likelihood and Bayesian inference recovered the monophyly of all subfamilies, except that Timarcha was split from Chrysomelinae in some analyses. Three previously recognized major clades of Chrysomelidae were broadly supported: the ‘chrysomeline’ clade consisting of (Chrysomelinae (Galerucinae + Alticinae)); the ‘sagrine’ clade with internal relationships of ((Bruchinae + Sagrinae) + (Criocerinae + Donaciinae)), and the ‘eumolpine’ clade comprising (Spilopyrinae (Cassidinae (Eumolpinae (Cryptocephalinae + Lamprosomatinae)))). Relationships among these clades differed between data treatments and phylogenetic algorithms, and were complicated by two additional deep lineages, Timarcha and Synetinae. Various topological tests favoured the PhyloBayes software as the preferred inference method, resulting in the arrangement of (chrysomelines (eumolpines + sagrines)), with Timarcha placed as sister to the chrysomeline clade and Synetinae as a deep lineage splitting near the base. Whereas mitogenomes provide a solid framework for the phylogeny of Chrysomelidae, the basal relationships do not agree with the topology of existing molecular studies and remain one of the most difficult problems of Chrysomelidae phylogenetics.
Nie, Rui-e; Andújar, Carmelo ; Gómez-Rodríguez, Carola ; Bai, Ming; Xue, Huai-Jun; Tang, Min; Yang, Chen-Tao.; Tang, Pu; Yang, Xing-Ke; Vogler, Alfried P.
Antiprotozoal activities of marine polyether triterpenoids
Chagas disease and leishmaniasis are tropical neglected diseases caused by kinetoplastids protozoan parasites of Trypanosoma and Leishmania genera, and a public health burden with high morbidity and mortality rates in developing countries. Among difficulties with their epidemiological control, a major problem is their limited and toxic treatments to attend the affected populations; therefore, new therapies are needed in order to find new active molecules. In this work, sixteen Laurencia oxasqualenoid metabolites, natural compounds 1–11 and semisynthetic derivatives 12–16, were tested against Leishmania amazonensis, Leishmania donovani and Trypanosoma cruzi. The results obtained point out that eight substances possess potent activities, with IC values in the range of 5.40–46.45 µM. The antikinetoplastid action mode of the main metabolite dehydrothyrsiferol (1) was developed, also supported by AFM images. The semi-synthetic active compound 28-iodosaiyacenol B (15) showed an IC 5.40 µM against Leishmania amazonensis, turned to be non-toxic against the murine macrophage cell line J774A.1 (CC > 100). These values are comparable with the reference compound miltefosine IC 6.48 ± 0.24 and CC 72.19 ± 3.06 μM, suggesting that this substance could be scaffold for development of new antikinetoplastid drugs.
Díaz-Marrero, Ana R. ; López-Arencibia, Atteneri; Bethencout-Estrella, Carlos J.; Cen-Pacheco, Francisco; Sifaoui, Ines; Hernández Creus, Alberto; Duque-Ramírez, María Clara; Souto, María L.; Hernández Daranas, Antonio ; Lorenzo-Morales, Jacob; Piñero, José E.; Fernández, José J.
Environmental temperatures shape thermal physiology as well as diversification and genome-wide substitution rates in lizards
Climatic conditions changing over time and space shape the evolution of organisms at multiple levels, including temperate lizards in the family Lacertidae. Here we reconstruct a dated phylogenetic tree of 262 lacertid species based on a supermatrix relying on novel phylogenomic datasets and fossil calibrations. Diversification of lacertids was accompanied by an increasing disparity among occupied bioclimatic niches, especially in the last 10 Ma, during a period of progressive global cooling. Temperate species also underwent a genome-wide slowdown in molecular substitution rates compared to tropical and desert-adapted lacertids. Evaporative water loss and preferred temperature are correlated with bioclimatic parameters, indicating physiological adaptations to climate. Tropical, but also some populations of cool-adapted species experience maximum temperatures close to their preferred temperatures. We hypothesize these species-specific physiological preferences may constitute a handicap to prevail under rapid global warming, and contribute to explaining local lizard extinctions in cool and humid climates.
Garcia-Porta, Joan; Irisarri, Iker; Kirchner, Martin; Rodríguez, Ariel; Kirchhof, Sebastian; et al.
Geochemical signals related to the 2011–2012 El Hierro submarine eruption
On 10 October 2011, a submarine volcanic eruption began 2 km south of the island of El Hierro (Canary Islands, Spain). It was the first eruption after 40 years of quiescence in the Canarian archipelago. Since mid-July 2011, a multiparametric network has been deployed by the Instituto Geográfico Nacional (hereinafter IGN)to monitor volcanic processes. The data recorded by five stations, measuring the air and soil temperature, CO and radon concentration in the air inside four galleries and one well, are the focus of the present paper. Two important anomalies were found that improve comprehension of the volcanic process. First, a clear increase in CO concentration in one gallery (from 825 ± 19 to 2305 ± 35 ppm)which seems to be mainly controlled by ground deformation measured during the unrest period preceding the eruption. Second, a relationship between radon concentration in air in a well located in the northern side of the island with seismic energy release (several peaks with up to 115% increase), and ground deformation (50% growth in background level), also before the eruption onset. Lastly, changes in radon concentration during eruptive period seem to be modulated by seismic activity (increases up to 233%), which in turn is likely related to variations in magma rheology.
Torres-González, Pedro; Moure-García, David; Luengo-Oroz, Natividad; Villasante-Marcos, Victor; Iribarren, Ilaskiñe; Blanco, M. José; Soler, Vicente ; Jiménez-Abizanda, García-Fraga, José
Chance and predictability in evolution: The genomic basis of convergent dietary specializations in an adaptive radiation
The coexistence of multiple eco-phenotypes in independently assembled communities makes island adaptive radiations the ideal framework to test convergence and parallelism in evolution. In the radiation of the spider genus Dysdera in the Canary Islands, species diversification occurs concomitant with repeated events of trophic specialization. These dietary shifts, to feed primarily on woodlice, are accompanied by modifications in morphology (mostly in the mouthparts), behaviour and nutritional physiology. To gain insight into the molecular basis of this adaptive radiation, we performed a comprehensive comparative transcriptome analysis of five Canary Island Dysdera endemics representing two evolutionary and geographically independent events of dietary specialization. After controlling for the potential confounding effects of hemiplasy, our differential gene expression and selective constraint analyses identified a number of genetic changes that could be associated with the repeated adaptations to specialized diet of woodlice, including some related to heavy metal detoxification and homeostasis, the metabolism of some important nutrients and venom toxins. Our results shed light on the genomic basis of an extraordinary case of dietary shift convergence associated with species diversification. We uncovered putative molecular substrates of convergent evolutionary changes at different hierarchical levels, including specific genes, genes with equivalent functions and even particular amino acid positions. This study improves our knowledge of rapid adaptive radiations and provides new insights into the predictability of evolution.
Vizueta, Joel; Macías-Hernández, Nuria; Arnedo, Miquel A.; Rozas, Julio; Sánchez-Gracia, Alejandro
Sheep and goats raised in mixed flocks have diverse immune status around parturition
Several physiological and metabolic changes take place in dairy ruminants around parturition (late pregnancy, parturition, and early lactation). Dairy species are genetically selected for their higher milk production compared with non-dairy species. This fact causes a constant stress that impairs the immune status of the animal, with consequences for its welfare and performance. In the present study, we assessed the immune status of high-yield dairy sheep and goats by quantifying IgG and IgM concentrations, as well as chitotriosidase (ChT) and complement system [total complement system (TC) and alternative complement pathway (AC)] activity in blood plasma around parturition. We also measured IgG and IgM concentrations and ChT activity in colostrum and milk during the first 40 d postpartum. The lowest blood IgG concentration was at parturition in both species. We detected no differences in blood IgG concentrations between species. Blood IgM concentrations were constant in both species throughout the study period. However, blood IgM concentrations were greater in sheep than in goats. Blood ChT activity was greater in goats than in sheep, and both species showed constant activity of this enzyme throughout the study period. We observed no differences in complement system (TC and AC) activity between sheep and goats. In addition, both TC and AC activity were constant in both species throughout the experiment. In general, IgG and IgM concentrations were greater in sheep colostrum than in goat colostrum, but these differences disappeared after d 4 (IgG) and d 3 (IgM) postpartum. In both species, the highest IgG and IgM concentrations were measured in colostrum, gradually decreasing during the first days postpartum. Chitotriosidase activity decreased in both species from colostrum to milk, although goats always showed greater ChT activity than sheep. Both sheep and goats seemed to be more susceptible to infectious diseases around parturition. As well, goats showed greater ChT activity in blood, colostrum, and milk than sheep. This fact may give these animals additional protection against parasite and fungal infections.
Hernández-Castellano, Lorenzo E.; Moreno-Indias, Isabel; Sánchez-Macías, Davinia; Morales-delaNuez, Antonio; Torres, Alexandr; Argüello, Anastasio; Castro, Noemí
Antimicrobial activity of poly(3,4-ethylenedioxythiophene) n-doped with a pyridinium-containing polyelectrolyte
In spite of p-doped conducting polymers having been widely studied in the last decades and many applications having been developed, studies based on n-doped conducting polymers are extremely scarce. This fact is even more evident when it comes to conducting polymers n-doped with polycations, even though polyanions, such as poly(styrenesulfonate), are often used to obtain p-doped conducting polymers. In this work poly(pyridinium-1,4-diyliminocarbonyl-1,4-phenylene-methylene chloride), abbreviated as P(Py-1,4-P), has been used to prepare n-doped poly(3,4-ethylenedioxythiophene) (PEDOT) electrodes by applying a reduction potential to a de-doped PEDOT film in a P(Py-1,4-P) water solution. The utilization of this cationic polyelectrolyte as an n-dopant agent results in drastic superficial changes, as is observed by comparing the morphology, topography and wettability of p-doped, de-doped and n-doped PEDOT. Cytotoxicity, cell adhesion and cell proliferation assays, which have been conducted using epithelial and fibroblast cell lines, show that the amount of P(Py-1,4-P) in the re-doped PEDOT films is below that required to observe a cytotoxic harmful response and that n-doped PEDOT:P(Py-1,4-P) films are biocompatible. The non-specific bacteriostatic properties of n-doped PEDOT:P(Py-1,4-P) films have been demonstrated against E. coli and S. aureus bacteria (Gram-negative and Gram-positive, respectively) using bacterial growth curves and adhesion assays. Although the bacteriostatic effect is in part due to the conducting polymer, as is proved by results for p-doped and de-doped PEDOT, the incorporation of P(Py-1,4-P) through the re-doping process greatly enhances this antimicrobial behaviour. Thus, only a small concentration of this cationic polyelectrolyte (∼0.1 mM) is needed to inhibit bacterial growth.
Sánchez-Jiménez, Margarita; Estrany, Francesc; Borràs, Nuria; Maiti, Binoy; Díaz Díaz, David ; Del Valle, Luis J.; Alemán, Carlos
5-(1 H-1,2,3-Triazol-5-yl)isophthalic Acid: A Versatile Ligand for the Synthesis of New Supramolecular Metallogels
The gelation ability of 5-(1H-1,2,3-triazol-5-yl)isophthalic acid (click-TIA) in the presence of different metal acetates has been studied in different solvents and ligand/metal ratios. This manuscript is focused on the metallogel obtained from the combination of click-TIA and copper(II) acetate, which has been used as a model system in terms of characterization and gelation studies. Sonication treatment of the initial mixture of compounds and the nature of the counter anion were found to be critical factors for the supramolecular assembly of the metal/click-TIA complexes and, hence, for the formation of stable and homogeneous metallogels. The gel materials have been characterized with a variety of techniques including infrared, rheology, UV-vis spectroscopy, powder X-ray diffraction, and scanning electron microscopy.
Häring, Marleen; Nandi, Sujay Kumar; Rodríguez-López, Julio; Haldar, Debasish; Martín, Victor S.; Lozano-Gorrín, Antonio Diego D.; Saldías, César; Díaz Díaz, David
To what extent are bryophytes efficient dispersers?
Bryophytes are typically seen as extremely efficient dispersers. Experimental evidence suggests that efficient short-distance dispersal coupled with random long-distance dispersal (LDD) leads to an inverse isolation effect. Under the latter, a higher genetic diversity of colonizing propagules is expected with increasing isolation, counteracting differentiation beyond the range of short-distance dispersal. This expectation is tested from a review of evidence on spatial genetic structure and analyses of isolation-by-distance (IBD) at different scales. A decay of the IBD signal, characterized by non-significant slopes between kinship coefficients and geographic distance was observed beyond 100 m. A second slope shift was observed at distances larger than 1 km, with a proportion of significant slopes in more than one third of the datasets. The decay of the IBD signal beyond 100 m, which reflects efficient LDD, is consistent with the inverse isolation hypothesis. Persistence of a significant IBD signal at medium ranges in one third of the analysed cases suggests, however, that the inverse isolation effect is not a rule in bryophyte spore dispersal. Furthermore, the higher proportion of significant IBD patterns observed at scales over 100 km likely marks the limits of regional dispersal, beyond which an increasingly smaller proportion of spores travel. Synthesis. We discuss the differences between experimental and genetic estimates of spore dispersal and conclude that geographic distance remains a significant proxy of spore colonization rates, with major consequences for our understanding of actual migration capacities in bryophytes, and hence, our capacity to model range shifts in a changing world.
Vanderpoorten, Alain; Patiño, Jairo; Désamoré, Aurélie; Laenen, Benjamin; Górski, Piotr; Papp, Beata; Holá, Eva; Korpelainen, Helena; Hardy, Olivier
The discovery of barretonus (Curculionidae: Cossoninae) in the canary islands: Barcoding, morphology and description of new species
The genus Barretonus Roudier, 1958 is a genus endemic to Macaronesia, described from the archipelago of Madeira where four species are known to date. In this study, two new species of Barretonus, B. auarita García & Oromí sp. nov. (from La Palma, Canary Islands) and B. daute García & Andújar sp. nov. (from Tenerife, Canary Islands), are described based on morphological and molecular evidence (mtCOI), and additional notes on their biology and ecology are provided. Morphology of the new species is compared with the other representatives of the genus, and their phylogenetic relationships with a Barretonus representative from Madeira and species from additional genera within the Cossoninae are shown. This work increases the number of species of the genus Barretonus from four to six anophthalmous or microphthalmous species with an endogean lifestyle, and incorporates the Canary Islands into its Macaronesian range of distribution.
García, Rafael; Andújar, Carmelo; Oromí, Pedro; Emerson, Brent C.; López, Heriberto
Use of a bis-1,2,3-triazole gelator for the preparation of supramolecular metallogels and stabilization of gold nanoparticles
In this work we have applied a double isosteric substitution of the amide groups presented in the gelator N,N′-((1S,2S)-cyclohexane-1,2-diyl)didodecanamide (C-Cyc) by 1,2,3-triazole rings affording the corresponding isosteric gelator (click-C-Cyc). This approach enabled the preparation of different metallogels and fine-tuning their properties. In particular, C-Cyc was found to form stable and reversible metallogels with Pd and Au in DMSO and DMF, whereas click-C-Cyc formed metallogels in these solvents only in the presence of Au. Comparing the gels made of C-Cycvs.click-C-Cyc in the presence of HAuCl·3HO, the former showed higher T values and 5-8 times slower gelation kinetics. Furthermore, the incorporation of EtN as reducing agent in the formulations facilitated the in situ formation of non-aggregated gold nanoparticles embedded in the viscoelastic networks made of either pure isosteres or with an equimolar mixture of click-C-Cyc and C-Cyc. So-formed NPs showed similar size distribution regardless the gold concentration and the nature of the gelator system.
Tautz, Markus; Saldías, César; Lozano-Gorrín, Antonio Diego; Díaz Díaz, David