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Digital Annex for the following thesis: Vila-Viçosa, C. (2023). Natural History, Biogeography and Evolution of the Iberian white oak syngameon (Quercus L. Sect. Quercus). Ph.D. Thesis, Faculdade de Ciências da Universidade do Porto, Portugal Abstract: The genus Quercus L. is one of the most diverse and important group of woody plants, particularly when considering that they are the trees that rule the Northern Hemisphere forests. Oaks have an intricate Biogeography that criss-crosses diverse climatic and edaphic gradients, encompassing a huge ambiguity in terms of species delimitation. Frequently, the taxonomic proposals brought by traditional Linnaean Botany are either insufficient or rather inflate the number of species and nomenclatural assignments, which are further diluted into inconsistent taxonomic ranks, varying from species to subspecies and varieties. The supremacy given to morphological characters that are inherently fragile and plastic, spread across the distribution areas of distinct lineages, may carry ambiguity on the identification and proper species delimitation. From the oaks that are distributed across the Western Palearctic region, the ones that are deciduous or brevi-deciduous present higher levels of ambiguity in terms of species number and their delimitation. This ambiguity is particularly strong in the circummediterranean region and in the transitional areas between the two major biogeographic Regions of the western Palearctic region, the Euro-Siberian and Mediterranean. This degree of uncertainty, which increases towards the Southern European Peninsulas, is amplified by the ease that the different species of oaks tend to hybridize among them. The present work provides a holistic framework that covers multiple areas, from the taxonomic and evolutive study of this genus, to biogeography and molecular characterization. Its major objective was to resolve the species delimitation of the Iberian deciduous and marcescent oaks and putative introgression among them, enhancing the available knowledge about species diversity, which can foster suitable species and forest conservation. A specific objective was to cross-reference the natural history revision and the different taxonomic treatments brought by distinct authors, with personal observations. These data were then incorporated into ecological modelling and molecular characterization, which in the end fed a newly updated taxonomic proposal. In Section A we obtained results from extensive field, herbaria, and literature review, updating the nomenclature of the Portuguese and western Mediterranean oaks. Section B was supported by Section A’s in-depth review and enabled finer species distribution models, nurturing both hindcast (since ca. 20 Kyr) and forecast (2070-2100) exercises of the range dynamics of Mediterranean oaks species. The study of past and future range shifts solved important pending biogeographic questions, especially related to past range-shifts. Such past-range shifts improved our knowledge on species responses to climate dynamics and allowed a better anticipation of future responses of range shifts driven by climate change. Section C encompassed the molecular characterization of Iberian white oak species and their hybrids, whose delimitation is often faltering when one intends to infer about species rank, or hypothesize about the participation of parent taxon in natural hybrid swarms. This work allowed us to solve the phylogenetic backbone of western Palearctic white oaks, suggesting a significant segregation of the Iberian pedunculate oaks and unveiling two subsections inside Section Quercus. These subsections are biogeographically well-segregated and present diverse levels of introgression among species. Results demonstrated the efficiency of RADSeq for rebuilding the reticulate phylogeny of the Eurasian white oaks, showcasing the significance of the Iberian Peninsula as a major hotspot for oak diversity. We implemented a circular approach to these methods, which retro-fed themselves in terms of insight generation, enabling a powerful strategy to solve the evolutionary history of this difficult groups of plants. We estimate that the reticulate historical biogeography of the western Palearctic white oaks deserves further scrutiny by adding vicariant oak populations from northern Africa, the Near East and southern European Peninsulas. Methods should again follow this similar additive and sequential process of adjoining deep Natural History examination, with extensive fieldwork in type populations and genome-wide molecular surveys, in order to solve this group of plants. With the present work, we were able to significantly improve on the depiction of the basic unit of Biodiversity (the Species), in the complex Quercus genus. We provided tools to enable further efforts for the conservation of the Mediterranean oak forests, which overwhelm one of the most important (and one of the most threatened) Biomes for plant conservation at the global scale.

The CARBO-ACID research cruise (EUROFLEETS+ SEA02_10) was carried out on the RV Ramón Margalef between August 2nd and August 11st, with departing from Vigo – Spain and ending in Lisbon – Portugal. The main objective of this cruise was to collect data and samples to study the potential effects of ocean acidification on carbonate marine organisms (coccolithophores, pteropods, planktonic and benthic foraminifera, and corals) along the Iberian margin. With this objective, oceanographic data and water samples, plankton, cold-water corals and sediment samples were collected during an upwelling season, along two transects coinciding with the two persistent upwelling filaments off the Iberia Margin: the Cape Finisterra and the Cape Roca. In this dataset is guiven all the acquired data recollected onboad. During the CARBO-ACID cruise we did a total of 7 stations, 4 stations along the Cape Finisterra transect (from W to E: CA3, CA2, CA7, CA8) and 3 stations at the Cape Roca (from W to E: CA6, CA5, CA4) transect (Fig). At each station we usually started with a multibeam survey, a CTD and Rosette cast. These initial operations allowed to identify the different water masses present in this area, characterize their physical properties and to recover seawater samples at specific depth levels. The seawater samples were onboard subsampled, preserved in cold conditions or with chemicals and/ or filtered for several further analysis in the shore-based laboratories: DNA, chlorophyll, fitoplankton, coccolithophores, pH, alkalinity, stable isotopic composition, trace elements concentration and Suspend Particulate Matter. Subsequently to these operations, at each station, two vertical tows with a plankton multinet (with 5 nets) were done on the top 700 m of the water column to sample the planktonic communities of the different water depths. After this, sediment samples were recovered with a box-corer to study the past oceanographic conditions, between the pre-industrial Era and the Present, with multi-proxies used in paleoceanography and sedimentology. A total of 10 box-cores were recollected and each of them was onboard sub-sampled for eDNA, enzymes and benthic foraminifera. Fifteen shipek grab samples were recollected at the Fontanelas seamount (Estremadura Spur), station CA6, to characterize the sedimentary cover and to evaluate the presence of deep cold-water corals. Preliminary results show that the stations CA7, CA8 and CA4, located close to the coast, as expected, are the most influenced by the coastal upwelling, exhibiting colder surface water, higher values of fluorescence, and more zooplankton content reflecting higher phyto-zooplankton concentrations, as typical of the upwelling waters. At station CA4 temperature was higher and fluorescence showed lower values, indicative of less phytoplankton, and interpreted as indicating a different upwelling source water from that upwelled further north. Based on the CTD data, the Cape Roca transect is more influenced by the subtropical East North Atlantic Central Water (ENACWst), while the Cape Finisterra transect is more under the influence of the subpolar branch (ENACWsp). Seafloor sediment samples showed significant differences between the stations. Along the northern transect (Cape Finisterra) the seafloor sediments show an increase in grain size from the offshore to the coast. The offshore stations CA3 and CA2 revealed finer grained sediments, CA8 were composed of coarser sand and the station CA7, the shallowest station 77 m, presented the sediment composed mainly of shell fragments and coarse grain sand. Along the southern transect (Cape Roca), the offshore station CA6 (Fontanelas seamount) has coarser sandy sediments with rock clasts and cold-water coral fragments, and the stations CA5 and CA4 with fine sand to muddy sediments. The detailed CA6 bathymetry allowed to verify the existence of small plateaus on the slope of the Fontanelas seamount, where the fossil cold-water corals fragments were found, suggesting that this area is a very interesting system deserving further study with a ROV, and to characterize the corals fields and verify if there are live corals. These recollected data and samples will allow not only to reconstruct the pH variability under different environmental conditions, but also to estimate the biogeochemical changes along the coastal ocean waters as the anthropogenic influence increases. These results will contribute to better understand and model the effects on the biota under the future expected oceans pH changes.

Species distribution models (SDMs) are becoming an important tool for marine conservation and management. Yet while there is an increasing diversity and volume of marine biodiversity data for training SDMs, little practical guidance is available on how to leverage distinct data types to build robust models. We explored the effect of different data types on the fit, performance and predictive ability of SDMs by comparing models trained with four data types for a heavily exploited pelagic fish, the blue shark (Prionace glauca), in the Northwest Atlantic: two fishery-dependent (conventional mark-recapture tags, fisheries observer records) and two fishery-independent (satellite-linked electronic tags, pop-up archival tags). We found that all four data types can result in robust models, but differences among spatial predictions highlighted the need to consider ecological realism in model selection and interpretation regardless of data type. Differences among models were primarily attributed to biases in how each data type, and the associated representation of absences, sampled the environment and summarized the resulting species distributions. Outputs from model ensembles and a model trained on all pooled data both proved effective for combining inferences across data types and provided more ecologically realistic predictions than individual models. Our results provide valuable guidance for practitioners developing SDMs. With increasing access to diverse data sources, future work should further develop truly integrative modeling approaches that can explicitly leverage strengths of individual data types while statistically accounting for limitations, such as sampling biases.  Please see the README document ("README.md") and the accompanying published article: Braun, C. D., M. C. Arostegui, N. Farchadi, M. Alexander, P. Afonso, A. Allyn, S. J. Bograd, S. Brodie, D. P. Crear, E. F. Culhane, T. H. Curtis, E. L. Hazen, A. Kerney, N. Lezama-Ochoa, K. E. Mills, D. Pugh, N. Queiroz, J. D. Scott, G. B. Skomal, D. W. Sims, S. R. Thorrold, H. Welch, R. Young-Morse, R. Lewison. In press. Building use-inspired species distribution models: using multiple data types to examine and improve model performance. Ecological Applications. Accepted. DOI: < article DOI will be added when it is assigned >

Integrated multitrophic aquaculture (IMTA) is a versatile technology emerging as an ecological and sustainable solution for traditional monoculture aquacultures in terms of effluent treatment. Nevertheless, IMTA is still poorly applied in aquaculture industry due to, among other reasons, the lack of effective, low-investment and low-maintenance solutions. In this study, one has developed a practical and low maintenance IMTA-pilot system, settled in a semi-intensive coastal aquaculture. The optimisation and performance of the system was validated using Ulva spp., a macroalgae that naturally grows in the fishponds of the local aquaculture. Several cultivation experiments were performed at lab-scale and in the IMTA-pilot system, in static mode. The specific growth rate (SGR), yield, nutrient removal, N and C enrichment, protein and pigment content were monitored. Ulva spp. successfully thrived in effluent from the fish species sea bream (Sparus aurata) and sea bass (Dicentrarchus labrax) production tanks and significantly reduced inorganic nutrient load in the effluent, particularly, NH4+, PO43− and NO3−. The enrichment of nitrogen in Ulva spp.’s tissues indicated nitrogen assimilation by the algae, though, the cultivated Ulva spp. showed lower amounts of protein and pigments in comparison to the wild type. This study indicates that the designed IMTA-pilot system is an efficient solution for fish effluent treatment and Ulva spp., a suitable effluent remediator.

The aim of this paper is to investigate the threshold effect of foreign direct investment (FDI) on environmental degradation. In empirical analysis, FDI and environmental degradation are jointly determined under the given threshold variable and other exogenous variables. Using carbon dioxide (CO2) emissions per capita as a proxy for environmental degradation, the results show that increasing FDI worsens CO2 emissions after a threshold level of corruption has been reached. Our results demonstrate that increasing FDI will increase CO2 emissions when the degree of corruptibility is relatively high. The study suggests that further FDI and improved environmental quality are competing rather than compatible objectives in high-corruption countries and are compatible rather than competing objectives in low-corruption countries. Higher trade liberalization in low-corruption countries could contribute to negative environmental consequences because of the increased output or economic activity which results from increased trade. The robustness estimation confirms the evidence that pollution and economic development increase together up to a certain income level, after which the trend reverses.

Abstract Scenedesmus obliquus biomass was used as a feedstock for comparing the biological production of hydrogen by two different types of anaerobic cultures: a heat-treated mixed culture from a wastewater treatment plant and Clostridium butyricum DSM 10702. The influence of the incubation temperature and the carbon source composition were evaluated in order to select the best production profile according to the characteristics of the microalgal biomass. C. butyricum showed a clear preference for monomeric sugars and starch, the latter being the major storage compound in microalgae. The highest H 2 production reached by this strain from starch was 468 mL/g, whereas the mixed culture incubated at 37 °C (LE37) produced 241 mL/g. When the mixed culture was incubated at 58 °C (LE58), a significant increase in the H 2 production occurred when xylose and xylan were used as carbon and energy source. The highest H 2 yield reached by the LE37 culture or in co-culture with C. butyricum was 1.52 and 2.01 mol/mol of glucose equivalents, respectively. However, the ratio H 2 /CO 2 (v/v) of the biogas produced in both cases was always lower than the one produced by the pure strain. In kinetic assays, C. butyricum attained 153.9 mL H 2 /L h from S. obliquus biomass within the first 24 h of incubation, with a H 2 yield of 2.74 mol/mol of glucose equivalents. H 2 production was accompanied mainly by acetate and butyrate as co-products. In summary, C. butyricum demonstrated a clear supremacy for third generation bioH 2 production from S. obliquus biomass.

The European Union Water Framework Directive (WFD), a new regulation aiming to achieve and maintain a clean and well-managed water environment, refers to phytoplankton as one of the biological quality elements that should be regularly monitored, and upon which the reference conditions of water quality should be established. However, the use of phytoplankton as a biological quality element will result in several constraints, which are analyzed in this article with examples from Portuguese waters. Specifically, the establishment of reference conditions of water quality may be difficult in some water bodies for which no historical data exists. The sampling frequency proposed for phytoplankton monitoring does not seem suitable to assess phytoplankton succession, and may preclude the detection of algal blooms. Finally, the use of chlorophyll a as a proxy of phytoplankton biomass and abundance has been proposed by some authors, but it may overlook blooms of pico- and small nanophytoplankton, and overestimate the importance of large microphytoplankton. Furthermore, most studies in Portugal have used only inverted microscopy for phytoplankton observation and quantification; this method does not permit the distinction between autotrophic and heterotrophic cells, especially in samples preserved with Lugol's solution, and does not allow the observation of smaller-sized cells. Finally, some techniques, such as remote sensing and chemotaxonomic analysis, are proposed to be used as supplements in phytoplankton monitoring programs.

Nowadays, the issues related with environment preservation assume an increasing importance. Progressively, more sustainable solutions/techniques are being developed to combat environmental destruction. The decision to include themes related to the environment in the curriculum of technological courses in higher education aims to promote more sustainable behaviors and in an indirect way, increase the environmental literacy of the population. Thus, this study aims to evaluate the environmental literacy focusing on four topics, i.e., air pollution, water pollution, global warming, and energy resources. For this purpose, a questionnaire was developed and applied to a convenience sample, formed by individuals of both genders, aged between 20 and 81 years old. The questionnaire intended to collect data to characterize the sample and assess the literacy regarding environmental issues. In order to carry out the environmental literacy assessment, the respondents were asked to express their degree of agreement with some statements related with the environmental themes mentioned above. The data collected was analyzed using data mining tools. The results suggest that the population’s literacy is satisfactory in relation to some issues, but insufficient in relation to others, equally important, but less disseminated.