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Gomphonema alavariense sp. nov. is a new freshwater diatom described in Portugal from phytobenthos samples in an urban pond located in Aveiro (Portugal). This new diatom is illustrated and discussed based on different samples collected in July–August 2017 from Santo António Park pond in the city centre. This taxon was compared with other Gomphonema taxa and the morphological features are documented through light and scanning electron micrographs. Gomphonema alavariense sp. nov. is characterized by showing solitary cells, rhombic-lanceolate to lanceolate valves with rounded apices, a narrow, linear axial area, and slightly asymmetrical central area. This taxon may be confused with G. affine var. rhombicum in terms of length, striae density and outline. Gomphonema alavariense sp. nov. was present in freshwater with low dissolved oxygen concentrations, high conductivity, neutral to slightly alkaline pH and high nitrate concentrations.

One of the most common anthropogenic impacts on river ecosystems is the effluent discharge from wastewater treatment plants. The effects of this contamination on stream biota may be intensified in Mediterranean climate regions, which comprise a drought period that leads to flow reduction, and ultimately to stagnant pools. To assess individual and combined effects of flow stagnation and sewage contamination, biofilm and gastropod grazers were used in a 5-week experiment with artificial channels to test two flow velocity treatments (stagnant flow/basal flow) and two levels of organic contamination using artificial sewage (no sewage input/sewage input). Stressors’ effects were determined on biofilm total biomass and chlorophyll (Chl) content, on oxygen consumption and growth rate of the grazers (Theodoxus fluviatilis), and on the interaction grazer-biofilm given by grazer’s feeding activity (i.e., biofilm consumption rate). The single effect of sewage induced an increase in biofilm biomass and Chl-a content, simultaneously increasing both grazers’ oxygen consumption and their feeding activity. Diatoms showed a higher sensitivity to flow stagnation, resulting in a lower content of Chl-c. Combined stressors interacted antagonistically for biofilm total biomass, Chl-b contents, and grazers’s feeding rate. The effect of sewage increasing biofilm biomass and grazing activity was reduced by the presence of flow stagnation (antagonist factor). Our findings suggest that sewage contamination has a direct effect on the functional response of primary producers and an indirect effect on primary consumers, and this effect is influenced by water flow stagnation.

Freshwater benthic diatoms, routinely used for bioassessment, are potential metal indicators because of their known capacity to reflect changes in water chemistry. Nevertheless, the effect of metals on benthic freshwater diatoms is still poorly understood. Metals, such as Zn and Cu, are not only essential as micronutrients, but also one of the main anthropogenic pressures in aquatic ecosystems. However, metal stress cannot be detected with the current European standard methods using bioindicators for environmental assessment, so new tools need to be developed. We studied the effects of Zn and Cu on the freshwater diatom Tabellaria flocculosa. This diatom isolated from a Zn and Cu contaminated stream, was exposed to different concentrations of Zn and Cu, and the metal effects were assessed by measuring physiological ((growth, fluorescence, pigments’ content (Chl a, c, carotenoids)) and morphological parameters (teratological forms). We found that at environmental concentrations occurring in European streams, Zn and Cu showed toxic effects on T. flocculosa. Toxic effects induced by Zn were only observed at high concentrations (500 and 1000 µg/L), resulting in fluorescence changes, an increase of Chl c cellular content and an increase of teratological forms (with an increase of more heavily deformed valves with increasing metal concentrations). Increasing Cu exposure induced several changes, such as increasing the cellular content of Chl a and c, while carotenoids’ cellular content decreased. Cu also increased the number of T. flocculosa frustule deformations. Cu did not affect the cellular fluorescence. Our results thus show that the frequency of diatom teratologies could potentially be used to assess metal stress for the studied metals Zn and Cu. Additionally, the analysis of algal pigments could potentially separate the effects of different metals.

The south of the Iberian Peninsula has a high number of saline ponds where electric conductivity (EC) is an important factor that affects directly aquatic organisms, influencing their distribution and abundance. Environmental factors (such as pH, EC and temperature) were measured and diatom assemblages were sampled in 15 saline shallow ponds in the south of Spain along a range of electric conductivity (1.4 mS to 51.6 mS cm-1) between spring of 2004 to early summer of 2006. Three groups of ponds were defined based on conductivity (oligosaline 1.4 to 5.3 mScm-1, mesosaline 10.9 to 17.3 mScm-1 and euhaline 32.3 to 51.6 mScm-1) and diatom assemblages were studied. PERMANOVA analysis showed significant differences in diatom community composition between the three groups of ponds. Multidimensional scaling analysis (nMDS) showed distinct clusters of diatom assemblages in oligosaline and mesosaline ponds. Dominant diatom species in the eusaline ponds were Tryblionella pararostrata, Halamphora cf. petrusa, Halamphora sp.1 and Cocconeis euglypta; in the mesosaline ponds Navicula veneta, Nitzschia elegantula and Planothidium delicatulum were dominant taxa and the oligosaline ponds were dominated by Navicula veneta, Pseudostaurosira brevistriata and Nitzschia inconspicua. A new diatom species was described in ponds with high electric conductivity (32-56 mS cm-1) and named N. maiorpargemina.A detailed description of N. maiorpargemina sp. nov. is presented in this study based on light and scanning electron microscopy after comparison with morphologically and ecologically related taxa.

This work assesses the effects of river regulation on the diversity of different instream and riparian biological communities along a relieve gradient of disturbance in regulated rivers. Two case studies in Portugal were used, with different river regulation typology (downstream of run-of-river and reservoir dams), where regulated and free-flowing river stretches were surveyed for riparian vegetation, macrophytes, bryophytes, macroalgae, diatoms and macroinvertebrates. The assessment of the regulation effects on biological communities was approached by both biological and functional diversity analysis. Results of this investigation endorse river regulation as a major factor differentiating fluvial biological communities through an artificial environmental filtering that governs species assemblages by accentuating species traits related to river regulation tolerance. Communities' response to regulation gradient seem to be similar and insensitive to river regulation typology. Biological communities respond to this regulation gradient with different sensibilities and rates of response, with riparian vegetation and macroinvertebrates being the most responsive to river regulation and its gradient. Richness appears to be the best indicator for general fluvial ecological quality facing river regulation. Nevertheless, there are high correlations between the biological and functional diversity indices of different biological groups, which denotes biological connections indicative of a cascade of effects leading to an indirect influence of river regulation even on non-responsive facets of communities' biological and functional diversities. These results highlight the necessary holistic perspective of the fluvial system when assessing the effects of river regulation and the proposal of restoration measures. published

The relevance of molecular composition of diatom assemblages to detect river impairment caused by different intensive land uses (industrial, agricultural, and urbanization) was tested in this study with data from two rivers (Ferreira and Sousa rivers) and 21 sampling sites located in the north of Portugal. The Water Framework Directive (WFD) gives the legal basis for the use of this ecological indicator for water quality assessment (Vasselon et al. 2017 ). However, the morphological identification and count of diatoms using the light microscope requires a high level of expertise, is time-consuming and costly (Valentin et al. 2019). DNA metabarcoding combined with high-throughput sequencing techniques (HTS), offers a promising alternative to classic methodologies, reducing time and costs (Mortágua et al. 2019 ). Thus, here we compared the response of the two approaches in terms of ecological assessments (IPS Ecological Quality Ratios) to the different types of pressures felt in the 21 sites. Diatoms were sampled at 21 sites located in the North of Portugal in autumn of 2019 (Fig. 1). Samples were submitted in parallel to the molecular and morphological analyses. The eDNA was extracted, PCR amplified (312 bp rbcL DNA barcode), and finally sequenced (Illumina MiSeq). The Mothur software was used to obtain the Operational Taxonomic Units (OTUs), which were then taxonomically assigned to the species through the R-Syst::diatom version 7.1 (Rimet et al. 2018) reference library. EQR values indicated a good correlation between morphological and molecular methods (Fig. 2). PCA analysis (Fig. 3 ) revealed that in urban, agricultural, and industrial areas there is a greater concentration of nutrients (phosphorus and nitrogen), organic matter, and heavy metals due to the discharge of urban/industrial effluents, while in places considered natural (without any type of anthropogenic pressure) we find low levels of these pollutants and high concentrations of dissolved oxygen (DO). The BEST (BIO-ENV) analysis (Tables 1, 2) shows in the case of the morphological approach, the combination of 4 environmental variables (NO₃⁻, Li, K, and Cu) is highly correlated with the biological patterns, and in the molecular approach the combination of only 2 of the environmental variables (Li and K), explains the distribution of diatom communities composition and has a slightly higher correlation. The morphological methodology seems to demonstrate a better response to urban pressures, mainly to effluent discharges, while the molecular one demonstrates a more diffuse response with special emphasis on good correlation with variables such as zinc and nitrate, which may also be related to effluent discharges and use of fertilizers in agriculture. However, it is necessary to improve the reference library so that there is a better response of the molecular methodology to the existing pressures.

During the past decade genetic approaches have been developed to monitor biodiversity in aquatic ecosystems. These enable access to taxonomic and genetic information from biological communities using DNA from environmental samples (e.g. water, biofilm, soil) and methods based on high-throughput sequencing technologies, such as DNA metabarcoding. Within the context of the Water Framework Directive (WFD), such approaches could be applied to assess Biological Quality Elements (BQE). These are used as indicators of the ecological status of aquatic ecosystems as part of national monitoring programs of the european network of 110,000 surface water monitoring sites with 79.5% rivers and 11% lake sites (Charles et al. 2020). A high-throughput method has the potential to increase our spatio-temporal monitoring capacity and to accelerate the transfer of information to water managers with the aim to increase protection of aquatic ecosystems. Good progress has been made with developing DNA metabarcoding approaches for benthic diatom assemblages. Technological innovation and protocol optimization have allowed robust taxonomic (species) and genetic (OTU, ESV) information to be obtained from which diatom quality indices can be calculated to infer ecological status to rivers and lakes. Diatom DNA metabarcoding has been successfully applied for biomonitoring at the scale of national river monitoring networks in several countries around the world and can now be considered technically ready for routine application (e.g. Apothéloz-Perret-Gentil et al. 2017, Bailet et al. 2019, Mortágua et al. 2019, Vasselon et al. 2019, Kelly et al. 2020, Pérez-Burillo et al. 2020, Pissaridou et al. 2021). However, protocols and methods used by each laboratory still vary between and within countries, limiting their operational transferability and the ability to compare results. Thus, routine use of DNA metabarcoding for diatom biomonitoring requires standardization of all steps of the metabarcoding procedure, from the sampling to the final ecological status assessment in order to define good practices and standards. Following previous initiatives which resulted in a CEN technical report for biofilm sampling and preservation (CEN 2018), a set of experiments was initiated during the DNAqua-Net WG2 diatom workshop (Cyprus, 2019) to focus on DNA extraction and PCR amplification steps in order to evaluate: i) the transferability and reproducibility of a protocol between different laboratories; ii) the variability introduced by different protocols currently applied by the scientific community. 19 participants from 14 countries performed DNA extraction and PCR amplification in parallel, using i) the same fixed protocol and ii) their own protocol. Experiments were performed by each participant on a set of standardized DNA and biofilm samples (river, lake, mock community). In order to specifically test the variability of DNA extraction and PCR amplification steps, all other steps of the metabarcoding process were fixed and the preparation of the Miseq sequencing was performed by only one laboratory. The variability within and between participants will be evaluated on DNA extracts quantity, taxonomic (genus, species) and genetic richness, community structure comparison and diatom quality index scores (IPS). We will also evaluate the variability introduced by different DNA extraction and PCR amplification protocols on diatom quality index scores and the final ecological status assessment. The results from this collaborative work will not serve to define “one protocol to rule them all”, but will provide valuable information to define guidelines and minimum requirements that should be considered when performing diatom metabarcoding for biomonitoring.

Review The biological assessment of rivers i.e., their assessment through use of aquatic assemblages, integrates the effects of multiple-stressors on these systems over time and is essential to evaluate ecosystem condition and establish recovery measures. It has been undertaken in many countries since the 1990s, but not globally. And where national or multi-national monitoring networks have gathered large amounts of data, the poor water body classifications have not necessarily resulted in the rehabilitation of rivers. Thus, here we aimed to identify major gaps in the biological assessment and rehabilitation of rivers worldwide by focusing on the best examples in Asia, Europe, Oceania, and North, Central, and South America. Our study showed that it is not possible so far to draw a world map of the ecological quality of rivers. Biological assessment of rivers and streams is only implemented officially nation-wide and regularly in the European Union, Japan, Republic of Korea, South Africa, and the USA. In Australia, Canada, China, New Zealand, and Singapore it has been implemented officially at the state/province level (in some cases using common protocols) or in major catchments or even only once at the national level to define reference conditions (Australia). In other cases, biological monitoring is driven by a specific problem, impact assessments, water licenses, or the need to rehabilitate a river or a river section (as in Brazil, South Korea, China, Canada, Japan, Australia). In some countries monitoring programs have only been explored by research teams mostly at the catchment or local level (e.g., Brazil, Mexico, Chile, China, India, Malaysia, Thailand, Vietnam) or implemented by citizen science groups (e.g., Southern Africa, Gambia, East Africa, Australia, Brazil, Canada). The existing large-extent assessments show a striking loss of biodiversity in the last 2–3 decades in Japanese and New Zealand rivers (e.g., 42% and 70% of fish species threatened or endangered, respectively). A poor condition (below Good condition) exists in 25% of South Korean rivers, half of the European water bodies, and 44% of USA rivers, while in Australia 30% of the reaches sampled were significantly impaired in 2006. Regarding river rehabilitation, the greatest implementation has occurred in North America, Australia, Northern Europe, Japan, Singapore, and the Republic of Korea. Most rehabilitation measures have been related to improving water quality and river connectivity for fish or the improvement of riparian vegetation. The limited extent of most rehabilitation measures (i.e., not considering the entire catchment) often constrains the improvement of biological condition. Yet, many rehabilitation projects also lack pre-and/or post-monitoring of ecological condition, which prevents assessing the success and shortcomings of the recovery measures. Economic constraints are the most cited limitation for implementing monitoring programs and rehabilitation actions, followed by technical limitations, limited knowledge of the fauna and flora and their life-history traits (especially in Africa, South America and Mexico), and poor awareness by decision-makers. On the other hand, citizen involvement is recognized as key to the success and sustainability of rehabilitation projects. Thus, establishing rehabilitation needs, defining clear goals, tracking progress towards achieving them, and involving local populations and stakeholders are key recommendations for rehabilitation projects (Table 1). Large-extent and long-term monitoring programs are also essential to provide a realistic overview of the condition of rivers worldwide. Soon, the use of DNA biological samples and eDNA to investigate aquatic diversity could contribute to reducing costs and thus increase monitoring efforts and a more complete assessment of biodiversity. Finally, we propose developing transcontinental teams to elaborate and improve technical guidelines for implementing biological monitoring programs and river rehabilitation and establishing common financial and technical frameworks for managing international catchments. We also recommend providing such expert teams through the United Nations Environment Program to aid the extension of biomonitoring, bioassessment, and river rehabilitation knowledge globally info:eu-repo/semantics/publishedVersion

The European Water Framework Directive (2000/60/EC) includes biological assessment of water bodies that has been implemented for many years. Indicator organisms such as diatoms respond to geological and hydrological features of rivers by modifying their structure. Therefore, when implementing the WFD, it was necessary to establish type-specific reference conditions to be able to measure the deviations of sampled communities due to anthropogenic impact.HTS-related eDNA metabarcoding has been developed to complement or even replace traditional approaches for its rapid, low-cost and highly accurate identification of communities for assessment of rivers’ ecological status (e.g. Mortágua et al., 2019; Pérez-Burillo et al. 2020) and proved to provide even more in-depth information about biological elements. The use of this information without assignment to species is being addressed once it eliminates the limiting factor of the reference database incompleteness and may provide new ecological information (e.g. Feio et al., 2020; Rivera et al., 2020). Since WFD requires the establishment of reference conditions for each water body type, for eDNA methods’ implementation it will be essential to review, confirm or reformulate, and perhaps create new typologies. Hereupon, the aim of this study is to analyze diatom communities from different typologies of Portuguese rivers resulting from DNA metabarcoding data and compare it with current typology system. To do so, we will verify the consistency of biological groups included in each type, validate the molecular data, analyze the correspondence of OTU/ISU/ESV to environmental characteristics of rivers. A total of 154 sampling sites were selected from central Portugal and northern Portugal in 2017 and 2019. The biofilm was collected for morphological identification and DNA sequencing of diatoms. Reference sites were selected for 4 river types (mountain, littoral, small and medium-large northern rivers) based on a set of pressure information (water quality, hydromorphology, land use and riparian zones). Diatom inventories were obtained from molecular and morphological analysis. DNA sequences were treated using Mothur software which processed two bioinformatic strategies in order to obtain the final ISU and OTU tables, while ESVs were treated with DADA2 package from R. Identification and counting of diatom valves took place under the light microscope concerning the morphological approach. We expect results to validate the molecular data for each typology either when assigning to species or not, and to understand whether it is necessary to establish new typologies for future use of the molecular approach in ecological assessment of rivers. Directive, W. F. (2000). Water Framework Directive. Journal reference OJL, 327, 1-73. Feio, M. J., Serra, S. R., Mortágua, A., Bouchez, A., Rimet, F., Vasselon, V., & Almeida, S. F. P. (2020). A taxonomy-free approach based on machine learning to assess the quality of rivers with diatoms. Science of the Total Environment, 722, 137900. https://doi.org/10.1016/j.scitotenv.2020.137900 Mortágua, A., Vasselon, V., Oliveira, R., Elias, C., Chardon, C., Bouchez, A., ... & Almeida, S. F. P. (2019). Applicability of DNA metabarcoding approach in the bioassessment of Portuguese rivers using diatoms. Ecological indicators, 106, 105470. https://doi.org/10.1016/j.ecolind.2019.105470 Pérez-Burillo, J., Trobajo, R., Vasselon, V., Rimet, F., Bouchez, A., & Mann, D. G. (2020). Evaluation and sensitivity analysis of diatom DNA metabarcoding for WFD bioassessment of Mediterranean rivers. Science of the Total Environment, 727, 138445. https://doi.org/10.1016/j.scitotenv.2020.138445 Rivera, S. F., Vasselon, V., Bouchez, A., & Rimet, F. (2020). Diatom metabarcoding applied to large scale monitoring networks: Optimization of bioinformatics strategies using Mothur software. Ecological indicators, 109, 105775. https://doi.org/10.1016/j.ecolind.2019.105775

This study presents major and trace element data and 87Sr/86Sr ratios for surface waters from a small watershed draining the Aljustrel sulphide mining area (South Portugal). The watershed is located in a geologically complex area comprising two main compartments: the northern compartment is dominated by Cenozoic formations and does not bear any mining activity, whilst the southern compartment is underlain by rocks from both the Cenozoic sedimentary cover and the mineralized sequences of the Variscan Paleozoic basement of the South Portuguese Zone. Based on the available geochemical and isotopic data, the analysed water samples can be roughly divided into five main groups: (a) group 1 corresponds to AMD-contaminated water drainages from the southern part of the catchment, showing pH 8, marked depletions in SO4 and heavy metals and displaying Sr isotopic compositions similar to rainwater (87Sr/86Sr = 0.7097–0.7098); (c) group 3 includes mixed-type stream waters with pH and elemental/isotopic signatures intermediate between those of groups 1 and 2; (d) group 4 comprises the samples collected at two lime-treated water dams from the mining area, that are clearly distinguished from the impacted waters of group 1 by their elevated Ca, Mg and Sr concentrations and low 87Sr/86Sr ratios (87Sr/86Sr = 0.7085–0.7091); (e) group 5 includes clean waters from two dams located upstream of the ore processing site, which are dominantly supplied by direct precipitation and surface runoff and have pH > 6, very low SO4 and metal concentrations and 87Sr/86Sr ratios varying between 0.7094 and 0.7108. Variations in the 87Sr/86Sr ratios and Sr concentrations of the analysed waters are attributed to mixing of two main end-members: atmospheric meteoric waters (local rainfall/runoff) with low Sr contents and 87Sr/86Sr ratios ≈ 0.7095 and more radiogenic waters derived from the Aljustrel mining area. The highly radiogenic signatures of the southern tributaries appear to have been controlled by weathering of plagioclase from the acid volcanic rocks of the Paleozoic basement, enhanced by mining activities (pyrite oxidation), although released strontium from gangue carbonates could have locally contributed to the fluctuations of Sr contents and 87Sr/86Sr ratios observed in some of these water samples. By contrast, the low 87Sr/86Sr ratios and relatively high Sr (Cl and Na) concentrations shown by the surface waters draining the northern compartment suggest that these tributaries correspond to meteoric waters that have gained Cl, Na and Sr through wash-out of Cenozoic deposits containing finely disseminated halite layers. Finally, the decrease in the 87Sr/86Sr ratios and elevation of Sr contents observed in the samples from the lime-treated water dams support the involvement of an anthropogenic source of Sr with 87Sr/86Sr ratios lower than that of rainfall.

This work was cofunded by the European Union through the European Regional Development Fund, framed in COMPETE 2020 (Operational Programme Competitiveness and Internationalization), through the ICT project (UID/GEO/04683/2013) with reference POCI-01-0145-FEDER007690 and the Alentejo Observation and Prediction Systems–ALOP project (ALT20-03-0145-FEDER-000004), which awarded a postdoc grant to the first author. The Foundation for Science and Technology (FCT, Portugal) granted financial support to GeoBioTec through the UID/GEO/04035/2013 project. The government of Xunta de Galicia (I2C Posdoctoral Fellow–2014) awarded a postdoc grant to the last author. This research received support from the SYNTHESYS Project (http://www.synthesys.info/) titled, “Study and Revision of the Different Type Materials of Fragilaria/Synedra Genera (Bacillariophyceae) and Comparison with Portuguese Taxa” (GB-TAF-3736), financed by European Community Research Infrastructure Action under the FP7 “Capacities” Program.

Major threats of freshwater systems are river damming and habitat degradation, further amplified by climate change, another major driver of biodiversity loss. This study aims to understand the effects of climate change, and its repercussions on hydropower production, on the instream biota of a regulated river. Particularly, it aims to ascertain how mesohabitat availability downstream of hydropower plants changes due to modified flow regimes driven by climate change; how mesohabitat changes will influence the instream biota; and if instream biota changes will be similar within and between biological groups. We used a mesohabitat-level ecohydraulic approach with four biological elements – macrophytes, macroalgae, diatoms and macroinvertebrates – to encompass a holistic ecosystem perspective of the river system. The ecological preferences of the biological groups for specific mesohabitats were established by field survey. The mesohabitat availability in three expected climate change-driven flow regime scenarios was determined by hydrodynamic modeling. The biota abundance/cover was computed for the mesohabitat indicator species of each biological group. Results show that climate-changed flow regimes are characterized by a significant water shortage during summer months already for 2050. Accordingly, the regulated rivers' hydraulics are expected to change towards more homogeneous flow conditions where run habitats should prevail. As a result, the biological elements are expected to face abundance/cover modifications ranging from decreases of 76% up to 67% increase, depending on the biological element and indicator taxa. Diatoms seem to endure the greatest range of modifications while macrophytes the slightest (15% decrease to 38% increase). The greatest modifications would occur on decreasing abundance/cover responses. Such underlies an important risk to fluvial biodiversity in the future, indicting climate change as a significant threat to the fluvial system in regulated rivers.

Contemporary large-scale river ecology is grounded on the existence of patterns in the distribution of aquatic communities, structured by prevailing abiotic conditions. Here, we investigated the existence of functional consistent associations of traits (i.e., traits appearing consistently together at different sites and the same river type) between different biological elements of the aquatic community, assuming that species traits confer them advantages for certain environmental conditions but also within the aquatic community. If this is true, these trait associations should be consistently found in water bodies with similar characteristics (river types), defining different types of ecosystem functioning. To test this, 79 least-disturbed sites, belonging to five well-defined Portuguese river types and covering the longitudinal river gradient were used: headwaters of semi-arid streams, mountainous streams and northern-Atlantic climate streams, middle reaches and lowland large rivers. For each river type, we analyzed the strongest associations (via the Bray–Curtis coefficient) between diatoms, benthic invertebrates and macrophytes and traits that could be relevant to their interactions (e.g., invertebrate trophic groups, mobility/fixation ability of diatoms, macrophyte affinity to water) against a priori predictions. The strongest associations of traits changed over the river continuum with an increase in their complexity (number of associations) from headwaters to middle reaches and a decrease in lowland large rivers. These changes were not related to total richness, which was similar for all river types and over the continuum (ca. 100 taxa). In the three types of headwaters, there were also clear differences in associations among aquatic elements. The importance of riparian trees in small streams was not as high as expected while instream macrophytes were more relevant than predicted. This study revealed the existence of predictable functional associations that could serve as a basis for the functional assessment of running waters.

Background: Due to their isolation, islands offer excellent areas for the study of distribution of benthic diatoms. On the other hand, diatoms bearing canal raphe have received less attention compared to other groups of diatoms such as Navicula, Pinnularia or Amphora. Questions: Is it possible that thermal springs on islands offer a refuge for infrequent diatom species? Studied species: Platichthys furnensis C. Delgado, V. Gonçalves & S.F.P. Almeida sp. nov. Study site and dates: The species here described was collected in the epilithon of a thermal spring in São Miguel Island (Azores Archipelago, Portugal) in September 2015. Methods: This new taxon was compared to other diatom species of the genera Nitzschia, Tryblionella, Entomoneis and Hantzschia and to the other species of the genus Platichthys. The morphology is documented by light and scanning electron images and discussed in detail. Results: Platichthys furnensis was found in a thermal pool, a similar habitat to the one where P. krammeri type was collected in Chile in 1940. P. furnensis has many structures that are characteristic of the recently described genus Platichthys, including raised canal raphe and fibulae, compressed valve face, steep valve face and numerous open copulae. Conclusions: The description of the new taxon is interesting because it is the first species within Platichthys to be described from the Northern Hemisphere. Fundação para a Ciência e a Tecnologia | Ref. UID/BIA/50027/2013 European Commission | Ref. POCI-01-0145-FEDER-006821 Fundação para a Ciência e a Tecnologia | Ref. UID/GEO/ 04035/2019

Thermal springs provide extreme ecological conditions for aquatic communities owing to their high water temperature and particular water chemistry. The thermal springs and their connected watercourses provide a thermal laboratory by offering a wide range of temperatures within short spatial distances. To date, the information on how the biodiversity of these springs is related to water temperature or chemistry is limited. We studied the effects of water temperature on diatom community diversity and structure with the objective to supply a baseline for the conservation of thermal springs. We sampled 31 sites of 16 thermal springs across a temperature gradient between 18.5 and 63.0°C in the north-western Iberian Peninsula and related diatom richness and structure to water temperature. A total of 124 diatom species were identified in springs with water temperature between 18.5 and 42.4°C. Community diversity decreased with increasing temperature and the highest species richness was found at temperatures ≤ 25°C. Three diatom assemblages were defined, discriminated by temperature ranges (≤25°C, 25–35°C and ≥35°C). Water temperature was found to be an important driver of diatom community composition in the thermal systems studied. Temperature affects diatom distribution decreasing diversity with the increase in global water temperature.

International audience; Diatoms are a compulsory biological quality element in the ecological assessment of rivers according to the Water Framework Directive. The application of current official indices requires the identification of individuals to species or lower rank under a microscope based on the valve morphology. This is a highly time-consuming task, often susceptible of disagreements among analysts. In alternative, the use of DNA metabarcoding combined with High-Throughput Sequencing (HTS) has been proposed. The sequences obtained from environmental DNA are clustered into Operational Taxonomic Units (OTUs), which can be assigned to a taxon using reference databases, and from there calculate biotic indices. However, there is still a high percentage of unassigned OTUs to species due to the incompleteness of reference libraries. Alternatively, we tested a new taxonomy-free approach based on diatom community samples to assess rivers. A combination of three machine learning techniques is used to build models that predict diatom OTUs expected in test sites, under reference conditions, from environmental data. The Observed/Expected OTUs ratio indicates the deviation from reference condition and is converted into a quality class. This approach was never used with diatoms neither with OTUs data. To evaluate its efficiency, we built a model based on OTUs lists (HYDGEN) and another based on taxa lists from morphological identification (HYDMORPH), and also calculated a biotic index (IPS). The models were trained and tested with data from 81 sites (44 reference sites) from central Portugal. Both models were considered accurate (linear regression for Observed and Expected richness: R2 ≈ 0.7, interception ≈ 0.8) and sensitive to global anthropogenic disturbance (Rs2 > 0.30 p < 0.006 for global disturbance). Yet, the HYDGEN model based on molecular data was sensitive to more types of pressures (such as, changes in land use and habitat quality), which gives promising insights to its use for bioassessment of rivers.

A great investment has been done in the last decades in the development of numerical and qualitative assessment methods to classify the ecological quality of water bodies. Yet, in spite of all attempts to avoid subjectivity, expert judgment is still used at numerous steps of the ecological classification and is considered by some authors as indispensible for management purposes. Thus, the aim of this study is to test the hypothesis that expert judgment, when done by the adequate experts (limnologists/river ecologist) with experience in the study area (i.e., natural conditions and expected communities), could be as good as quantitative indices and measures (i.e., result in the same classification), but quicker and with lower cost. For that we compared the classifications (on 13 aspects of rivers ecosystems) attributed by two experts to 20 sites (10 each) located in their study areas, with the classifications of ecological quality based on biological indices (for invertebrates and diatoms), hydromorphology and water chemistry, calculated by an independent team. Our results show that assessments made by experts and those calculated through indices (biological quality and hydromorphology) are globally very similar (RELATE test; Rho = 0.442; p

Although diatoms are important bioindicators of ecological quality, their ecological traits are still not well understood. A major issue is that of substrate preferences, which may result in differences in production, and assemblage structure and composition, and which should therefore be taken into account for ecological quality assessment studies. Thus, in this work, the periphyton grown on sand and ceramic tiles in indoor controlled channels were compared to understand whether substrate differences lead to differences in: periphyton production (chlorophyll-a), chlorophyll-b and c concentrations, diatom assemblages (diversity-Shannon-Wiener, cell density, taxonomic composition, trait proportions), and ecological quality assessments (IPS-‘Indice de Polluosensibilite Specifique’). A combined inoculum of periphyton from four Portuguese streams was introduced to the running channels (six sand and six tile) and left to colonize for 35 days. Epilithic (tiles) and epipsammic (sand) assemblages were sampled at days 14 and 35. We verified that there were no differences in chlorophyll-a concentration over time and between substrates. On both sampling occasions, the epipsammic assemblages had higher concentration of chlorophyll-c and diatom density but without significant differences over time in each substrate. The taxonomic composition was different between substrates and over time. However, these differences were not reflected in ecological quality assessment. The diversity was also similar between substrates in both sampling occasions, but it was higher at day 14. Mobile and stalked species were more abundant over the entire study and differed significantly between substrates, with the epipsammic assemblages presenting higher abundances of both traits. We concluded that the colonizing substrate influences diatom assemblages but not the ecological quality assessment.

Contemporary bioassessment methods for water bodies require the description of “reference conditions” representing an absence or only “very minor” presence of human impacts on hydromorphological, physical and chemical properties. However, minimally disturbed reference sites are lacking in many European regions and other parts of the world because of pervasive anthropogenic influences. Here we describe the use of environmental filters modelling, incorporating boosted trees (BT), to derive reference data for abiotic variables and biological communities (diatoms and macroinvertebrates) for rivers in a highly disturbed region (Portuguese central-western lowland area), where minimally disturbed reference sites are non-existent. We also revise quality class boundaries for diatom and macroinvertebrate bioassessment in this region, and develop a new multimetric diatoms index (MDI). The new index includes not only the ‘ Indice de Polluosensibilite Specifique’ (IPS) based on species’ sensitivity to organic pollution and nutrients, but also the numbers of total and sensitive taxa. Our approach predicted significantly different communities under reference conditions from those observed, with a higher median reference number of taxa per site than the observed number (69 against 27 for diatoms; 53 against 22 for macroinvertebrates). In addition, the predicted communities for both biological groups were more similar among sites than the observed communities. Adjustment of index calculation and quality class boundaries to incorporate the new reference data resulted in more stringent site assessments that were better correlated with human pressures than assessments with previous methods. This study brings new insight to solve the problem of an absence of minimally disturbed reference sites.

Intraspecific variability occurs in all types of organisms and is a driving force to speciation, conferring genotypic and phenotypic differences that enable adaptive responses to sub-lethal stimuli such as exposure to pollutants (including cadmium, Cd). Thus, differences in biochemical parameters are expected among isolates of the same species. Studying the extent of these differences throughout a stress range, will provide information for the development of approaches to mitigate habitat contamination. This work was designed to identify possible differences in Cd tolerance of five isolates of the freshwater diatom Nitzschia palea from different sampling sites. Each isolate was exposed to five increasing Cd concentrations during 10 days. Growth inhibition was assessed and intracellular accumulation of Cd was quantified. Superoxide dismutase and catalase activities were determined. Glutathione as well as lipid peroxidation (LPO) and intracellular protein content were quantified. The results obtained identified intraspecific differences among isolates. These differences were associated with different approaches of coping with Cd stress. Higher intracellular Cd concentrations induced lower tolerance in isolates, since antioxidant mechanisms were unable to fight effectively against higher oxidative stress. Reversely, lower intracellular accumulation of Cd induced lower oxidative damage and allowed cells to better tolerate exposure to Cd. LPO emerged as an excellent marker of oxidative stress in N. palea and its use can differentiate isolates according to their tolerance.

The biological responses of acidobiontic species to extremely acidic waters, as those related with Acid Mine Drainage (AMD) processes, are relevant not only to understand the toxicological degree of the waters as well as the mechanisms responsible for the survival and adaptation of such organisms. Therefore, the objective of this study was to evaluate the effect of acidic pH and metals in biofilms collected in two sites a lentic permanent acid pond (AP) and a temporary acid stream (AS) affected by Acid Mine Drainage (AMD), and taking as control (C), an uncontaminated site with circumneutral pH. The results showed typical conditions of AMD-contaminated waters in sites AS and AP (pH 3 and high concentrations of Al, Cu, Fe, Mn, Pb and Zn), which are responsible for a substantial decrease in biomass and biodiversity of biofilms (with a dominance of acidobiontic species, such as Pinnularia aljustrelica and Eunotia exigua, comparing to the control site. Also expressed by low values of photosynthetic pigments and reduced amount of proteins. In addition, such extreme acidic conditions also induced biofilms' stress, increasing antioxidant [catalase (CAT), superoxide dismutase (SOD) and biotransformation glutathione S-transferases (GSTs)] enzymes activity and also membrane damage (peroxidized lipids). However, despite the quite similar physico-chemical conditions of both AMD-contaminated sites, differences in the toxicity status between them were indirectly detected through some of the biochemical parameters [GSTs, SOD and CAT], indicating site AS has the most toxic, which fact was attributed to the higher concentrations of Pb

Freshwaters are constantly facing ecosystem functioning alterations and loss of biodiversity driven by multiple anthropogenic and natural stressors, that by acting simultaneously create complex interactions, affecting the quantity and quality of water resources. Stream biofilms are complex communities, which are exposed to these alterations and, in addition, are naturally stressed by invertebrate grazing. Therefore, they are expected to reflect these impacts through shifts in community structure, composition and function. Here we used a mesocosm system to assess the single and interacting effect of major anthropogenic stressors acting in Mediterranean streams (i.e. flow stagnation and sewage contamination) in combination with a biological natural stressor (i.e. grazing) on the main assemblages composing biofilm (i.e. bacteria, fungi and algae) by assessing communities’ relative abundance through Denaturing Gradient Gel Electrophoresis (DGGE) operational taxonomic units (OTUs). Biofilm was submitted to the three stressors, in a full-factorial design (2 flow conditions × 2 contamination conditions × 2 grazing settings) in a 5-week experiment. Molecular data showed that the combined effect of anthropogenic stressors (flow stagnation and sewage contamination) induced unequal OTUs responses on biofilm assemblages, with antagonistic effects for bacteria, synergistic for fungi and additive for algae. Sewage and grazing interaction were significant for all groups revealing a negative effect (antagonistic) on bacteria and algae diversity but positive on fungi diversity (synergistic). The same overall response pattern was also found for the triple co-occurring stressors, which increased fungi diversity while decreasing algae and bacteria. In stream ecosystems in which low flow conditions and sewage contamination prevail in the presence of natural herbivory, algae and bacterial diversity may be severely affected, while fungal diversity may be surprisingly enhanced. Consequently, shifts in the relative proportions could led to the unbalancing of ecosystem processes (e.g., photosynthesis, nutrient mineralization) defined by the microbial communities.

Aljustrel mining area (South Portugal) is a part of the Iberian Pyrite Belt and encloses six sulfide mineral masses. This mine is classified of high environmental risk due to the large tailings’ volume and acid mine drainage (AMD)-affected waters generated by sulfides’ oxidation. The use of biological indicators (e.g., diatoms) revealed to be an important tool to address the degree of AMD contamination in waters. Multivariate analysis has been used as a relevant approach for the characterization of AMD processes. Cluster analysis was used to integrate the significant amount and diversity of variables (physicochemical and biological), discriminating the different types of waters, characterized by the high complexity occurring in this region. The distinction of two main marked phenomena was achieved: (1) the circumneutral-Na-Cl water type (sites DA, PF, BX, BF, RO, CB), expressing the geological contributions of the Cenozoic sediments of Sado river basin, with high diatom diversity (predominating brackish diatoms as Entomoneis alata); and (2) the acid–metal-sulfated water type (sites BM, JU, RJ, AA, MR, BE, PC, AF), reflecting both the AMD contamination and the dissolution of minerals (e.g., silicates) from the hosting rocks, potentiated by the extremely low pH. This last group of sites showed lower diatom diversity but with typical diatoms from acid- and metal-contaminated waters (e.g., Pinnularia aljustrelica). In addition to these two water types, this hierarchical classification method also allowed to distinguish individual cases in subclusters, for example, treated dams (DC, DD), with alkaline substances (lime/limestone), that changed the physicochemical dynamics of the contaminated waters.

This work presents a combination of geochemical, mineralogical, and biological data obtained in water reservoirs located in one of the most paradigmatic mining regions, suffering from acid mine drainage (AMD) problems: the Iberian Pyrite Belt (IPB). Four water reservoirs located in the Spanish sector of the IBP, storing water for different purposes, were selected to achieve an environmental classification based on the effects of AMD: two mining dams (Gossan and Águas Ácidas), a reservoir for industrial use (Sancho), and one with water used for human supply (Andévalo). The results indicated that the four reservoirs are subject to the effect of metallic loads from polluted rivers, although with different levels: Águas Ácidas > Gossan > Sancho ≥ Andévalo. In accordance, epipsammic diatom communities have differences in the respective composition and dominant taxa. The dominant diatoms in each reservoir indicated acid water: Pinnularia acidophila and Pinnularia aljustrelica were found in the most acidic dams (Gossan and Águas Ácidas, with pH, The authors thank to António Azevedo for his help in XRD analysis and to Elisabete Vivas for her assistance with the preparation of clay fraction. Financial support for this research was provided by DGCICYT National Plan, Project CGL2010-21268-C02-01 and Project RNM-6570.The authors are grateful to two anonymous reviewers for their valuable comments and suggestions., info:eu-repo/semantics/publishedVersion

International audience; Organic contaminants, and herbicides in particular, represent a risk for aquatic ecosystems. The primary target of herbicides are producers, the base of food webs, but frequently they end up far from the application point affecting non-target species. Its presence can work as sub-lethal stimulus, which sort the genetic and phenotypic differences within a species. Intraspecific variation allows adaptation to changes in the environment but also to new niches due to variations in species' sensitivity and biochemical response to a certain chemical. A better understanding of these variations can lead to the development of improved strategies for ecosystem protection. This research aimed to compare a sensitive and a tolerant strain of the freshwater diatom Nitzschia palea to atrazine. Strains were exposed to three concentrations within their tolerance range, during 96 h. The activity of the antioxidant enzymes superoxide dismutase, catalase, glutathione-S-transferases and glutathione peroxidases was determined. In addition, chlorophylls a and c, carotenoids, reduced glutathione, proteins and lipid peroxidation were quantified. Both strains displayed different strategies to deal with atrazine toxicity: while the sensitive strain decreased the oxidative stress, increasing the activity of antioxidant enzymes such as superoxide dismutase, the tolerant strain invested in conjugation pathways and carotenoids' maintenance.

Metals are a recognised threat to aquatic organisms but the impact of metals such as copper (Cu) on benthic freshwater diatoms is poorly understood, even if diatoms are commonly used as water quality indicators. Our study aimed to elucidate the cellular targets of Cu toxicity and the mechanisms cells resort to counteract toxicity and to increase tolerance to Cu. A concerted approach analysing the biochemical, physiological and metabolome alterations in diatom cells was conducted by exposing the freshwater diatom Tabellaria flocattosa to 0, 0.3, 6 and 10 mu g Cu/L. Cu was already toxic to T. flocculosa at concentrations common in environments and which are not usually considered to be contaminated (0.3 mu g Cu/L). Under Cu impact, the metabolome of T. flocculosa changed significantly, especially at high concentrations (6 and 10 mu g Cu/L). Cu toxicity was counteracted by increasing extracellular immobilization (EPS, frustulins), antioxidant (SOD, CAT) and detoxifying (GSTs) enzymes activity and low molecular weight antioxidants (GSH). These mechanisms were fuelled by higher energy production (increased ETS activity). At the highest Cu concentration (10 mu g/L), these processes were specially enhanced in an attempt to restrain the oxidative stress generated by high intracellular Cu concentrations. However, these mechanisms were not able to fully protect cells, and damage in membranes and proteins increased. Moreover, the decrease of hydroxylamine and unsaturated fatty acids and the increase of saturated fatty acids, 2-palmiloylglycerol, glycerol and diterpenoid compounds should be tested as new specific markers of Cu toxicity in future studies. This information can support the prediction of diatom behaviour in different Cu contamination levels, including highly impacted environments, such as mining scenarios, and may assist in environmental risk assessment policies and restoration programs. (C) 2018 Elsevier B.V. All rights reserved.

Modern ecological assessments of running waters are based on the a priori definition of ecological benchmarks, given by reference-quality sites. Such benchmarks are established at the level of ecoregions, typologies, or site. Yet, in highly disturbed regions, such as coastal areas of European countries, the assessment of streams’ water quality based on the reference condition concept is very difficult, due to the lack of undisturbed sites. Among others, the reduced number of reference sites may have as a consequence the definition of imprecise ecological benchmarks. Here we tested the hypotheses that (1) the increase in the number of potential reference sites (2) the definition of more precise abiotic thresholds using the least disturbed condition approach (LDC), and (3) the use of diatom assemblages, as the most ubiquitous element in lowland areas, would result in refinement and eventual sub-division of existing river types of a highly disturbed area, such as the Portuguese centre-western region. For this purpose, abiotic data characterising natural conditions of 55 sites from a littoral highly disturbed region were used in a hierarchical classification analysis that revealed the existence of three different sub-groups. In addition, a three-step approach was used to define thresholds for the pressure variables in LDC. Based on these new thresholds, sites in LDC were selected. A hierarchical classification performed to the LDC diatom spring assemblages revealed the existence of two sub-groups, concordant with two of the abiotic sub-groups. Several species contributed to the dissimilarity between the two sub-groups ( e.g. , Achnanthidium minutissimum and Karayevia oblongella ). Differences between the sub-groups were also found in the trait proportions of stalked species. New benchmark values for these two sub-groups, based on the scores of the official diatom index, the Indice de Polluosensibilite Specifique (IPS), were different from the previous reference value used. Yet, no biological benchmark values were established for one of the groups due to the absence of sites in the LDC. Our study suggests that streambed substrate is an important characterisation variable in the river type definition and highlights that, in spite of the potential refinement in reference conditions and typology obtained, an alternative approach that does not require the use of reference sites should be explored in the future.

Freshwater ecosystems are under threatening anthropogenic pressures worldwide, namely by metals. Diatoms are used as water quality indicators, but the influence of micronutrients such as Zn and its impacts are poorly understood. Thus, our study aimed to elucidate the tolerance level, the cellular targets and the responses to counteract Zn toxicity of freshwater diatoms by exposing Tabellaria flocculosa, isolated from a Zn contaminated stream. Biochemical, physiological and metabolomic approaches were used. It was demonstrated that Zn is toxic to T. flocculosa at concentrations occurring in contaminated environments. At low stress (30 μg Zn/L) few alterations in the metabolome were observed, but the enzymatic (SOD, CAT) and molecular (GSH, GSSG) antioxidant systems were induced, protecting cells from oxidative stress. At moderate stress (500 μg Zn/L) the main changes occurred in the metabolome (increases in fatty acids, amino acids, terpenoids, glycerol and phosphate, decreases in sucrose and lumichrome) with a moderate increase in cell damage (LPO and PC). The concerted action of all these mechanisms resulted in a non-significant decrease of growth, explaining the survival of this T. flocculosa strain in an environment with this Zn concentration. At the highest stress level (1000 μg Zn/L) the metabolome was identical to 500 μg Zn/L, and the induction of antioxidant systems and extracellular ion chelation (exopolysaccharides, frustulins) were the main responses to the increase of Zn toxicity. However, these mechanisms were unable to effectively abrogate cellular damage and growth reduction was observed. Moreover, the decrease in sucrose and especially in lumichrome should be tested as new specific markers of Zn toxicity. The information obtained in this study can assist in environmental risk assessment policies, support the prediction of diatom behaviour in highly impacted Zn environments, such as mining scenarios, and may help develop new indices, which include alterations induced by metals.

Periphytic communities are good indicators of river quality due to their general sensitivity to several pollutants. The primary objective of this study was to develop and optimize an ecotoxicological testing methodology using the freshwater benthic diatom Navicula libonensis. This species was selected due to its ubiquity and suitability for use under laboratory conditions. In the most suitable test medium (Chu10) the diatom demonstrated comparable sensitivity to potassium dichromate and 3,5-dichlorophenol using growth rate as the reference parameter, with median effect concentrations (ErC50) in the same order of magnitude (0.119 and 0.799 mg L(-1)) respectively. Yield-based estimates did not confirm this pattern and potassium dichromate was one order of magnitude more toxic than 3,5-dichlorophenol. The sensitivity of N. libonensis to the reference chemicals was higher than that published in the literature for several standard planktonic microalgae. This advantage, as well as the ability to grow the species in the laboratory, supports further efforts towards the standardisation of a toxicity testing protocol. In addition, the functional role of benthic diatoms in lotic ecosystems justifies their inclusion in risk assessment test batteries to better cover an environmental compartment that has so far been neglected.

International audience; Benthic diatom communities are used in the ecological assessment of Portuguese rivers through the calculation of an autecological index, the IPS (Indice de Polluosensibilite Specifique), officially adopted for Portugal. This index requires a high level of taxonomic expertise for morphological identification of individuals at species level. Advances in genomics, such as the DNA metabarcoding combined with high-throughput sequencing techniques offer a promising alternative to this classical approach, decreasing expertise requirement and therefore reducing time and costs. The aim of this study was to test the potential of DNA metabarcoding of diatoms in the bioassessment of Portuguese rivers, by comparing the classifications of the IPS obtained with morphological and molecular approaches. A total of 88 samples were collected in four river types and a quality gradient in central Portugal in the spring of 2017, following the Water Framework Directive (WFD) standards. The morphological approach comprised taxonomic identification of at least 400 valves, under the light microscope. Molecular approach included eDNA extraction followed by PCR amplification and DNA high-throughput sequencing (Illumina MiSeq) using a diatom-specific 312 bp rbcL DNA barcode. Sequences were analysed with Mothur software, producing Operational Taxonomic Units (OTUs) that were taxonomically assigned using the R-Syst::diatom reference library. We also tested the effect of a correction factor (CF) based on biovolume for the quantification of molecular data. When comparing the number of species identified, morphological, molecular without and with CF inventories revealed 306, 125 and 111 species, respectively. Molecular data showed 67% of unassigned OTUs. These results were mainly due to the incompleteness of the reference library, which currently represents the major difficulty in taxonomic assignment of DNA sequences in metabarcoding. Regarding IPS values, the results indicated a good correlation between morphological and molecular methods, particularly when applying the CF. Furthermore, Principal Coordinates analysis plot based on species abundances revealed a quality gradient in all 3 methodologies (morphological, molecular without and with CF). These results support the hypothesis that DNA metabarcoding is a valid approach for ecological quality assessment of Portuguese rivers. Yet, there is still work to be done on this new methodology to enable a smooth transition between the traditional and this new approach without misspend accumulated knowledge from the last decades on water quality assessment.

Volcanic areas are hostile environments where the release of toxic gases is added to the low pH and high temperature, subjecting living organisms to extreme conditions. High-temperature environments, particularly those associated with volcanic activity have been evolving on earth throughout time. The aim of this study is to contribute to increasing knowledge of these high-temperature events and describe their associated diatom assemblages. The fumarolic fields of Sao Miguel Island in the Azores Archipelago were selected as a case study due to its particular characteristics and were studied seasonally during 2007. Temperature, pH and conductivity variables were measured in situ. Other environmental data were available in the literature. Diatom diversity, assessed using the Shannon–Wiener Index (H′), ranged between 0.02 and 4. Statistical analyses revealed that temperature is a significant driver for the distribution of diatom species. Achnanthidium exiguum Grunow, Nitzschia cf. thermalis var. minor (Hilse)...

This study compares regional differences in benthic diatom communities exposed to similar stresses in Canada and Portugal. Diatoms were sampled in the Água Forte Stream, Aljustrel (SW Portugal) and in the Little River, New Brunswick (SE Canada), both streams surround the respective zinc mine and are subject to similar metal (e.g. Cd, Cu, Fe, Zn) and acidic (Água Forte pH = 1.9–2.9 vs. Little River pH = 2.2–5.5) stresses. In this kind of extreme environment, diatoms are frequently the main algae group in the streams, widely used as bioindicators. Diatom communities in the Água Forte Stream were dominated mostly by Pinnularia aljustrelica and Eunotia exigua (5% teratological forms), whereas communities in the Little River were more diverse (e.g. Achnanthidium minutissimum, Nitzschia palea, Eunotia sp.). Shannon-Wiener Index (H′) and percentage of taxa relative abundance were used to characterize the diversity and species composition of the diatom communities. Using canonical correspondence analysis (CCA), it was found that regional variation in acceptable in-stream concentrations of metals, conductivity and pH were the primary drivers of benthic diatom community. Mine remediation to decrease metal concentrations and increase pH in streams will increase diatom diversity even in highly impacted streams such as Água Forte.

Epub 2017 Jul 06; Diatoms are used as indicators of freshwater ecosystems integrity. Developing diatom-based tools to assess impact of herbicide pollution is expected by water managers. But, defining sensitivities of all species to multiple herbicides would be unattainable. The existence of a phylogenetic signal of herbicide sensitivity was shown among diatoms and should enable prediction of new species sensitivity. However, diatoms present a cryptic diversity that may lead to variation in their sensitivity to herbicides that would need to be taken into account. Using bioassays, the sensitivity to four herbicides (Atrazine, Terbutryn, Diuron, Isoproturon) was evaluated for 11 freshwater diatom taxa and intraspecific variability was assessed for two of them (Nitzschia palea and Achnanthidium spp.). Intraspecific variability of herbicide sensitivity was always smaller than interspecific variability, but intraspecific variability was more important in N. palea than in Achnanthidium spp. Indeed, one species showed no intraspecific phylogenetic signal (N. palea) whereas the other did (Achnanthidium spp.). On one hand, species boundaries are not set properly for Achnanthidium spp. which encompass several taxa. On the other hand, there is a higher phenotypic plasticity for N. palea. Finally, a phylogenetic signal of herbicide sensitivity was measured at the interspecific level, opening up prospects for setting up reliable biomonitoring tools based on sensitivity prediction, insofar as species boundaries are correctly defined.

The surrounding streams of the Aljustrel mining area (Alentejo Region, Southern Portugal) are strongly affected by acid mine drainage (AMD), as a result of the weathering of the tailing's sulphide minerals. Benthic diatoms (Bacillariophyceae) and water samples were collected seasonally, during one year (2008/09), at 13 sampling sites (5 impacted and 8 unimpacted). Benthic diatom assemblages were studied using Pulse Amplitude Modulated (PAM) fluorometry to assess the effects of the metals/acidity on the photosynthetic efficiency. The information provided by Chl-a fluorescence parameters (e. g. alpha, ETRmax) is an early warning signal of biological effects after acute exposure to chemicals, damaging either directly or indirectly Photosystem II (PSII). Statistical analysis (Two-way and Factorial ANOVAs) found significant differences on Chl-a fluorescence parameters of impacted and unimpacted sites. Photosynthetic efficiency and biomass (Chl-a) were lower for benthic diatom assemblages in impacted streams (Agua Forte and Agua Azeda), especially during summer, due to very low pH (2.3) and the concentration effect of Cu (348 ppm), Fe (6173 ppm) and Zn (1728 ppm) in waters and sediments. Diatom diversity was low at these sites, mainly dominated by Pinnularia aljustrelica, the main contributor to the high similarity between impacted sites (81 % of similarity by SIMPER). This is a typical species from acidic waters with high metal concentrations. Maximum values of Chl-a fluorescence parameters (alpha, ETRmax) were found for winter at the unimpacted sites in the Canal da Barrada and Roxo streams due to: 1) higher distance from mine input, 2) pH differences, 3) the existence of brackish/marine species (e. g. Entomoneis paludosa and Haslea spicula), supported by high ions content in the waters. When the analysis was based on the structure of diatom assemblages dominated by Achnanthidium minutissimum, two groups were highlighted: JU-PB and RJ-BF (uncontaminated), indicating seasonal differences. This study confirmed PAM fluorescence as an effective method for the rapid and non-destructive estimation of photosynthetic performance in diatom communities of AMD impacted streams from pyritic mining areas. The authors are grateful to the Biology and Geosciences Departments of University of Aveiro, Portugal and to the Fundação para a Ciência e Tecnologia, Portugal (grant number SFRH/ BD/36137/2007) for their financial support during this study. We thank Helena Novais for the valuable help with the software (ArcGis) to do the sites’ location map. We thank the two anonymous reviewers and the editor for critical comments on the manuscript. published

A new benthic freshwater diatom, Fragilaria rinoi Almeida & C.Delgado sp. nov., is described from river periphyton samples in Portugal. Fragilaria rinoi sp. nov. is illustrated and discussed based on populations collected from the Vouga, Mondego and Lis river basins in central Portugal and compared with the type material of Fragilaria vaucheriae (Kütz.) J.B.Petersen. The morphological features of the new diatom species are documented through light and scanning electron micrographs, including a comparative analysis with related species of the genus (F. candidagilae Almeida, C.Delgado, Novais & S.Blanco, F. intermedia Grunow in Van Heurck, F. neointermedia Tuji & D.M.Williams, F. recapitellata Lange-Bert. & Metzeltin, F. perminuta (Grunow) Lange-Bert., F. vaucheriae and F. microvaucheriae C.E.Wetzel & Ector). Fragilaria rinoi sp. nov. is characterized by solitary cells without spines, lanceolate valves with slightly rostrate apices, a narrow, linear axial area, and a large, unilateral central area. Fragilaria rinoi sp. nov. may be confused with F. microvaucheriae in terms of length, striae density and outline, although a morphometric analysis revealed that F. rinoi sp. nov. is significantly wider. Fragilaria rinoi sp. nov. is present in rivers with high dissolved oxygen concentrations, medium to high conductivity, neutral to slightly alkaline pH and high mean values of nitrates and ammonium.

Aljustrel mines were classified as having high environmental hazard due to their large tailings volume and high metal concentrations in waters and sediments. To assess acid mine drainage impacted systems whose environmental conditions change quickly, the use of biological indicators with short generation time such as diatoms is advantageous. This study combined geochemical and diatom data, whose results were highlighted in 3 groups: Group 1, with low pH (1.9-5.1) and high metal/metalloid (Al, As, Cd, Co, Cu, Fe, Mn, Ni, Pb, Zn; 0.65-1032 mg/L) and SO4 (405-39124 mg/L) concentrations. An acidophilic species, Pinnularia aljustrelica, was perfectly adapted to the adverse conditions; in contrast, teratological forms of Eunotia exigua were found, showing that metal toxicity affected this species. The low availability of metals/metalloids in sediments of this group indicates that metals/metalloids of the exchangeable fractions had been solubilized, which in fact enables metal/metalloid diatom uptake and consequently the occurrence of teratologies; Group 2, with sites of near neutral pH (5.0-6.8) and intermediate metal/metalloid (0.002-6 mg/L) and SO4 (302-2179 mg/L) concentrations; this enabled the existence of typical species of uncontaminated streams (Brachysira neglectissima, Achnanthidium minutissimum); Group 3, with samples from unimpacted sites, showing low metal/metalloid (0-0.8 mg/L) and SO4 (10-315 mg/L) concentrations, high pH (7.0-8.4) and Cl contents (10-2119 mg/L) and the presence of brackish to marine species (Entomoneis paludosa). For similar conditions of acidity, differences in diversity, abundance and teratologies of diatoms can be explained by the levels of metals/metalloids. (C) 2015 The Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences. Published by Elsevier B.V.

The ubiquity of diatom distribution, species richness, short generation time, and specific sensitivity to several environmental stressors such as metals, make diatoms particularly useful for scientific studies. Anthropogenic activities have increased the concentration of metals in air, soil and water. Due to their toxicity and persistent character, the effects of metals on organisms have been extensively studied. In this work, the association of cadmium to different extracellular molecules of Nitzschia palea cells was investigated. Cells were grown in the absence and presence (0.2 mg l(-1)) of cadmium in Chu no. 10 medium. Extracellular polysaccharides were extracted, and subsamples were used for polysaccharide and Cd determination. The frustules were broken mechanically under liquid nitrogen and the intracellular and frustule fractions separated. Frustulins, a protein family found on the outmost frustule layer, constituting a protection coating to environmental stress, were extracted. In each fraction proteins were quantified by the BCA method and separated by gel electrophoresis (SDS-PAGE). Cadmium associated to each fraction was quantified by Inductively Coupled Plasma Mass Spectrometry (ICP-MS) analysis. Exposure of Nitzschia palea to cadmium decreased extracellular polysaccharides by 52.8 % and increased 6 times the amount of frustulins. Cadmium was mostly retained extracellularly: 85.4 % was bound to the frustulin fraction, and 11.1 % to polysaccharides. The ability of Nitzschia palea to increase the production of frustulins due to the presence of Cd, the extracellular location of this frustulin coating and the ability of these proteins to bind Cd, suggests a new cellular defense mechanism to metals unknown until now.

According to the Water Framework Directive phytobenthos (diatoms) and macrophytes are part of the same biological element, the aquatic flora, which should be used in monitoring programs for rivers ecological assessment. However, they have been treated separately over time, and different indices are used to reach an Ecological Quality Status. Here we build a predictive model (the AQUAFLORA) based on communities composed of diatoms and macrophytes, with the aim of determining if these two elements of the aquatic flora could be combined in a single model to evaluate the streams ecological quality. For that we used biological and environmental information from 200 reference and 171 disturbed sites distributed through the entire Portuguese mainland territory. After clustering analysis, 5 biological reference groups significantly different (ANOSIM Global R = 0.685; p

Biosilica from diatoms is formed at ambient conditions under the control of biological and physicochemical processes. The changes in growth and biosilica formation through uptake of different concentrations of Cd2+ by the diatom Nitzschia palea (Kutzing) W. Smith was investigated, correlating Cd2+ effects to changes in the biosilica nanostructure and the relative content of the encapsulated biomolecules. Diatom growth rates at different Cd2+ concentrations (as 1, 2, 3, 4, and 5 X 10-1 mg L-1 CdCl2) were studied in order to determine the concentrations at which sublethal effects were visible, allowing the harvest of sufficient diatom cells for further experiments. We found a clear correlation between the Cd2+ concentrations and both the nanostructure of the biosilica and content of encapsulated peptides. Cd2+ induced biosilica deformation was assessed by scanning electron microscopy and attenuated total reflectance-Fourier Transformed Infrared Spectroscopy (FTIR), revealing that micromorphological changes in frustule features (striae, costae, pores) and nanostructural modifications (structure of the silica and conformation of the encapsulated peptides) occurred at applied Cd2+ concentrations of 2 and 3 X 10-1 mg L-1. In particular the FTIR contribution of peptides decreased at elevated Cd2+ concentrations, whereas shifts in wave number of several relevant organic bonds as C = O stretching (1765 cm-1) and possibly hydrated sulfate (1160, 1110 and 980 cm-1) were assigned. Additional analysis of the amide I band showed a relative increase in beta-sheet structure (16801620 cm-1) when Cd2+ concentration increased. Cadmium uptake clearly affected the molecular ordering of the biosilica in Nitzschia palea, most probably by interfering in biological or physicochemical processes involved in diatom biosilicification.

To understand whether seasons influence the ecological quality assessment of streams on the basis of diatoms, a study was undertaken in two lowland water courses located in northwest Portugal, between autumn 2008 and summer 2009. Temporal variation in the chemical pollution of these streams was small as revealed by a number of physical and chemical parameters analyzed. PERMANOVA global test revealed significant variation in water temperature among seasons. The diatom communities also showed a temporal variation although not all the seasons were statistically different. The multidimensional scaling analysis showed that the main differences in the diatom communities were between two groups of seasons: autumn/winter and spring/summer. Species such as Cocconeis pseudolineata, Gomphonema parvulum var. exilissimum, Fragilaria vaucheriae, Encyonema minutum, and Nitzschia recta were more abundant in spring/summer, while species such as Mayamaea atomus and Nitzschia pusilla were more abundant in autumn/winter. The BIOENV routine confirmed that the biological and temperature patterns are highly correlated. Despite the effects observed on diatom communities, these differences were buffered by the EQR (IPS) values which do not reflect seasonal differences. Therefore, the use of the index IPS seems to allow the monitoring of the streams’ ecological quality throughout the year without the interference of the natural temporal variability of diatom communities.

A predictive model for diatoms based on an adaptation of the River Invertebrate Prediction and Classification System/Australian River Assessment System approaches was evaluated as an effective tool for measuring stream ecological quality. This type of model was originally developed in UK and later in Australia and is extensively used to obtain ecological quality assessments with macroinvertebrates. The first step for the model construction was the definition of six consistent reference biological groups (ANOSIM: Global R = 0.77; P

A new benthic freshwater diatom species belonging to the genus Pinnularia Ehrenb. has been recorded in the Agua Forte stream surrounding the Aljustrel mining area in southern Portugal. Pinnularia aljustrelica Luis, Almeida et Ector sp. nov. is described as a new species based on light and scanning electron microscopy observations and on its particular habitat in an acidic environment due to acid mine drainage, high metal concentrations (As, Cd, Cu, Fe, Mn, Pb and Zn) and high sulphate and conductivity. The taxa most similar to P. aljustrelica are P. acidophila Hofmann et Krammer, P. acoricola Hustedt and P. acoricola var. lanceolata Hustedt, so type materials of these taxa were studied for comparative purposes. Although, the ecology of the three similar taxa is also quite similar, the new species has a combination of particular morphological characteristics studied under LM and SEM that separates it from the rest. Pinnularia aljustrelica has a general shape relatively similar to P. acidophila and P. acoricola, but the valve outline is not as linear as in P. acidophila and not as oval as in P. acoricola.

Lousal mine is a typical “abandoned mine” with all sorts of problems as consequence of the cessation of the mining activity and lack of infrastructure maintenance. The mine is closed at present, but the heavy metal enriched tailings remain at the surface in oxidizing conditions. Surface water and stream sediments revealed much higher concentrations than the local geochemical background values, which the “Contaminated Sediment Standing Team” classifies as very toxic. High concentrations of Cu, Pb, Zn, As, Cd and Hg occurred within the stream sediments downstream of the tailings sites (up to: 817 mg kg −1 As, 6.7 mg kg −1 Cd, 1568 mg kg −1 Cu, 1059 mg kg −1 Pb, 82.4 mg kg −1 Sb, 4373 mg kg −1 Zn). The AMD waters showed values of pH ranging from 1.9 to 2.9 and concentrations of 9249 to 20,700 mg L −1 SO 4 −2 , 959 to 4830 mg L −1 Fe and 136 to 624 mg L −1 Al. Meanwhile, the acid effluents and mixed stream waters also carried high contents of SO 4 2−, Fe, Al, Cu, Pb, Zn, Cd, and As, generally exceeding the Fresh Water Aquatic Life Acute Criteria. Negative impacts in the diatom communities growing at different sites along a strong metal pollution gradient were shown through Canonical Correspondence Analysis: in the sites influenced by Acid Mine Drainage (AMD), the dominant taxon was Achnanthidium minutissimum . However, Pinnularia acoricola was the dominant species when the environmental conditions were extremely adverse: very low pH and high metal concentrations (sites 2 and 3). Teratological forms of Achnanthidium minutissimum (Kutzing) Czarnecki, Brachysira vitrea (Grunow) Ross in Hartley, Fragilaria rumpens (Kutzing) G. W. F. Carlson and Nitzschia hantzschiana Rabenhorst were found. A morphometric study of B. vitrea showed that a decrease in size was evident at the most contaminated sites. These results are evidence of metal and acidic pollution.

Gomphonema specimens with wide axial areas collected from streams in Northern Portugal were examined by light and scanning electron microscopy. Three species were found and identified as G. rhombicum, G. uniserhombicum and G. ibericum. No specimens were found with the features of G. clevei sensu stricto, and previous records from the region with the latter name are regarded as representing one or a mixture of the three species examined herein. The three species differed in the fine details of the striae; double alternating poroids in G. rhombicum, slit-like with irregular outlines in G. uniserhombicum and single or divided slit-like poroids in G. ibericum. Differences in size and shape of the valve and axial area allow discrimination of at least medium-sized to large specimens of the three species. Gomphonema rhombicum was the most common species, sometimes occurring as dominant in the epilithon. Both G. uniserhombicum and G. ibericum were found more sparsely and always in communities containing G. rhombi...