Escherichia coli Phylogenetic and Antimicrobial Pattern as an Indicator of Anthropogenic Impact on Threatened Freshwater Mussels.

Freshwater bivalves are widely used as accumulation indicators and monitoring tools for assessing contaminant effects on different levels of biological integration. This pilot study aimed to explore the phylogenetic diversity of Escherichia coli isolated from freshwater mussels (Margaritifera margaritifera and Potomida littoralis) and characterize their phenotypes and antibiotic resistance profiles. Samples were collected in the Rabaçal and Tua Rivers, in the Douro basin, Portugal-two sites representing different levels of anthropogenic contamination. Antimicrobial susceptibility testing was performed via the disk diffusion method with 21 antibiotics. Results showed that 31% of isolates were multidrug-resistant (MDR). Thus, freshwater mussels provide an effective and time-integrated approach for identifying/quantifying fecal indicators, including MDR bacteria. PCR-based assays were designed for assessing phylogenetic E. coli groups. Among the E. coli isolates, the highest prevalence (44%) was observed in group D or E, followed by group E or Clade I (25%), group A (19%), and group B1 (13%). E. coli isolated from M. margaritifera predominantly exhibited a higher prevalence of phylogroups D or E, whereas E. coli from P. littoralis showed associations with phylogroups E or clade I, B1, A, and D or E. Our results provide new insights into the phylogenetic diversity of E. coli in freshwater bivalves. Additionally, the findings highlight the possible linkage of phylogroups with the host species, the geographical location in the water stream, and human activity. Using E. coli as a bioindicator isolated from freshwater mussels helps us grasp how human activities affect the environment. This study has important implications for those interested in safeguarding water resources, especially in tackling antibiotic resistance in aquatic ecosystems.

Streams in the Mediterranean Region are not for mussels: Predicting extinctions and range contractions under future climate change.

Climate change is becoming the leading driver of biodiversity loss. The Mediterranean region, particularly southwestern Europe, is already confronting the consequences of ongoing global warming. Unprecedented biodiversity declines have been recorded, particularly within freshwater ecosystems. Freshwater mussels contribute to essential ecosystem services but are among the most threatened faunal groups on Earth. Their poor conservation status is related to the dependence on fish hosts to complete the life cycle, which also makes them particularly vulnerable to climate change. Species Distribution Models (SDMs) are commonly used to predict species distributions, but often disregard the potential effect of biotic interactions. This study investigated the potential impact of future climate on the distribution of freshwater mussel species while considering their obligatory interaction with fish hosts. Specifically, ensemble models were used to forecast the current and future distribution of six mussel species in the Iberian Peninsula, including environmental conditions and the distribution of fish hosts as predictors. We found that climate change is expected to severely impact the future distribution of Iberian mussels. Species with narrow ranges, namely Margaritifera margaritifera and Unio tumidiformis, were predicted to have their suitable habitats nearly lost and could potentially be facing regional and global extinctions, respectively. Anodonta anatina, Potomida littoralis, and particularly Unio delphinus and Unio mancus, are expected to suffer distributional losses but may gain new suitable habitats. A shift in their distribution to new suitable areas is only possible if fish hosts are able to disperse while carrying larvae. We also found that including the distribution of fish hosts in the mussels' models avoided the underprediction of habitat loss under climate change. This study warns of the imminent loss of mussel species and populations and the urgent need of management actions to reverse current trends and mitigate irreversible damage to species and ecosystems in Mediterranean regions.

Identifying freshwater priority areas for cross-taxa interactions.

Information about biotic interactions (e.g. competition, predation, parasitism, diseases, mutualism, allelopathy) is fundamental to better understand species distribution and abundance, ecosystem functioning, and ultimately guide conservation efforts. However, conservation planning often overlooks these important interactions. Here, we aim to demonstrate a new framework to include biotic interactions into Marxan. For that, we use freshwater mussels and fish interaction (as mussels rely on fishes to complete their life cycle) in the Douro River basin (Iberian Peninsula) as a case study. While doing that, we also test the importance of including biotic interactions into conservation planning exercises, by running spatial prioritisation analysis considering either: 1) only the target species (freshwater mussels); 2) freshwater mussels and their obligatory hosts (freshwater fishes); 3) freshwater mussels, fishes and their interactions. With this framework we found that biotic interactions tend to be underrepresented when the data on both freshwater mussels and fishes is not simultaneously included in the spatial prioritisation. Overall, the priority areas selected across all scenarios are mostly located in the western part of the Douro River basin, where most freshwater mussels and fishes still occur. Given the low overlap of priority areas identified here and the current Natura 2000 network, our approach may be useful for establishing (or enlarging) protected areas, especially in light of the EU Biodiversity Strategy for 2030. Also, this work may provide guidance for future habitat restoration and management of main threats to freshwater biodiversity.

A roadmap for the conservation of freshwater mussels in Europe.

Europe has a long history of human pressure on freshwater ecosystems. As pressure continues to grow and new threats emerge, there is an urgent need for conservation of freshwater biodiversity and its ecosystem services. However, whilst some taxonomic groups, mainly vertebrates, have received a disproportionate amount of attention and funds, other groups remain largely off the public and scientific radar. Freshwater mussels (Bivalvia, Unionida) are an alarming example of this conservation bias and here we point out six conceptual areas that need immediate and long-term attention: knowledge, threats, socioeconomics, conservation, governance and education. The proposed roadmap aims to advance research, policy and education by identifying the most pressing priorities for the short- and long-term conservation of freshwater mussels across Europe.

Multiresistant bacteria: Invisible enemies of freshwater mussels.

Freshwater mussels are among the most endangered groups of fauna anywhere in world. The indiscriminate use of antibiotics has led to the emergence of resistant strains. These antibiotic-resistant bacteria play a key role in increasing the risk allied with the use of surface water and in spread of resistance genes. Two endangered freshwater mussel species, Margaritifera margaritifera and Potomida littoralis, were sampled at 4 sampling sites along a 50 km stretch of River Tua. Water samples were taken at same sites. Of the total of 135 isolates, 64.44% (39.26% from water and 25.19% from mussels) were coliform bacteria. Site T1, with the lowest concentration of coliform bacteria, and site T2 were the only ones where M. margaritifera was found. No E. coli isolates were found in this species and the pattern between water and mussels was similar. P. littoralis, which was present at T3/T4 sites, is the one that faces the highest concentration of bacterial toxins, which are found in treated wastewater effluents and around population centers. Sites T3/T4 have the isolates (water and mussels) with the highest resistance pattern, mainly to ß-lactams. Water and P. littoralis isolates (T3/T4) showed resistance to penicillins and their combination with clavulanic acid, and to cephalosporins, precisely to a fourth generation of cephalosporin antibiotics. The analysis provides important information on the risk to water systems, as well as the need to investigate possible management measures. It is suggested that future studies on the health status of freshwater bivalves should incorporate measures to indicate bacteriological water quality.

Alarming decline of freshwater trigger species in western Mediterranean key biodiversity areas.

Theidentification of key biodiversity areas (KBA) was initiated by the International Union for Conservation of Nature in 2004 to overcome taxonomic biases in the selection of important areas for conservation, including freshwater ecosystems. Since then, several KBAs have been identified mainly based on the presence of trigger species (i.e., species that trigger either the vulnerability and or the irreplaceability criterion and thus identify a site as a KBA). However, to our knowledge, many of these KBAs have not been validated. Therefore, classical surveys of the taxa used to identify freshwater KBAs (fishes, molluscs, odonates, and aquatic plants) were conducted in Douro (Iberian Peninsula) and Sebou (Morocco) River basins in the Mediterranean Biodiversity Hotspot. Environmental DNA analyses were undertaken in the Moroccan KBAs. There was a mismatch between the supposed and actual presence of trigger species. None of the trigger species were found in 43% and 50% of all KBAs surveyed in the Douro and Sebou basins, respectively. Shortcomings of freshwater KBA identification relate to flawed or lack of distribution data for trigger species. This situation results from a misleading initial identification of KBAs based on poor (or even inaccurate) ecological information or due to increased human disturbance between initial KBA identification and the present. To improve identification of future freshwater KBAs, we suggest selecting trigger species with a more conservative approach; use of local expert knowledge and digital data (to assess habitat quality, species distribution, and potential threats); consideration of the subcatchment when delineating KBAs boundaries; thoughtful consideration of terrestrial special areas for conservation limits; and periodic field validation.

Alarming decline of freshwater trigger species in western Mediterranean Key Biodiversity Areas Resumen La identificación de las áreas clave de biodiversidad (ACB) fue iniciada por la Unión Internacional para la Conservación de la Naturaleza en 2004 con el objetivo de sobreponerse a los sesgos taxonómicos en la selección de áreas importantes para la conservación, incluyendo los ecosistemas de agua dulce. Desde entonces, varias ACB han sido identificadas principalmente con base en la presencia de especies desencadenantes (es decir, especies que desencadenan el criterio de vulnerabilidad o de carácter irremplazable y por lo tanto identifican a un sitio como una ACB). Sin embargo, a nuestro conocimiento, muchas de estas ACB no han sido validadas. Por lo tanto, los censos clásicos de taxones utilizados para identificar las ACB de agua dulce (peces, moluscos, odonatos y plantas acuáticas) fueron realizados en las cuencas de los ríos Duero (Península Ibérica) y Sebou (Marruecos) en el Punto Caliente de Biodiversidad del Mediterráneo. Realizamos análisis de ADN ambiental en las ACB de Marruecos. Hubo una discrepancia entre la supuesta presencia y la actual presencia de especies desencadenantes. Ninguna de las especies desencadenantes se encontró en 43% y 50% de las ACB censadas en las cuencas del Duero y del Sebou, respectivamente. Las deficiencias en la identificación de las ACB de agua dulce están relacionadas con la carencia de datos o datos erróneos sobre la distribución de las especies desencadenantes. Esta situación resulta en una identificación inicial engañosa de las ACB con base en información ecológica deficiente (o incluso incorrecta) o también puede deberse al incremento en las perturbaciones humanas ocurridas entre la identificación de la ACB y el presente. Para mejorar la identificación de ACB de agua dulce en el futuro, sugerimos que la selección de especies desencadenantes se realice con un enfoque más conservador; que se usen el conocimiento local de los expertos y los datos digitales (para evaluar la calidad del hábitat, la distribución de las especies y las amenazas potenciales); que se consideren las subcuencas cuando se delimiten las fronteras de las ACB; que se consideren cuidadosamente las áreas de especies terrestres para los límites de conservación; y que se realicen validaciones periódicas de campo.

Distribution and Potential Availability of As, Metals and P in Sediments from a Riverine Reservoir in a Rural Mountainous Catchment (NE Portugal).

A geochemical investigation was carried out on the bottom sediments of a riverine reservoir, located in a mountainous rural region (NE Portugal), with the aim of evaluating the contents of As, metals and P and their potential availability. The elements contents were detected in the following ranges (µg g-1): As (18-64); Cr (32-128); Cu (39-93); Ni (18-80); Pb (49-160); Zn (207-334); P (1705-2681). The reducible fraction is the most significant in the retention of the elements. Based on their potential relative mobility, the detected metals could be classed as follows: Zn > As, Pb > Cu > Cr, Ni. The results on geochemical partitioning were revealed to be important when the Sediment Quality Guidelines (SQGs) were considered. Arsenic, Cr, Ni, Pb and Zn showed total contents exceeding the values of Probable Effect Level (PEL), but only As occurred in the most potentially available form; Cr and Ni can be considered relatively unavailable, since these are mainly associated with the residual phase. Locally, oxygen depletion could release P into the water column due to the higher concentrations in Fe-P and CDB-P fractions. The potential availability of As, metals and P in sediments indicates that the quality of sediments accumulated in small reservoirs should be considered in management policies.

The role of anthropogenic habitats in freshwater mussel conservation.

Anthropogenic freshwater habitats may provide undervalued prospects for long-term conservation as part of species conservation planning. This fundamental, but overlooked, issue requires attention considering the pace that humans have been altering natural freshwater ecosystems and the accelerated levels of biodiversity decline in recent decades. We compiled 709 records of freshwater mussels (Bivalvia, Unionida) inhabiting a broad variety of anthropogenic habitat types (from small ponds to large reservoirs and canals) and reviewed their importance as refuges for this faunal group. Most records came from Europe and North America, with a clear dominance of canals and reservoirs. The dataset covered 228 species, including 34 threatened species on the IUCN Red List. We discuss the conservation importance and provide guidance on how these anthropogenic habitats could be managed to provide optimal conservation value to freshwater mussels. This review also shows that some of these habitats may function as ecological traps owing to conflicting management practices or because they act as a sink for some populations. Therefore, anthropogenic habitats should not be seen as a panacea to resolve conservation problems. More information is necessary to better understand the trade-offs between human use and the conservation of freshwater mussels (and other biota) within anthropogenic habitats, given the low number of quantitative studies and the strong biogeographic knowledge bias that persists.

Water security and watershed management assessed through the modelling of hydrology and ecological integrity: A study in the Galicia-Costa (NW Spain).

Water management is a crucial tool for addressing the increasing uncertainties caused by climate change, biodiversity loss and the conditions of socioeconomic limits. The multiple factors affecting water resources need to be successfully managed to achieve optimal governance and thus move towards water security. This study seeks to obtain a holistic vision of the various threats that affect the ecological integrity of the basins that form the hydrological district of Galicia-Costa, through the method of partial least squares path modelling (PLS-PM). The data is analysed overall for the hydrological years from 2009 to 2015. The independent latent variables are "Anthropogenic" (comprising the percentage of water bodies with edges alongside artificial surfaces, the percentage connected to artificial land use patches, the edge density of artificial surfaces and population density) and "Nature" (edge density of forestry land uses, edge length of land water bodies alongside forested areas and the percentage of land occupied by the largest patch of forest). The dependent latent variables are "SWP", which represents surface water parameters (biological oxygen demand, chlorides, conductivity and dissolved iron) and "Ecological Integrity" (METI Bioindicator). The connections between latent variables are uantified through path coefficients (ß). From an overall perspective, the PLS-PM results reveal that 69.0% of "SWP" is predicted by the independent variables (R2 = 0.690), "Anthropogenic" contributes by increasing SWP (ß = 0.471), while "Nature" decreases the concentration of SWP (ß = -0.523), which indicates the polluting parameters in the water. The variables "Anthropogenic" (ß = -0.351) and "SWP" (ß = -0.265) lower the quality of "Ecological Integrity". This variable must be managed through soil conservation measures for the benefit of water security. This study has been able to identify and quantify the variables that increase contaminant concentration and decrease ecological integrity, providing a promising methodology that facilitates protection and correction measures to guarantee water safety.

Small hydropower plants as a threat to the endangered pearl mussel Margaritifera margaritifera.

Freshwater mussels are undergoing rapid global declines due to habitat loss and fragmentation, among other factors, but little is known about the effects of small hydropower plants. Here we assessed the impact of small hydropower plants on the abundance and size structure of the imperilled pearl mussel Margaritifera margaritifera. For this, we sampled 66 sites in three Portuguese rivers (Mente, Rabaçal and Tuela) located upstream and downstream of dams and within the reservoirs. Pearl mussels were significantly more abundant upstream than downstream of dams (97.4% more) or within reservoirs (98.5% more). In addition, juveniles were mostly found upstream of dams. The most significant environmental alterations that explained the observed patterns were related to changes in sediment characteristics (accumulation of fine sediments and organic matter in reservoirs) and water chemistry, most notably suspended solids (highest values in reservoirs) and dissolved oxygen (lowest values in reservoirs). Overall, results show that small hydropower plants can deeply affect pearl mussel populations: specimens almost disappeared from the areas within the reservoirs and sites located downstream only retained adults without signs of recent recruitment. Future management measures devoted to the conservation of pearl mussels should take into account the results reported here to avoid the construction of new dams in pearl mussel rivers; improve management of the river flow in downstream areas; and consider the decommissioning of dams in pearl mussel rivers with a particular attention devoted to the re-naturalization of river sections under the influence of the reservoir and downstream areas.

Mesozoic mitogenome rearrangements and freshwater mussel (Bivalvia: Unionoidea) macroevolution.

Using a new fossil-calibrated mitogenome-based approach, we identified macroevolutionary shifts in mitochondrial gene order among the freshwater mussels (Unionoidea). We show that the early Mesozoic divergence of the two Unionoidea clades, Margaritiferidae and Unionidae, was accompanied by a synchronous split in the gene arrangement in the female mitogenome (i.e., gene orders MF1 and UF1). Our results suggest that this macroevolutionary jump was completed within a relatively short time interval (95% HPD 201-226 Ma) that coincided with the Triassic-Jurassic mass extinction. Both gene orders have persisted within these clades for ~200 Ma. The monophyly of the so-called "problematic" Gonideinae taxa was supported by all the inferred phylogenies in this study using, for the first time, the M- and F-type mitogenomes either singly or combined. Within Gonideinae, two additional splits in the gene order (UF1 to UF2, UF2 to UF3) occurred in the Mesozoic and have persisted for ~150 and ~100 Ma, respectively. Finally, the mitogenomic results suggest ancient connections between freshwater basins of East Asia and Europe near the Cretaceous-Paleogene boundary, probably via a continuous paleo-river system or along the Tethys coastal line, which are well supported by at least three independent but almost synchronous divergence events.

A tale of shells and claws: The signal crayfish as a threat to the pearl mussel Margaritifera margaritifera in Europe.

The freshwater pearl mussel Margaritifera margaritifera is a highly threatened species in Europe. Several mechanisms may be responsible for the decline in distribution and abundance of European pearl mussel populations, but almost no quantitative data exists about the possible negative impacts of invasive alien species (IAS). In this study, we clearly demonstrate that the invasive signal crayfish Pacifastacus leniusculus predates pearl mussels, using a laboratorial experiment followed by in situ validation in four rivers in the North of Portugal (Mente, Rabaçal, Tuela and Baceiro Rivers; Douro Basin). In the laboratory, the crayfish had a clear preference for small-sized pearl mussels but no differences in predation were found in mesocosms with and without sediment. In addition, we clearly demonstrated that the signal crayfish predates pearl mussels in natural conditions and detected a significant density dependent effect (i.e., sites with more crayfish presented higher number of pearl mussel shells with marks of predation). Given the recent introduction of the signal crayfish and the potential negative impacts on pearl mussel populations we also investigated its autoecology (distribution, abundance, size structure and sex-ratio) in the four studied rivers. Significant differences in average abundance and size of the crayfish were detected between sites and the sex-ratio was highly skewed to females. In view of the widespread distribution of signal crayfish (and other invasive crayfish species) in Europe, future management actions devoted to the conservation of pearl mussels should take in consideration the possible negative effects of these predators, especially on juveniles.

A Gill Histopathology Study in two Native Fish Species from the Hydrographic Douro Basin.

Gill histopathology is a valuable tool to evaluate ecosystems health, because the gill is a key site of waterborne pollutant uptake and the first target organ to their toxicity. Consequently, this makes it important in biomonitoring programs. This study aims to evaluate gill histopathological differences in Douro basin native fish species and determine possible associations with water quality and the ecological status classifications. Two native fish species (Pseudochondrostoma duriense and Luciobarbus bocagei) were sampled in four points of the Douro basin: two reference points, Ameixiosa (Paiva River) and Covelas (Bestança River), both classified with an excellent ecological status; and two disturbed points, Castro Daire (Paiva River) and Alvações do Corgo (Corgo River), categorized with a good and a moderate ecological status, respectively. Gill histopathological differences were qualitative and quantitatively analyzed. The histological analysis showed that, in all sampling locations, both species presented some degree of gill differences, such as epithelial lifting, lamellar fusion, and/or necrosis. The histopathological differences evaluation emphasized some variances in the responses between the two species. In nase, the filament and lamellar epithelium proliferation were the histopathological differences that better reflected the river ecological status classification, proving their usefulness in biomonitoring programs.

Acoustic barriers as an acoustic deterrent for native potamodromous migratory fish species.

This study focused on the use of sound playbacks as acoustic deterrents to direct native potamodromous migratory species away from all kind of traps. The effects of two acoustic treatments, a repeated sine sweep up to 2 kHz (sweep-up stimulus) and an intermittent 140 Hz tone, were tested in three fish species native to Iberia: Salmo trutta, Pseudochondrostoma duriense and Luciobarbus bocagei. In contrast with S. trutta, the endemic cyprinids P. duriense and L. bocagei exhibited a strong repulse reaction to the frequency sweep-up sound. The 140 Hz stimulus did not seem to alter significantly the behaviour of any of the studied species. These results highlight the potential of acoustic stimuli as fish behavioural barriers and their application to in situ conservation measures of native Iberian fish populations, to protect them from hydropower dams. In addition, this study shows that acoustic deterrents can be used selectively on target species.

Invasive crayfishes as a threat to freshwater bivalves: Interspecific differences and conservation implications.

Freshwater bivalves have suffered major global declines, being the introduction of invasive alien species (IAS) an important, but not well studied, mechanism of threat. This study assessed the predator-prey relationship between two non-native crayfish species (Procambarus clarkii and Pacifastacus leniusculus) and three native (Anodonta anatina, Potomida littoralis and Unio delphinus) and one non-native (Corbicula fluminea) freshwater bivalve species through experiments in laboratory and validation under natural conditions (Sabor River basin, Portugal). All native bivalve species were preyed both in laboratory and in the field; however, both crayfish species were unable to prey C. fluminea. Predation was dependent on crayfish and bivalve species but was not affected neither by crayfish nor bivalve sizes. In the laboratory, the most preyed species by both crayfishes was A. anatina. On average, this species was preyed at least 12% more than other species, when crayfishes had a choice. Similar results were found in the field. We also found signs of competition between both crayfishes, being P. clarkii more dominant and aggressive as this species, on average, manipulated the bivalves 63.6% more times and 24:33 min longer than P. leniusculus, and initiated 55.8% more agnostic bouts. Our results support the idea that P. clarkii and P. leniusculus can affect native freshwater bivalves, but clear interspecific differences were detected. Both crayfishes may have direct and indirect impacts on bivalve populations by increasing mortality or by reducing their fitness. In addition, since both crayfishes do not prey C. fluminea, they offer this IAS another advantage over native bivalves. Given the widespread distribution of both P. clarkii and P. leniusculus and the threatened status of many freshwater bivalves, the dynamics and impacts of this relationship should be taken in account in the implementation of management measures devoted to the conservation of native freshwater bivalves.

Expansion and systematics redefinition of the most threatened freshwater mussel family, the Margaritiferidae.

Two Unionida (freshwater mussel) families are present in the Northern Hemisphere; the Margaritiferidae, representing the most threatened of unionid families, and the Unionidae, which include several genera of unresolved taxonomic placement. The recent reassignment of the poorly studied Lamprotula rochechouartii from the Unionidae to the Margaritiferidae motivated a new search for other potential species of margaritiferids from members of Gibbosula and Lamprotula. Based on molecular and morphological analyses conducted on newly collected specimens from Vietnam, we here assign Gibbosula crassa to the Margaritiferidae. Additionally, we reanalyzed all diagnostic characteristics of the Margaritiferidae and examined museum specimens of Lamprotula and Gibbosula. As a result, two additional species are also moved to the Margaritiferidae, i.e. Gibbosula confragosa and Gibbosula polysticta. We performed a robust five marker phylogeny with all available margaritiferid species and discuss the taxonomy within the family. The present phylogeny reveals the division of Margaritiferidae into four ancient clades with distinct morphological, biogeographical and ecological characteristics that justify the division of the Margaritiferidae into two subfamilies (Gibbosulinae and Margaritiferinae) and four genera (Gibbosula, Cumberlandia, Margaritifera, and Pseudunio). The systematics of the Margaritiferidae family is re-defined as well as their distribution, potential origin and main biogeographic patterns.

Conservation status of freshwater mussels in Europe: state of the art and future challenges.

Freshwater mussels of the Order Unionida provide important ecosystem functions and services, yet many of their populations are in decline. We comprehensively review the status of the 16 currently recognized species in Europe, collating for the first time their life-history traits, distribution, conservation status, habitat preferences, and main threats in order to suggest future management actions. In northern, central, and eastern Europe, a relatively homogeneous species composition is found in most basins. In southern Europe, despite the lower species richness, spatially restricted species make these basins a high conservation priority. Information on freshwater mussels in Europe is unevenly distributed with considerable differences in data quality and quantity among countries and species. To make conservation more effective in the future, we suggest greater international cooperation using standardized protocols and methods to monitor and manage European freshwater mussel diversity. Such an approach will not only help conserve this vulnerable group but also, through the protection of these important organisms, will offer wider benefits to freshwater ecosystems.

Phylogeny of the most species-rich freshwater bivalve family (Bivalvia: Unionida: Unionidae): Defining modern subfamilies and tribes.

Freshwater mussels of the order Unionida are key elements of freshwater habitats and are responsible for important ecological functions and services. Unfortunately, these bivalves are among the most threatened freshwater taxa in the world. However, conservation planning and management are hindered by taxonomic problems and a lack of detailed ecological data. This highlights the urgent need for advances in the areas of systematics and evolutionary relationships within the Unionida. This study presents the most comprehensive phylogeny to date of the larger Unionida family, i.e., the Unionidae. The phylogeny is based on a combined dataset of 1032bp (COI+28S) of 70 species in 46 genera, with 7 of this genera being sequenced for the first time. The resulting phylogeny divided the Unionidae into 6 supported subfamilies and 18 tribes, three of which are here named for the first time (i.e., Chamberlainiini nomen novum, Cristariini nomen novum and Lanceolariini nomen novum). Molecular analyses were complemented by investigations of selected morphological, anatomical and behavioral characters used in traditional phylogenetic studies. No single morphological, anatomical or behavioral character was diagnostic at the subfamily level and few were useful at the tribe level. However, within subfamilies, many tribes can be recognized based on a subset of these characters. The geographical distribution of each of the subfamilies and tribes is also presented. The present study provides important advances in the systematics of these extraordinary taxa with implications for future ecological and conservation studies.

The role of calcium concentration in the invasive capacity of Corbicula fluminea in crystalline basins.

The natural variation of environmental factors in freshwater basins determines their biodiversity. Among them, calcium is a key physiological compound for freshwater invertebrates. It is required for shell formation, muscle contraction, it mediates gene expression and allows counteracting acidosis during stress periods, among other functions. Although the distribution of different freshwater species has been suggested to be linked with the environmental calcium concentration, as yet, no research studies have confirmed this. Identifying whether environmental calcium concentrations might determine the invasion success of alien species would be critical in developing and implementing effective management strategies to control them. Here, a multidisciplinary approach integrating field surveys, analytical chemistry techniques, molecular biology analyses and a lab-scale experiment was taken to decipher whether the environmental calcium concentration might hamper the establishment of Corbicula fluminea in northwestern Iberian rivers. A Principal Component Analysis on water chemistry variables from 13 water bodies identified environmental calcium concentration, among others, as one key factor that best characterized the distribution area of C. fluminea. The calcium content in animals' bodies from two representative rivers was dependent on the environmental calcium concentration of freshwater basins; the lower the concentration, the lower the body's content. The expression of stress- and calcium homeostasis-related genes was higher in C. fluminea from low calcium concentration environments than in those from calcium-rich freshwater basins. Finally, under experimental conditions, lower water calcium concentrations decreased C. fluminea growth rates. The present data suggest, for the first time, that environmental calcium concentration may act as a determinant factor on the invasion success of C. fluminea in freshwater environments. Our results provide new clues for the identification of basins with increased risk of potential invasion by C. fluminea based on environmental calcium levels.

A multiple index integrating different levels of organization.

Many methods in freshwater biomonitoring tend to be restricted to a few levels of biological organization, limiting the potential spectrum of measurable of cause-effect responses to different anthropogenic impacts. We combined distinct organisational levels, covering biological biomarkers (histopathological and biochemical reactions in liver and fish gills), community based bioindicators (fish guilds, invertebrate metrics/traits and chironomid pupal exuviae) and ecosystem functional indicators (decomposition rates) to assess ecological status at designated Water Framework Directive monitoring sites, covering a gradient of human impact across several rivers in northern Portugal. We used Random Forest to rank the variables that contributed more significantly to successfully predict the different classes of ecological status and also to provide specific cut levels to discriminate each WFD class based on reference condition. A total of 59 Biological Quality Elements and functional indicators were determined using this procedure and subsequently applied to develop the integrated Multiple Ecological Level Index (MELI Index), a potentially powerful bioassessment tool.