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.

Diversity, biogeography, evolutionary relationships, and conservation of Eastern Mediterranean freshwater mussels (Bivalvia: Unionidae).

Located at the junction between Europe, Africa, and Asia, with distinct evolutionary origins and varied ecological and geographical settings, together with a marked history of changes in orogeny and configuration of the main river basins, turned the Eastern Mediterranean into a region of high diversity and endemism of freshwater taxa. Freshwater mussels (Bivalvia, Unionidae) from the Western Palearctic have been widely studied in their European range, but little attention has been dedicated to these taxa in the Eastern Mediterranean region and their diversity and phylogeography are still poorly understood. The present study aims to resolve the diversity, biogeography, and evolutionary relationships of the Eastern Mediterranean freshwater mussels. To that end, we performed multiple field surveys, phylogenetic analyses, and a thorough taxonomic revaluation. We reassessed the systematics of all Unionidae species in the region, including newly collected specimens across Turkey, Israel, and Iran, combining COI + 16S + 28S and COI phylogenies with molecular species delineation methods. Phylogeographical patterns were characterized based on published molecular data, newly sequenced specimens, and species distribution data, as well as ancestral range estimations. We reveal that Unionidae species richness in the Eastern Mediterranean is over 70% higher than previously assumed, counting 19 species within two subfamilies, the Unioninae (14) and Gonideinae (5). We propose two new species, Anodonta seddonisp. nov. and Leguminaia anatolicasp. nov. Six additional taxa, Unio delicatusstat. rev., Unio eucirrusstat. rev., Unio huetistat. rev., Unio sesirmensisstat. rev., Unio terminalisstat. rev. removed from the synonymy of Unio tigridis, as well as Unio damascensisstat. rev. removed from the synonymy of Unio crassus, are re-described. The nominal taxa Unio rothi var. komarowi O. Boettger, 1880 and Unio armeniacus Kobelt, 1911 are proposed as new synonyms of Unio bruguierianus, and Anodonta cyrea Drouët, 1881 and Anodonta cilicica Kobelt & Rolle, 1895 as new synonyms of Anodonta anatina. Also, the presence of Unio tumidus in the Maritza River is confirmed. The phylogeographic patterns described here are interpreted concerning major past geological events. Conservation needs and implications are presented, together with populations and species conservation priorities.

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.

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.

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.

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.

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.

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.

Phylogeny, phylogeography, and evolution in the Mediterranean region: News from a freshwater mussel (Potomida, Unionida).

The Potomida genus (Bivalvia, Unionida) has a Circum-Mediterranean distribution and like other freshwater mussel species, its populations have suffered dramatic declines. Although this genus is currently considered as monotypic, it has a long history of taxonomic revisions and presently many aspects of its systematics and evolutionary history are unclear. We sampled a total of 323 individuals from 39 different sites across the Potomida genus distribution, and sequenced two mitochondrial (16S rDNA and Cytochrome c Oxidase Subunit I) and one nuclear (28S rDNA) genes to clarify its phylogeny and phylogeographic history. Our results show that the genus includes two well-supported clades, one comprising solely the western Mediterranean species Potomida littoralis, and the other including two eastern Mediterranean species, the Greek endemic P. acarnanica and the Anatolian and Middle Eastern P. semirugata. We suggest that Potomida started radiating during the upper Miocene, and that both vicariance and dispersal events shaped the diversification and distribution of the genus along the Mediterranean region. P. littoralis is further divided in two mitochondrial lineages, one restricted to Europe and the other occurring mostly in North Africa. Moreover, some European basins present both lineages in sympatry. The conservation status of the three recognized species should be reevaluated, particularly P. acarnanica, since it is restricted to two Greek river basins presenting a high risk of extinction. Overall, our results clarify some important gaps in knowledge concerning the phylogeny, phylogeography and evolution of the Potomida genus in the Mediterranean region with important taxonomical, ecological and conservational implications.

The complete mitochondrial genome of Potomida acarnanica (Kobelt, 1879).

Freshwater mussels (Bivalvia, Unionida) play essential roles in the well-functioning of ecosystems, even providing essential services to humans. However, these bivalves face numerous threats (e.g. habitat loss and fragmentation, pollution, introduction of invasive species, and climate change) which have already led to the extinction of many populations. This underscores the need to fully characterize the biology of these species, particularly those, such as Potomida acarnanica, that are still poorly studied. This study presents the first mitogenome of P. acarnanica (Kobelt, 1879), an endemic species of Greece with a distribution limited to only two river basins. The mitochondrial genome of a P. acarnanica specimen, collected at Pamisos River (Peloponnese, Greece), was sequenced by Illumina high-throughput sequencing. This mitogenome (16,101 bp) is characterized by 13 protein-coding genes, 22 transfer RNA and 2 ribosomal RNA genes. The size of this mitogenome is within the range of another Potomida mitogenome already published for the species Potomida littoralis. In the phylogenetic inference, P. acarnanica was recovered as monophyletic with P. littoralis mitogenome in the Lamprotulini tribe, as expected. This genomic resource will assist in genetically characterizing the species, potentially benefiting future evolutionary studies and conservation efforts.

Ecological status of a Margaritifera margaritifera (Linnaeus, 1758) population at the southern edge of its distribution (River Paiva, Portugal).

An important population of the critically endangered pearl mussel Margaritifera margaritifera (Linnaeus, 1758) was surveyed at the edge of its southern distribution (River Paiva, Portugal). Although an earlier study suggested that this population had a very low number of individuals (<500), a narrow distribution, and was mainly comprised by old specimens our data contradict these findings. Our assessment estimated a population with probably more than 5,000 individuals distributed across 80 km of the river length. From the 32 sites surveyed, 19 contained M. margaritifera with higher abundances verified in the middle and upper parts of the river (a maximum of 78 ind. per 100 m of river stretch was recorded). The pearl mussels showed a clear preference for areas near the banks, in shallow water, sandier and gravel sediments, and a high degree of riparian vegetation cover. The population structure was skewed with a very high percentage of large (and old) animals but 3.7 % of the individuals collected were juveniles (<60 mm in length); therefore, this population can be considered functional. Environmental characterization indicated that this river is still in excellent or good condition although some areas showed deterioration due to discharge of domestic effluents. The main conservation requirements of M. margaritifera in the River Paiva include maintaining the water quality (and if possible stopping the discharge of domestic effluents), increasing riparian vegetation cover, removing several weirs to increase connectivity, and increasing trout density.

The Crown Pearl V2: an improved genome assembly of the European freshwater pearl mussel Margaritifera margaritifera (Linnaeus, 1758).

Contiguous assemblies are fundamental to deciphering the composition of extant genomes. In molluscs, this is considerably challenging owing to the large size of their genomes, heterozygosity, and widespread repetitive content. Consequently, long-read sequencing technologies are fundamental for high contiguity and quality. The first genome assembly of Margaritifera margaritifera (Linnaeus, 1758) (Mollusca: Bivalvia: Unionida), a culturally relevant, widespread, and highly threatened species of freshwater mussels, was recently generated. However, the resulting genome is highly fragmented since the assembly relied on short-read approaches. Here, an improved reference genome assembly was generated using a combination of PacBio CLR long reads and Illumina paired-end short reads. This genome assembly is 2.4 Gb long, organized into 1,700 scaffolds with a contig N50 length of 3.4 Mbp. The ab initio gene prediction resulted in 48,314 protein-coding genes. Our new assembly is a substantial improvement and an essential resource for studying this species' unique biological and evolutionary features, helping promote its conservation.

PacBio Hi-Fi genome assembly of the Iberian dolphin freshwater mussel Unio delphinus Spengler, 1793.

Mussels of order Unionida are a group of strictly freshwater bivalves with nearly 1,000 described species widely dispersed across world freshwater ecosystems. They are highly threatened showing the highest record of extinction events within faunal taxa. Conservation is particularly concerning in species occurring in the Mediterranean biodiversity hotspot that are exposed to multiple anthropogenic threats, possibly acting in synergy. That is the case of the dolphin freshwater mussel Unio delphinus Spengler, 1793, endemic to the western Iberian Peninsula with recently strong population declines. To date, only four genome assemblies are available for the order Unionida and only one European species. We present the first genome assembly of Unio delphinus. We used the PacBio HiFi to generate a highly contiguous genome assembly. The assembly is 2.5 Gb long, possessing 1254 contigs with a contig N50 length of 10 Mbp. This is the most contiguous freshwater mussel genome assembly to date and is an essential resource for investigating the species' biology and evolutionary history that ultimately will help to support conservation strategies.

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.

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.

The gill transcriptome of threatened European freshwater mussels.

Genomic tools applied to non-model organisms are critical to design successful conservation strategies of particularly threatened groups. Freshwater mussels of the Unionida order are among the most vulnerable taxa and yet almost no genetic resources are available. Here, we present the gill transcriptomes of five European freshwater mussels with high conservation concern: Margaritifera margaritifera, Unio crassus, Unio pictorum, Unio mancus and Unio delphinus. The final assemblies, with N50 values ranging from 1069-1895 bp and total BUSCO scores above 90% (Eukaryote and Metazoan databases), were structurally and functionally annotated, and made available. The transcriptomes here produced represent a valuable resource for future studies on these species' biology and ultimately guide their conservation.

The Crown Pearl: a draft genome assembly of the European freshwater pearl mussel Margaritifera margaritifera (Linnaeus, 1758).

Since historical times, the inherent human fascination with pearls turned the freshwater pearl mussel Margaritifera margaritifera (Linnaeus, 1758) into a highly valuable cultural and economic resource. Although pearl harvesting in M. margaritifera is nowadays residual, other human threats have aggravated the species conservation status, especially in Europe. This mussel presents a myriad of rare biological features, e.g. high longevity coupled with low senescence and Doubly Uniparental Inheritance of mitochondrial DNA, for which the underlying molecular mechanisms are poorly known. Here, the first draft genome assembly of M. margaritifera was produced using a combination of Illumina Paired-end and Mate-pair approaches. The genome assembly was 2.4 Gb long, possessing 105,185 scaffolds and a scaffold N50 length of 288,726 bp. The ab initio gene prediction allowed the identification of 35,119 protein-coding genes. This genome represents an essential resource for studying this species' unique biological and evolutionary features and ultimately will help to develop new tools to promote its conservation.

Time travelling through local ecological knowledge regarding an endangered species.

European freshwater pearl mussel (Margaritifera margaritifera) populations are declining despite a growing effort to conserve them. Here we used a combination of local ecological knowledge (LEK) and field sampling to assess past and current distribution and conservation status of this endangered species in Cávado and Neiva Rivers (Portugal). We performed face-to-face interviews in both rivers and sampled the entire area where the respondents confirmed the historical presence of this species. Abiotic characterization, water quality and fish diversity were also assessed in both rivers. We found that freshwater pearl mussels are now possibly extinct in Cávado River but almost 50% of the respondents confirm its presence in the past, especially elders that lived in villages near its historical distribution. To the contrary, and although the species is still present in Neiva River, only 3.8% of the respondents remembered its presence in the past. In both rivers, respondents suggested pollution as the most important explanation for the freshwater pearl mussels decline. However, nowadays both rivers present excellent water quality and trout Salmo trutta (the freshwater pearl mussel fish host) is still abundant. Since we identified the areas where the species was present in a recent past, this information is vital for possible management actions with the aim of re-introduce or increase the abundance of M. margaritifera populations and/or for the rehabilitation of habitats in both rivers. We also highlight the vital importance of getting LEK, mainly from elders, in order to avoid shifting baseline syndromes and to get qualitative accurate information of past references and/or experience with historical conditions. Results reported here reinforce concern about the conservation status of freshwater pearl mussel populations in Portugal and can be used to guide future research and management initiatives to better conserve this species.

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.