Freshwater conservation assessments in (semi-)arid regions: testing river intermittence and buffer strategies using freshwater mussels (Bivalvia, Unionida) in Morocco

The IUCN Red List assessments are essentially based on population trends and range, namely Area of Occupancy (AOO) and Extent of Occupancy (EOO). Range estimations are based on fixed grids, but this is likely inappropriate for species living in river networks. Furthermore, AOO and EOO are measured using the whole hydrographic network, therefore disregarding temporary sections, which is particularly problematic in arid and semi-arid regions. Here we mapped the permanent hydrographic network of Morocco using satellite imagery, complemented with field surveys to collect samples for molecular analyses of the five freshwater mussel species present and assess their distribution. The phylogeographic patterns are described for each species and used to identify priority areas and evolutionary significant units for conservation. Permanent hydrographic river sections represent only 18.3% of the whole hydrographic network. A north-to-south gradient of genetic diversity, species richness and distribution range was found, being coherent with water availability and river intermittence. Isolated evolutionary units were detected in southern basins that should also receive particular attention in conservation planning. We propose the mean river width multiplied by the extent of the river network as the best and the most adequate way to estimate both EOO and AOO. Given the worldwide degradation of freshwater systems and biodiversity, an accurate (re)assessment of species conservation status supported with maps of intermittent water bodies will be essential for prioritizing and guiding conservation actions and management plans, especially in arid and semi-arid regions.This work was partially supported by the Portuguese Foundation for Science and Technology (FCT) under grant SFRH/BD/115728/2016 (MLL) and grant SFRH/BD/137935/2018 (AGS). This research was developed under ConBiomics: the missing approach for the Conservation of freshwater Bivalves Project N° NORTE-01-0145-FEDER-030286, co-financed by COMPETE 2020, Portugal 2020 and the European Union through the ERDF, and by FCT through national funds. This study was additionally conducted within the scope of project “Biodiversity and conservation of the critically endangered freshwater mussels in Morocco: ecogeographic, genetic and physiological information”, funded by Mohamed bin Zayed Species Conservation Fund (Reference 15256799) and project “Breeding the most endangered bivalve on Earth: Margaritifera marocana”, funded by IUCN SOS save our species fund (Reference 2015B-015). Official capture and sampling licenses were issued by the Université Cadi Ayyad (Faculté des Sciences, Semlalia, Marrakech, Maroc)

Microcondylaea bonellii as a new host for the European bitterling Rhodeus amarus

We report for the first time that the freshwater mussel Microcondylaea bonellii (Ferussac, 1827) functions as a suitable host for the European bitterling Rhodeus amarus (Bloch, 1782). Given the recent expansion of R. amarus in Europe, the possible physiological cost (e.g. competition for oxygen, reduction in water circulation, and consequent impairment of filter-feeding) of this interaction may further affect the already poor conservation status of M. bonellii populations.We acknowledge the two anonymous referees for the helpful suggestions that improve the clarity of our manuscript. This research was funded by FCT under project ConBiomics No NORTE-01-0145-FEDER-030286, cofinanced by COMPETE 2020, Portugal 2020 and the European Union through the ERDF

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.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.FCT - MAVA Foundation(2020.04637

Deglaciation records of 17O-excess in East Antarctica: reliable reconstruction of oceanic normalized relative humidity from coastal sites

We measured 17O and 18O in two Antarctic ice cores at EPICA Dome C (EDC) and TALDICE (TD), respectively, and computed 17O-excess with respect to VSMOW. The comparison of our 17O-excess data with the previous record obtained at Vostok (Landais et al., 2008a) revealed differences up to 35 ppm in 17O-excess mean level and evolution for the three sites. Our data show that the large increase depicted at Vostok (20 ppm) during the last deglaciation is a regional and not a general pattern in the temporal distribution of 17O-excess in East Antarctica. The EDC data display an increase of 12 ppm, whereas the TD data show no significant variation from the Last Glacial Maximum (LGM) to the Early Holocene (EH). A Lagrangian moisture source diagnostic revealed very different source regions for Vostok and EDC compared to TD. These findings combined with the results of a sensitivity analysis, using a Rayleigh-type isotopic model, suggest that normalized relative humidity (RHn) at the oceanic source region (OSR) is a determining factor for the spatial differences of 17O-excess in East Antarctica. However, 17O-excess in remote sites of continental Antarctica (e.g. Vostok) may be highly sensitive to local effects. Hence, we consider 17O-excess in coastal East Antarctic ice cores (TD) to be more reliable as a proxy for RHn at the OSR

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 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 seddoni sp. nov. and Leguminaia anatolica sp. nov. Six additional taxa, Unio delicatus stat. rev., Unio eucirrus stat. rev., Unio hueti stat. rev., Unio sesirmensis stat. rev., Unio terminalis stat. rev. removed from the synonymy of Unio tigridis, as well as Unio damascensis stat. 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

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

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

The identification 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.Financial support was provided by the Portuguese Foundation for Science and Technology (FCT) Grant to J.N. (2020.04637.BD). We thank the editor and 3 anonymous referees for the valuable suggestions made, which increased the clarity of our manuscript. This study was partially funded by the MAVA Foundation through the action plan Ensuring Integrated Resource Management in River Basins.info:eu-repo/semantics/publishedVersio

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.info:eu-repo/semantics/publishedVersio

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.A.G.-d.-S. was funded by the Portuguese Foundation for Science and Technology (FCT) under the grants SFRH/BD/137935/2018, EF (CEECIND/00627/2017) and MLL (2020.03608.CEECIND). This research was developed under ConBiomics: the missing approach for the Conservation of freshwater Bivalves Project No. NORTE-01-0145-FEDER- 030286, co-financed by COMPETE 2020, Portugal 2020 and the European Union through the ERDF, and by FCT through national funds. Additional strategic funding was provided by FCT UIDB/04423/2020 and UIDP/04423/2020. Authors’ interaction and writing of the article was promoted and facilitated by the COST Action CA18239: CONFREMU—Conservation of freshwater mussels: a pan- European approach.info:eu-repo/semantics/publishedVersio

Functional Interaction between CFTR and the Sodium-Phosphate Co-Transport Type 2a in Xenopus laevis Oocytes

A growing number of proteins, including ion transporters, have been shown to interact with Cystic Fibrosis Transmembrane conductance Regulator (CFTR). CFTR is an epithelial chloride channel that is involved in Cystic Fibrosis (CF) when mutated; thus a better knowledge of its functional interactome may help to understand the pathophysiology of this complex disease. In the present study, we investigated if CFTR and the sodium-phosphate co-transporter type 2a (NPT2a) functionally interact after heterologous expression of both proteins in Xenopus laevis oocytes.NPT2a was expressed alone or in combination with CFTR in X. laevis oocytes. Using the two-electrode voltage-clamp technique, the inorganic phosphate-induced current (IPi) was measured and taken as an index of NPT2a activity. The maximal IPi for NPT2a substrates was reduced when CFTR was co-expressed with NPT2a, suggesting a decrease in its expression at the oolemna. This was consistent with Western blot analysis showing reduced NPT2a plasma membrane expression in oocytes co-expressing both proteins, whereas NPT2a protein level in total cell lysate was the same in NPT2a- and NPT2a+CFTR-oocytes. In NPT2a+CFTR- but not in NPT2a-oocytes, IPi and NPT2a surface expression were increased upon PKA stimulation, whereas stimulation of Exchange Protein directly Activated by cAMP (EPAC) had no effect. When NPT2a-oocytes were injected with NEG2, a short amino-acid sequence from the CFTR regulatory domain that regulates PKA-dependent CFTR trafficking to the plasma membrane, IPi values and NPT2a membrane expression were diminished, and could be enhanced by PKA stimulation, thereby mimicking the effects of CFTR co-expression.We conclude that when both CFTR and NPT2a are expressed in X. laevis oocytes, CFTR confers to NPT2a a cAMPi-dependent trafficking to the membrane. This functional interaction raises the hypothesis that CFTR may play a role in phosphate homeostasis