M. foulquié, S. Coupé, N. Vicente, R. Bunet (2024). Population status of the Critically Endangered fan mussel Pinna nobilis in Thau Lagoon (France). Endangered Species Research, Volume 55, 21-36, https://doi.org/10.3354/esr01357

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​Since 2016, the noble pen shell Pinna nobilis, a unique and emblematic giant bivalve endemic to the Mediterranean Sea, has been affected by mass mortality events (MMEs), primarily due to the pathogen Haplosporidium pinnae. To date, all known populations located in the open sea have been decimated by epizootics, and P. nobilis is currently classified as a Critically Endangered species. The last refuge areas with P. nobilis populations are now in coastal lagoons. This study assesses the status, size, density, and spatial distributions of P. nobilis populations in Thau Lagoon (Occitany, France). At the end of January 2024, 1931 live individuals were recorded at 30 different surveyed sites. Our surveys indicate various size-class distributions ranging from young recruits to adults. Examining the spatial distribution of P. nobilis revealed high-density populations in specific areas (up to 25 ind. 100 m–2). P. nobilis is present from the edges of the lagoon to the central and deeper zones (up to 9 m deep). Although Thau Lagoon experienced a localized MME at the end of 2020, driven by the presence of H. pinnae, this study reveals the occurrence of new individuals in previously infected areas, indicating signs of population recovery and potential resilience. All these observations suggest that Thau Lagoon represents one of the very last sanctuaries for P. nobilis in the Mediterranean Sea.​

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A. Nikolaou, E. Papadimitriou, E. Kiourani, S. Katsanevakis (2024). Pinna nobilis refugia breached: Ongoing Mass Mortality Event in the Gulf of Kalloni (Aegean Sea). Mediterranean Marine Science, 25(3), 747-752, https://doi.org/10.12681/mms.38613

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The endemic Mediterranean fan mussel Pinna nobilis is on the brink of extinction due to an infectious disease that has caused an ongoing Mediterranean Mass Mortality Event (MME) since 2016. Currently, healthy P. nobilis populations can only be found in a few refuges, mainly transitional waters such as coastal lagoons and estuaries. In the eastern Mediterranean, the only remaining fan mussel population is located in the Gulf of Kalloni (Lesvos Island, Greece). This study reports on a severe mortality event that occurred during summer 2023, leaving the population decimated and limited mainly to young individuals. The estimated density of live individuals dropped from 1.22 to 0.23 individuals per 100 m², indicating an 81% population decline within approximately three months. The significant reduction in the P. nobilis population in the Gulf of Kalloni highlights the ongoing threat of MMEs. Continuous monitoring of P. nobilis and environmental variables, such as temperature and salinity, enhanced protection from anthropogenic pressures, and research into disease resistance mechanisms are crucial for the conservation of this critically endangered species.

P. Martínez-Martínez, E. Cortés-Melendreras, Y. Fernández-Torquemada, C. Barberá, Y. del-Pilar-Ruso, A. Izquierdo-Muñoz, P. Prado, F. Giménez-Casalduero (2024). Assessing habitat microscale conditions underlying with mortality and survival of Pinna nobilis in the Mar Menor coastal lagoon (SE Spain). Journal of Sea Research, Volume 200, 102518, ISSN 1385-1101, https://doi.org/10.1016/j.seares.2024.102518

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Pinna nobilis Linnaeus, 1758 is a Mediterranean endemic bivalve critically endangered due to a severe epidemic caused mainly by the parasite Haplosporidium pinnae. Presently, live populations in Spanish waters are restricted to the Ebro Delta (Catalonia) and the Mar Menor (Murcia). The coastal lagoon of the Mar Menor has faced several eutrophication crises since 2016, which pose a threat to the survival of P. nobilis. Since 2016, >95% of the specimens of this species have died due to recurrent episodes of eutrophication in the lagoon. An analysis of the mortality within the relict population of P. nobilis in the Mar Menor lagoon after the last population survey in 2019 has been carried out, together with a characterisation of some microscale factors that describe its current habitat in the lagoon. For this purpose, three sampling localities were selected along the east coast of the lagoon, where a study of both live and dead individuals was conducted. Additionally, a characterisation of the macrophyte beds and the sediment conditions surrounding these individuals was carried out, comparing the sediment near the recently dead and live individuals. All studied localities exhibit similar mixed macrophyte beds of Cymodocea nodosa (Ucria) Asch., 1870 and Caulerpa prolifera (Forsskål) J.V. Lamouroux, 1809. At the location where significantly less C. nodosa cover was observed, the mortality of P. nobilis individuals was higher, and the sediment near the dead individuals was also more disturbed (with a more negative Eh and higher percentage of organic matter). These results contribute to a better understanding of the dynamics of microscale factors following P. nobilis mortality events, facilitating the identification of potential survival areas.

Pilar Martínez-Martínez, Francisco Alonso-Sarría, Francisco Gomariz-Castillo, Enrique Reñé-Sánchez, Emilio Cortés-Melendreras, Yolanda Fernández-Torquemada, Francisca Giménez-Casalduero (2024). Looking for the surviving optimal areas for the threatened species Pinna nobilis in a highly anthropized coastal lagoon (Mar Menor, SE Spain). Ocean & Coastal Management, Volume 255, 107265, ISSN 0964-5691, https://doi.org/10.1016/j.ocecoaman.2024.107265

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Pinna nobilis, an endemic bivalve species in the Mediterranean Sea, is critically endangered mainly due to the devastating impact of the protozoan parasite Haplosporidium pinnae. The population within the Mar Menor lagoon (Murcia, Spain) represents one of only two surviving populations along the Spanish coast. This relict population has witnessed a significant decline since 2016, primarily due to eutrophication episodes and other anthropogenic impacts affecting the lagoon. It is imperative to identify the most suitable zones for the survival of this species, where adult individuals could be translocated from affected areas to enhance their chances of survival. In the present study, we identify these zones through a Multi-Criteria Evaluation considering several factors that pose a threat to the species. This evaluation was complemented using Geographic Information Systems based multi-criteria decision analysis (GIS-MCDA). A total of 18 factors were weighted and combined, resulting in a surface area of 307.92 hectares (2.28% of the lagoon) with over 70% high suitability for species survival.

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Francesca Carella, Patricia Prado, José Rafael García-March, José Tena-Medialdea, Emilio Cortés Melendreras, Antonio Porcellini, Antonia Feola (2024). Measuring immunocompetence in the natural population and captive individuals of noble pen shell Pinna nobilis affected by Pinna nobilis Picornavirus (PnPV). Fish & Shellfish Immunology, Volume 151, 109664, ISSN 1050-4648, https://doi.org/10.1016/j.fsi.2024.109664

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Mass Mortality Events (MMEs) affecting the noble pen shell Pinna nobilis have been reported since 2016. In this work, we used an in vitro flow cytometric assay to evaluate phagocytosis, coupled with cytology and Electron Microscopy (TEM), to define animal immunocompetence following infection by P. nobilis Picornavirus (PnPV). The study was performed on 27 animals in July 2021 and May 2022 on two natural population from the Ebro Delta (Catalonia, Spain) and animals maintained in captivity at facilities in Valencia and Murcia Aquarium. Hemolymph was collected in the field and in captivity as a non-destructive sampling method. Based on dimension and internal complexity, flow cytometry identified three haemocyte types, distinguished in granulocytes, hyalinocytes and a third type, biggest in size and with high internal complexity and granularity. Those cells corresponded at ultrastructure to hemocytes with advanced phases of PnPV infection and related to cytopathic effect of the replicating virus displaying numerous Double Membrane Vesicles (DMVs) and cells corpse fusion. The results showed that pen shell in captivity had significantly lower Total Hemocyte Count (THC) compared with natural population of Alfacs Bay (mean number of 7–9 x 104 vs 2-5 x 105 cells/mL, respectively). FACS (Fluorescence-activated cell sorting) based phagocytosis analysis demonstrate that animals in captivity at IMEDMAR-UCV and Murcia Aquarium, had scarce or absent ability to phagocyte the two stimuli (Staphylococcus aureus and Zymosan A) (10,2 % ± 1,7 of positives) if compared with the natural population in Alfacs Bay (28,5 % ± 5,6 of positive). Ultrastructure images showed that PnPV itself can lead to an alteration of the hemocyte cytoskeleton, impairing the capabilities to perform an active phagocytosis and an efficient phagolysosome fusion.

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Feria-Rodríguez, A., March, D., Mourre, B., Hendriks, I. E., & Vázquez-Luis, M. (2024).   Sink-source connectivity for restocking of Pinna nobilis in the western Mediterranean Sea. Marine Environmental Research, 197, 106428.  https://doi.org/10.1016/j.marenvres.2024.106428

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The critically endangered endemic bivalve Pinna nobilis from the Mediterranean Sea suffered a sudden population decline after a mass mortality event in early autumn 2016. Conservation efforts aimed at preventing extinction included safeguarding resistant individuals and implementing a breeding plan to contribute to the repopulation of the species. This study utilized a model combining Lagrangian dispersion and connectivity analyses to pinpoint optimal restocking sites in the Western Mediterranean. Our approach allowed to identify locations capable of sustaining and generating larvae for broader repopulation in key areas of the Western Mediterranean Sea prior to the mass mortality event. Six important repopulation locations from Murcia, Valencia and Balearic Islands were selected for reintroduction efforts. The results obtained in this study show how the network could be self-sufficient and able to self-replenish itself of recruits. Overall, our work can be used to direct the reintroduction of resistant animals in the Western Mediterranean Sea.

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Carella, F., Prado, P., de Vico, G., Palić, D., Villari, G., García-March, J. R., Tena-Medialdea, J., Cortés Melendreras, E., Giménez-Casalduero, F., Sigovini, M., & Aceto, S. (2023). A widespread picornavirus affects the hemocytes of the noble pen shell (Pinna nobilis), leading to its immunosuppression. Frontiers in Veterinary Science, 10. https://doi.org/10.3389/fvets.2023.1273521

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The widespread mass mortality of the noble pen shell (Pinna nobilis) has occurred in several Mediterranean countries in the past 7 years. Single-stranded RNA viruses affecting immune cells and leading to immune dysfunction have been widely reported in human and animal species. Here, we present data linking P. nobilis mass mortality events (MMEs) to hemocyte picornavirus (PV) infection. This study was performed on specimens from wild and captive populations. We sampled P. nobilis from two regions of Spain [Catalonia (24 animals) and Murcia (4 animals)] and one region in Italy [Venice (6 animals)]. Each of them were analyzed using transmission electron microscopy (TEM) to describe the morphology and self-assembly of virions. Illumina sequencing coupled to qPCR was performed to describe the identified virus and part of its genome. In 100% of our samples, ultrastructure revealed the presence of a virus (20 nm diameter) capable of replicating within granulocytes and hyalinocytes, leading to the accumulation of complex vesicles of different dimensions within the cytoplasm. As the PV infection progressed, dead hemocytes, infectious exosomes, and budding of extracellular vesicles were visible, along with endocytic vesicles entering other cells. The THC (total hemocyte count) values observed in both captive (eight animals) (3.5 × 104–1.60 × 105 ml−1 cells) and wild animals (14 samples) (1.90–2.42 × 105 ml−1 cells) were lower than those reported before MMEs. Sequencing of P. nobilis (6 animals) hemocyte cDNA libraries revealed the presence of two main sequences of Picornavirales, family Marnaviridae. The highest number of reads belonged to animals that exhibited active replication phases and abundant viral particles from transmission electron microscopy (TEM) observations. These sequences correspond to the genus Sogarnavirus—a picornavirus identified in the marine diatom Chaetoceros tenuissimus (named C. tenuissimus RNA virus type II). Real-time PCR performed on the two most abundant RNA viruses previously identified by in silico analysis revealed positive results only for sequences similar to the C. tenuissimus RNA virus. These results may not conclusively identify picornavirus in noble pen shell hemocytes; therefore, further study is required. Our findings suggest that picornavirus infection likely causes immunosuppression, making individuals prone to opportunistic infections, which is a potential cause for the MMEs observed in the Mediterranean.

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Coupé, S., Giantsis, I. A., Vázquez Luis, M., Scarpa, F., Foulquié, M., Prévot, J. M., Casu, M., Lattos, A., Michaelidis, B., Sanna, D., García-March, J. R., Tena-Medialdea, J., Vicente, N., & Bunet, R. (2023). The characterization of toll-like receptor repertoire in Pinna nobilis after mass mortality events suggests adaptive introgression. Ecology and Evolution, 13(8). https://doi.org/10.1002/ece3.10383

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The fan mussel Pinna nobilis is currently on the brink of extinction due to a multifactorial disease mainly caused to the highly pathogenic parasite Haplosporidium pinnae, meaning that the selection pressure outweighs the adaptive potential of the species. Hopefully, rare individuals have been observed somehow resistant to the parasite, stretching the need to identify the traits underlying this better fitness. Among the candidate to explore at first intention are fast-evolving immune genes, of which toll-like receptor (TLR). In this study, we examined the genetic diversity at 14 TLR loci across P. nobilis, Pinna rudis and P. nobilis × P. rudis hybrid genomes, collected at four physically distant regions, that were found to be either resistant or sensitive to the parasite H. pinnae. We report a high genetic diversity, mainly observed at cell surface TLRs compared with that of endosomal TLRs. However, the endosomal TLR-7 exhibited unexpected level of diversity and haplotype phylogeny. The lack of population structure, associated with a high genetic diversity and elevated dN/dS ratio, was interpreted as balancing selection, though both directional and purifying selection were detected. Interestingly, roughly 40% of the P. nobilis identified as resistant to H. pinnae were introgressed with P. rudis TLR. Specifically, they all carried a TLR-7 of P. rudis origin, whereas sensitive P. nobilis were not introgressed, at least at TLR loci. Small contributions of TLR-6 and TLR-4 single-nucleotide polymorphisms to the clustering of resistant and susceptible individuals could be detected, but their specific role in resistance remains highly speculative. This study provides new information on the diversity of TLR genes within the P. nobilis species after MME and additional insights into adaptation to H. pinnae that should contribute to the conservation of this Mediterranean endemic species.

Foulquie, M., Coupe, S., Vicente, N., & Bunet, R. (2023). First detection of Pinna nobilis infection by Haplosporidium pinnae in the sanctuary area of Thau lagoon, France. Mediterranean Marine Science, 24(3), 569–573. https://doi.org/10.12681/MMS.32300

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The noble pen shell Pinna nobilis is an endemic and emblematic giant bivalve whose populations have been recently affected by a mass mortality event (MME), primarily due to the spread of the pathogen Haplosporidium pinnae. Since the beginning of the MME in Spain in 2016, nearly one hundred percent of monitored open sea water populations have been decimated around the Mediterranean Sea. The only refuge areas with living P. nobilis populations were found in coastal lagoons. Today, the Thau lagoon in France is home to a vitally important population of P. nobilis and was thought to be safe from the parasite. Here, we report the first molecular detection of H. pinnae (isolate PN1) in tissue samples of moribund individuals from Thau lagoon.

Hernandis, S., Ibarrola, I., Tena-Medialdea, J., Albentosa, M., Prado, P., Vázquez-Luis, M., & García-March, J. R. (2023). Physiological responses of the fan mussel Pinna nobilis to temperature: ecological and captivity implications. Mediterranean Marine Science, 24(2). https://doi.org/10.12681/mms.31050

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The fan mussel Pinna nobilis is experiencing a mass mortality event throughout the Mediterranean Sea. The survival of the remaining isolated populations and the maintenance and reproduction of individuals in captivity could determine the future of the species. This paper examines the clearance rate (CR) and oxygen consumption (VO2) of fan mussel individuals measured under 5 different temperatures (8.5, 14, 18, 23 and 28ºC). The measurements of both physiological variables revealed a threshold limit of thermal tolerance at both extreme temperatures, indicating the critical situation of the remaining populations, which are located in coastal lagoons and shallow bays where these extreme temperatures usually occur. Besides, the high clearance rates observed highlight the significant ecosystem service provided by the species in terms of water filtration, especially in confined waters with low renewal rates. Routine control of the clearance rate as an early warning system is proposed for detecting ailing individuals. Such a system could also be used to improve captivity conditions.

Labidi, S., Vázquez-Luis, M., Catanese, G., Grau, A., Khammassi, M., ben Youssef, S., & Sghaier Achouri, M. (2023). First detection of the invasive protozoan Haplosporidium pinnae in the critically endangered bivalve Pinna nobilis in the Southern Mediterranean Sea (Bizerte Lagoon, Tunis) and update of its current status. Mediterranean Marine Science, 24(3), 470–481. https://doi.org/10.12681/MMS.31664

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Pinna nobilis (Linnaeus, 1758) populations have been severely damaged in the last few decades, and since early autumn 2016, a mass mortality event (MME) has drastically impacted populations in the Mediterranean Sea. Accordingly, the aim of the present study was to improve the knowledge on the status of P. nobilis populations in the Bizerte Lagoon (Tunisia) between 2016 and 2022. Before the MME, P. nobilis was found in the lagoon at depths from 1.5 to 6 m, with a density ranging from 2 to 30 ind/100 m². After the MME, mortality reached 100% in the monitored area, except in the eastern part of the Bizerte Lagoon near the Menzel Jemil site, where some living specimens were detected. Moreover, in 2022, successful recruitment was observed in the lagoon. Additionally, our results revealed the presence of Haplosporidium pinnae and Mycobacterium sp. in some living specimens sampled in the Bizerte Lagoon after the MME. This finding constitutes the first detection of both pathogens in Tunisia and in the entire Southern Mediterranean Sea.

Moro-Martínez, I., Vázquez-Luis, M., García-March, J. R., Prado, P., Mičić, M., & Catanese, G. (2023). Haplosporidium pinnae Parasite Detection in Seawater Samples. Microorganisms, 11(5). https://doi.org/10.3390/microorganisms11051146

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In this study, we investigated the presence of the parasite Haplosporidium pinnae, which is a pathogen for the bivalve Pinna nobilis, in water samples from different environments. Fifteen mantle samples of P. nobilis infected by H. pinnae were used to characterize the ribosomal unit of this parasite. The obtained sequences were employed to develop a method for eDNA detection of H. pinnae. We collected 56 water samples (from aquaria, open sea and sanctuaries) for testing the methodology. In this work, we developed three different PCRs generating amplicons of different lengths to determine the level of degradation of the DNA, since the status of H. pinnae in water and, therefore, its infectious capacity are unknown. The results showed the ability of the method to detect H. pinnae in sea waters from different areas persistent in the environment but with different degrees of DNA fragmentation. This developed method offers a new tool for preventive analysis for monitoring areas and to better understand the life cycle and the spread of this parasite.

Papadakis, O., Mamoutos, I., Ramfos, A., Catanese, G., Papadimitriou, E., Theodorou A., J., Batargias, C., Papaioannou, C., Kamilari, M., Tragou, E., Zervakis, V., & Katsanevakis, S. (2023). Status, distribution, and threats of the last surviving fan mussel populations in Greece. Mediterranean Marine Science, 24(3), 679–708. https://doi.org/10.12681/mms.35384

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Since the first confirmed records of mass mortality events (MME) in the Aegean Sea in 2018, Pinna nobilis populations in Greek seas have been decimated. To bolster recovery efforts, this study aimed to assess the status of fan mussel populations in the Aegean and Ionian seas and investigate potential recolonization through natural recruitment. From May 2022 to May 2023, 163 independent underwater visual surveys were conducted across various locations and depths along the Greek coastline. A total of 4348 P. nobilis individuals was recorded, of which 87.3% were found dead and 12.7% were alive. The sole surviving fan mussel populations were located in the semi-enclosed gulfs of Amvrakikos in the Ionian Sea and of Kalloni in the Aegean Sea, with estimated recent mortality rates (excluding potential poaching) of 7.7% and 6.3%, respectively. To track potential new recruitment, a network of larvae collectors was deployed in multiple locations. Additionally, an ocean circulation model (OCM) was developed to predict the export and fate of larvae from the surviving populations in the Marmara Sea towards the Aegean Sea. Beyond the MME, this study identified several other threats, which significantly endanger fan mussel survival. The findings of this study underscore the urgent need to implement protection measures and restoration actions to enhance the chances of P. nobilis survival and recovery in the Greek seas.

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Zotou, M., Papadakis, O., Catanese, G., Stranga, Y., Ragkousis, M., Kampouris E., T., Naasan Aga - Spyridopoulou, R., Papadimitriou, E., Koutsoubas, D., & Katsanevakis, S. (2023). New kid in town: Pinna rudis spreads in the eastern Mediterranean. Mediterranean Marine Science, 24(3), 709–721. https://doi.org/10.12681/mms.35343

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In the Mediterranean Sea, the genus Pinna encompasses two large fan-shaped bivalve species, Pinna nobilis and Pinna rudis. Historically, both species coexisted in the western Mediterranean until a mass mortality event (MME) brought P. nobilis to the brink of extinction. Notably, P. rudis remained unaffected by the MME, and its recent successful recruitment and further spread have been hypothesized to be linked to the local extinction of P. nobilis. Although P. rudis has been sparsely recorded in the eastern Mediterranean Sea (with some of these records being doubtful), reports emerging in the summer of 2023 from researchers and citizens have unveiled its sudden spread in the region. This study documents the recent presence of P. rudis within Greek waters through a dedicated survey and molecularly confirmation through two distinct molecular methods. Information derived from a citizen science initiative, following photo-identification of the species was also included. Furthermore, an updated review of the distribution of P. rudis in the Mediterranean Sea, integrating data from literature and online repositories is provided. This research confirms the recent spread of P. rudis in Crete, the Greek Ionian Sea, and Cyprus (first verified records of the species in the regions), in marine areas where P. nobilis has become extinct, further strengthening the hypothesis that P. rudis has benefited from the collapse of P. nobilis populations.

Cortés-Melendreras, E., Gomariz-Castillo, F., Alonso-Sarría, F., Giménez Martín, F. J., Murcia, J., Canales-Cáceres, R., Ramos Esplá, A. A., Barberá, C., & Giménez-Casalduero, F. (2022). The relict population of Pinna nobilis in the Mar Menor is facing an uncertain future. Marine Pollution Bulletin, 185, 114376. https://doi.org/10.1016/J.MARPOLBUL.2022.114376

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Pinna nobilis is undergoing one of the most dramatic events suffered by an endangered species. An emerging disease has relegated its populations to coastal lagoons or estuaries with salinities beyond the 36.5–39.7 psu range. The Mar Menor is one of two such locations on the Spanish coastline. Poor environmental conditions and eutrophication and anoxia events, that became critical in 2016, 2019 and 2021, have reduced its population in >99 %. In this work, the spatial distribution of the species within the lagoon and the factors determining its survival along the successive crises of eutrophication are studied using a two-stage (presence/absence estimation and density modelling) Species Distribution Model. A potential area of 200.97 ha and an average density of 1.05 ind.100 m2 is estimated for 2020. The viability of the Mar Menor population depends on management actions designed both for the species and to improve the lagoon environmental state.

Terradas-Fernández, M., Valverde-Urrea, M., López-Moya, F., & Fernández-Torquemada, Y. (2022). On the Presence and Ubiquity of the Exotic Batophora (J. Agardh) in the Mar Menor Lagoon (SE Spain). Water, 14(18), 2909. https://doi.org/10.3390/w14182909

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We first report the presence of a species of the genus Batophora in the Iberian Peninsula, in the Mar Menor lagoon (Murcia, SE Spain). We detected this macroalga in November 2021. However, according to some observations, it could have been present in the lagoon at least since 2016, being described as a “rare” form of Dasycladus vermicularis. A further survey made during May 2022 confirmed the presence of this species in much of the perimeter of the Mar Menor. Morphologic analyses are not conclusive regarding the species description, but most traits point to Batophora occidentalis or Batophora occidentalis var. largoensis. A consensus sequence from the rbcL barcode gene was compared by the Blastn tool showing its closeness to other Batophora specimens, yet the specific level was unresolved. The recent confirmation of a Batophora species in a littoral lagoon from Formentera (Balearic Islands) with similar morphologic characteristics could indicate that both putative introductions are connected. Further morphologic and genetic analyses are required in order to know the origin and expansion of this genus along the Mediterranean coasts.