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Monitoring of benthic eukaryotic communities in two tropical coastal lagoons through eDNA metabarcoding: a spatial and temporal approximation

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  • Basset, A., Elliott, M., West, R. J. & Wilson, J. G. Estuarine and lagoon biodiversity and their natural goods and services. Estuar. Coast. Shelf Sci. 132, 1–4 (2013).

    CAS 

    Google Scholar 

  • Newton, A. et al. Assessing, quantifying and valuing the ecosystem services of coastal lagoons. J. Nat. Conserv. 44, 50–65 (2018).

    Google Scholar 

  • Heck, K. L., Able, K. W., Roman, C. T. & Fahay, M. P. Composition, abundance, biomass, and production of macrofauna in a New England estuary: Comparisons among eelgrass meadows and other nursery habitats. Estuaries 18, 379–389 (1995).

    Google Scholar 

  • Franco, A. et al. Use of shallow water habitats by fish assemblages in a Mediterranean coastal lagoon. Estuar. Coast. Shelf Sci. 66, 67–83 (2006).

    Google Scholar 

  • Barbosa, F. A. R., Scarano, F. R., Sabará, M. & Esteves, F. A. Brazilian LTER: Ecosystem and biodiversity information in support of decision-making. Environ. Monit. Assess. 90, 121–133 (2004).

    CAS 
    PubMed 

    Google Scholar 

  • Esteves, F. et al. Neotropical coastal lagoons: An appraisal of their biodiversity, functioning, threats and conservation management. Braz. J. Biol. 68, 967–981 (2008).

    CAS 
    PubMed 

    Google Scholar 

  • Kjerfve, B. Coastal lagoons. Elsevier Oceanogr. Ser. 60, 1–8 (1994).

    Google Scholar 

  • Whitfield, A. K. Coastal lagoons—Critical habitats of environmental change. Mar. Biol. Res. 7, 416–417 (2011).

    Google Scholar 

  • Obolewski, K. et al. Patterns of salinity regime in coastal lakes based on structure of benthic invertebrates. PLoS ONE 13, 1–19 (2018).

    Google Scholar 

  • Schallenberg, M., Hall, C. J. & Burns, C. W. Consequences of climate-induced salinity increases on zooplankton abundance and diversity in coastal lakes. Mar. Ecol. Prog. Ser. 251, 181–189 (2003).

    Google Scholar 

  • Broman, E. et al. Salinity drives meiofaunal community structure dynamics across the Baltic ecosystem. Mol. Ecol. 28, 3813–3829 (2019).

    PubMed 
    PubMed Central 

    Google Scholar 

  • Bird, E. C. F. Physical setting and geomorphology of coastal lagoons. Elsevier Oceanogr. Ser. 60, 9–39 (1994).

    Google Scholar 

  • Barnes, N., Bamber, R. N., Moncrieff, C. B., Sheader, M. & Ferrero, T. J. Estuarine, Coastal and Shelf Science Meiofauna in closed coastal saline lagoons in the United Kingdom: Structure and biodiversity of the nematode assemblage. Estuar. Coast. Shelf Sci. 79, 328–340 (2008).

    Google Scholar 

  • Frühe, L. et al. Supervised machine learning is superior to indicator value inference in monitoring the environmental impacts of salmon aquaculture using eDNA metabarcodes. Mol. Ecol. 00, 1–19 (2020).

    Google Scholar 

  • Cordier, T. et al. Multi-marker eDNA metabarcoding survey to assess the environmental impact of three offshore gas platforms in the North Adriatic Sea (Italy). Mar. Environ. Res. 146, 24–34 (2019).

    CAS 
    PubMed 

    Google Scholar 

  • Balzano, S., Abs, E. & Leterme, S. C. Protist diversity along a salinity gradient in a coastal lagoon. Aquat. Microb. Ecol. 74, 263–277 (2015).

    Google Scholar 

  • Polinski, J. M., Bucci, J. P., Gasser, M. & Bodnar, A. G. Metabarcoding assessment of prokaryotic and eukaryotic taxa in sediments from Stellwagen Bank National Marine Sanctuary. Sci. Rep. 9, 14820 (2019).

    PubMed 
    PubMed Central 

    Google Scholar 

  • López-Escardó, D. et al. Metabarcoding analysis on European coastal samples reveals new molecular metazoan diversity. Sci. Rep. 8, 1–14 (2018).

    Google Scholar 

  • Günther, B., Knebelsberger, T., Neumann, H., Silke, L. & Arbizu, P. M. Metabarcoding of marine environmental DNA based on mitochondrial and nuclear genes. Sci. Rep. 8, 1–13 (2018).

    Google Scholar 

  • Park, D. S. & Razafindratsima, O. H. Anthropogenic threats can have cascading homogenizing effects on the phylogenetic and functional diversity of tropical ecosystems. Ecography (Cop.) 42, 148–161 (2019).

    Google Scholar 

  • Pan, Y., Yang, J., McManus, G. B., Lin, S. & Zhang, W. Insights into protist diversity and biogeography in intertidal sediments sampled across a range of spatial scales. Limnol. Oceanogr. 65, 1103–1115 (2020).

    Google Scholar 

  • Wangensteen, O. S., Palacín, C., Guardiola, M. & Turon, X. DNA metabarcoding of littoral hard-bottom communities: High diversity and database gaps revealed by two molecular markers. PeerJ 6, e4705 (2018).

    PubMed 
    PubMed Central 

    Google Scholar 

  • Polanco Fernández, A. et al. Comparing environmental DNA metabarcoding and underwater visual census to monitor tropical reef fishes. Environ. DNA 3, 1–15 (2020).

    Google Scholar 

  • Armeli Minicante, S. et al. Habitat heterogeneity and connectivity: Effects on the planktonic protist community structure at two adjacent coastal sites (the lagoon and the Gulf of Venice, Northern Adriatic Sea, Italy) revealed by metabarcoding. Front. Microbiol. 10, 1–16 (2019).

    Google Scholar 

  • Alves-De-Souza, C. et al. Does environmental heterogeneity explain temporal β diversity of small eukaryotic phytoplankton? Example from a tropical eutrophic coastal lagoon. J. Plankton Res. 39, 698–714 (2017).

    Google Scholar 

  • Grzebyk, D. et al. Insights into the harmful algal flora in northwestern Mediterranean coastal lagoons revealed by pyrosequencing metabarcodes of the 28S rRNA gene. Harmful Algae 68, 1–16 (2017).

    CAS 
    PubMed 

    Google Scholar 

  • Lallias, D. et al. Environmental metabarcoding reveals heterogeneous drivers of microbial eukaryote diversity in contrasting estuarine ecosystems. ISME J. 9, 1208–1221 (2015).

    PubMed 

    Google Scholar 

  • Avó, A. P. et al. DNA barcoding and morphological identification of benthic nematodes assemblages of estuarine intertidal sediments: Advances in molecular tools for biodiversity assessment. Front. Mar. Sci. 4, 1–16 (2017).

    Google Scholar 

  • Behera, P. et al. Salinity and macrophyte drive the biogeography of the sedimentary bacterial communities in a brackish water tropical coastal lagoon. Sci. Total Environ. 595, 472–485 (2017).

    CAS 
    PubMed 

    Google Scholar 

  • Alsaffar, Z. et al. The role of seagrass vegetation and local environmental conditions in shaping benthic bacterial and macroinvertebrate communities in a tropical coastal lagoon. Sci. Rep. 10, 1–17 (2020).

    Google Scholar 

  • Spalding, M. D. et al. Marine ecoregions of the world: A bioregionalization of coastal and shelf areas. Bioscience 57, 573–583 (2007).

    Google Scholar 

  • Lara-Lara, J. Los ecosistemas marinos. Cap. Nat. Méx. 1, 135–159 (2008).

    Google Scholar 

  • García-Grajales, J. & Buenrostro-Silva, A. E. Parque Nacional Lagunas de Chacahua, Oaxaca: Perspectivas a sus 75 años. Cienc. Ergo Sum. 21, 149–153 (2014).

    Google Scholar 

  • Zamorano, P., Barrientos-Luján, N. A. & Ahumada-Sempoal, M. Á. Moluscos bentónicos de dos sistemas lagunares de la costa chica de Oaxaca, México y su relación con parámetros fisicoquímicos. Cienc. y Mar. 14, 13–28 (2012).

    Google Scholar 

  • Sanay-González, R., MonrealGómez, M. A. & de León, D. A. S. Simulación de la circulación en el sistema lagunar Chacahua-Pastoría, Oaxaca, México. Cienc. y Mar. 10, 3–16 (2006).

    Google Scholar 

  • Comisión Nacional de Acuacultura y Pesca. Obras de dragado y escolleras en Boca de Oro, laguna de Corralero, Oaxaca (2010).

  • Sánchez-Meraz, B. & Martínez-Vega, J. A. Inmigración de Postlarvas de Camarón Litopenaeus sp. y Farfantepenaeus sp. a través la Boca El Oro del Sistema Lagunar Corralero-Alotengo, Oaxaca. Cienc. y Mar. 4, 29–46 (2000).

    Google Scholar 

  • Angel-Pérez, C., Serrano-Guzmán, S. J. & Ahumada-Sempoal, M. A. Ciclo reproductivo del molusco Atrina maura (Pterioidea: Pinnidae) en un sistema lagunar costero, al sur del Pacífico tropical mexicano. Rev. Biol. Trop. 55, 839–852 (2007).

    PubMed 

    Google Scholar 

  • Sánchez Méndez, E., Urbano Alonso, B., Sierra Hernández, S. & Garcés Salazar, J. L. Características malacológicas y sociales de la pesquería artesanal de moluscos en la Laguna de Chacahua, Oaxaca, México. Cienc. y Mar. 19, 3–11 (2015).

    Google Scholar 

  • Cowart, D. A. et al. Metabarcoding is powerful yet still blind: A comparative analysis of morphological and molecular surveys of seagrass communities. PLoS ONE 10, 1–26 (2015).

    Google Scholar 

  • Holman, L. E. et al. Detection of novel and resident marine species using environmental DNA metabarcoding of sediment and water. Sci. Rep. https://doi.org/10.1038/s41598-019-47899-7 (2019).

    Article 
    PubMed 
    PubMed Central 

    Google Scholar 

  • Bojorges-Baños, J. C. Riqueza y diversidad de especies de aves asociadas a manglar en tres sistemas lagunares en la región costera de Oaxaca, México. Rev. Mex. Biodivers. 82, 205–215 (2011).

    Google Scholar 

  • Ahumada-Sempoal, M. Á. & Ruiz-García, N. Características fisicoquímicas de la Laguna Pastoría, Oaxaca, México. Cienc. y Mar. 12, 3–17 (2008).

    Google Scholar 

  • Aylagas, E., Mendibil, I., Borja, Á. & Rodríguez-ezpeleta, N. Marine sediment sample pre-processing for macroinvertebrates metabarcoding: Mechanical enrichment and homogenization. Front. Mar. Sci. 3, 1–8 (2016).

    Google Scholar 

  • Hestetun, J. T., Lanzén, A., Skaar, K. S. & Dahlgren, T. G. The impact of DNA extract homogenization and replication on marine sediment metabarcoding diversity and heterogeneity. Environ. DNA 3, 997–1006 (2021).

    Google Scholar 

  • Comeau, M., Li, W. K. W., Carmack, E. C. & Lovejoy, C. Arctic ocean microbial community structure before and after the 2007 record sea ice minimum. PLoS ONE 6, 1–12 (2011).

    Google Scholar 

  • Caporaso, J. G. et al. QIIME allows analysis of high-throughput community sequencing data. Nat. Methods 7, 335–336 (2010).

    CAS 
    PubMed 
    PubMed Central 

    Google Scholar 

  • Edgar, R. C. Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26, 2460–2461 (2010).

    CAS 
    PubMed 

    Google Scholar 

  • Martin, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.journal 17, 10–12 (2011).

    Google Scholar 

  • Edgar, R. C. UPARSE: Highly accurate OTU sequences from microbial amplicon reads. Nat. Methods 10, 996–998 (2013).

    CAS 
    PubMed 

    Google Scholar 

  • Edgar, R. C., Haas, B. J., Clemente, J. C., Quince, C. & Knight, R. UCHIME improves sensitivity and speed of chimera detection. Bioinformatics 27, 2194–2200 (2011).

    CAS 
    PubMed 
    PubMed Central 

    Google Scholar 

  • Quast, C. et al. The SILVA ribosomal RNA gene database project: Improved data processing and web-based tools. Nucleic Acids Res. 41, 590–596 (2013).

    Google Scholar 

  • Ratnasingham, S. & Hebert, P. D. N. BOLD: The barcode of life data system. Mol. Ecol. Notes 7, 355–364 (2007).

    CAS 
    PubMed 
    PubMed Central 

    Google Scholar 

  • R Core Team. R: A Language and Environment for Statistical Computing (2018).

  • Anderson, M. J. Permutational multivariate analysis of variance (PERMANOVA). Wiley StatsRef Stat. Ref. Online. https://doi.org/10.1002/9781118445112.stat07841 (2017).

    Article 

    Google Scholar 

  • Ter Braak, C. J. F. Canonical correspondence analysis: A new eigenvector technique for multivariate direct gradient analysis. Ecology 67, 1167–1179 (1986).

    Google Scholar 

  • Ter Braak, C. J. F. The analysis of vegetation-environment relationships by canonical correspondence analysis*. Vegetatio 69, 69–77 (1987).

    Google Scholar 

  • Hammer, Ø., Harper, D. A. T. & Ryan, P. D. PAST: Paleontological statisticssofware package for education and data analysis. Palaeontol. Electron. 4, 1–9 (2001).

    Google Scholar 

  • Coan, E. V. & Valentich-Scott, P. Bivalve Seashells of Tropical West America. Marine Bivalve Mollusks from Baja California to Northern Peru (Santa Barbara Museum of Natural History, 2012).

    Google Scholar 

  • MolluscaBase. MolluscaBase. Mytella strigata (Hanley, 1843) (2022).

  • Aylagas, E., Borja, Á., Muxika, I. & Rodríguez-ezpeleta, N. Adapting metabarcoding-based benthic biomonitoring into routine marine ecological status assessment networks. Ecol. Indic. 95, 194–202 (2018).

    Google Scholar 

  • Cronin-O’Reilly, S. et al. Limited congruence exhibited across microbial, meiofaunal and macrofaunal benthic assemblages in a heterogeneous coastal environment. Sci. Rep. 8, 1–10 (2018).

    Google Scholar 

  • Forster, D. et al. Benthic protists: The under-charted majority. FEMS Microbiol. Ecol. 92, 1–11 (2016).

    Google Scholar 

  • Kim, H., Kim, H., Hwang, H. S. & Kim, W. Metagenomic analysis of the marine coastal invertebrates of South Korea as assessed by Ilumina MiSeq. Anim. Cells Syst. (Seoul) 21, 37–44 (2017).

    Google Scholar 

  • Brannock, P. M., Wang, L., Ortmann, A. C., Waits, D. S. & Halanych, K. M. Genetic assessment of meiobenthic community composition and spatial distribution in coastal sediments along northern Gulf of Mexico. Mar. Environ. Res. 119, 166–175 (2016).

    CAS 
    PubMed 

    Google Scholar 

  • Guardiola, M. et al. Spatio-temporal monitoring of deep-sea communities using metabarcoding of sediment DNA and RNA. PeerJ 4, e2807 (2016).

    PubMed 
    PubMed Central 

    Google Scholar 

  • Barnes, M. A. & Turner, C. R. The ecology of environmental DNA and implications for conservation genetics. Conserv. Genet. 17, 1–17 (2016).

    CAS 

    Google Scholar 

  • Bastida-Zavala, J. R. et al. Marine and coastal biodiversity of Oaxaca, Mexico. Check List 9, 329–390 (2013).

    Google Scholar 

  • Nascimento, F. J. A., Lallias, D., Bik, H. M. & Creer, S. Sample size effects on the assessment of eukaryotic diversity and community structure in aquatic sediments using high-throughput sequencing. Sci. Rep. https://doi.org/10.1038/s41598-018-30179-1 (2018).

    Article 
    PubMed 
    PubMed Central 

    Google Scholar 

  • In the Wrong Place: Alien Marine Crustaceans: Distribution, Biology and Impacts, Vol. 6 (2011).

  • Rodríguez-Almaraz, G. A. & García-Madrigal, M. D. S. Crustáceos exóticos invasores. Especies Acuáticas Invasoras en México 347–371 (2014).

  • Gómez, S., Fleeger, J. W., Rocha, A. & Foltz, D. Four new species of Cletocamptus Schmankewitsch, 1875, closely related to Cletocamptus deitersi (Richard) (Copepoda: Harpacticoida). J. Nat. Hist. 38, 2669. https://doi.org/10.1080/0022293031000156240 (2004).

    Article 

    Google Scholar 

  • Ciros Pérez, J., Silva Briano, M. & Elías Gutierrez, M. A new species of Macrothrix (Anomopoda: Macrothricidae) from Central Mexico. Hydrobiologia 319, 159–166 (1996).

    Google Scholar 

  • Fuentes-Reines, J. M., De Roa, E. Z., Morón, E., Gámez, D. & López, C. Conocimiento de la fauna de cladocera (Crustacea: Branchiopoda) de la ciénaga grande de Santa Marta, Colombia. Bol. Investig. Mar. y Costeras 41, 121–164 (2012).

    Google Scholar 

  • Thakur, R. K., Jindal, R., Singh, U. B. & Ahluwalia, A. S. Plankton diversity and water quality assessment of three freshwater lakes of Mandi (Himachal Pradesh, India) with special reference to planktonic indicators. Environ. Monit. Assess. 185, 8355–8373 (2013).

    CAS 
    PubMed 

    Google Scholar 

  • Band-Schmidt, C. J., Bustillos-Guzmán, J. J., López-Cortés, D. J., Núñez-Vázquez, E. & Hernández-Sandoval, F. E. The actual state of the study of harmful algal blooms in Mexico. Hidrobiológica 21, 381–413 (2011).

    Google Scholar 

  • Maciel-Baltazar, E. Dinoflagelados (Dinoflagellata) tóxicos de la costa de Chiapas, México, Pacífico centro oriental. UNED Res. J. 7, 39–48 (2015).

    Google Scholar 

  • Okolodkov, Y. B. & Gárete-Izárraga, I. An annotated checklist od dinoflagellates (Dinophyceae) from the Mexican Pacific. Acta Bot. Mex. 74, 1–154 (2006).

    Google Scholar 

  • Murray, S. A. et al. A fish kill associated with a bloom of Amphidinium carterae in a coastal lagoon in Sydney, Australia. Harmful Algae 49, 19–28 (2015).

    PubMed 
    PubMed Central 

    Google Scholar 

  • Gárate-Lizárraga, I. et al. Seasonality of the dinoflagellate Amphidinium cf. carterae (Dinophyceae: Amphidiniales) in Bahía de la Paz, Gulf of California. Mar. Pollut. Bull. 146, 532–541 (2019).

    PubMed 

    Google Scholar 

  • Varona-Cordero, F. & Gutiérrez, J. Seasonal phytoplankton composition of two coastal lagoons of the tropical Pacific. Hidrobiológica 16, 159–174 (2006).

    Google Scholar 

  • Hyeon, S. & Jin, H. Gyrodinium jinhaense n. sp., a new heterotrophic unarmored dinoflagellate from the coastal waters of Korea. J. Eukaryot. Microbiol. 66, 821–835 (2019).

    Google Scholar 

  • Onuma, R., Watanabe, K. & Horiguchi, T. Pellucidodinium psammophilum gen. & sp. nov. and Nusuttodinium desymbiontum sp. nov. (Dinophyceae), two novel heterotrophs closely related to kleptochloroplastidic dinoflagellates. Phycologia 54, 192–209 (2015).

    Google Scholar 

  • Elliott, M. & Whitfield, A. K. Challenging paradigms in estuarine ecology and management. Estuar. Coast. Shelf Sci. 94, 306–314 (2011).

    Google Scholar 

  • Sreenivasulu, G., Jayaraju, N. & Sundara Raja, R. Physico-chemical parameters of coastal water from Tupilipalem coast, Southeast coast of India. J. Coast. Sci. 2, 34–39 (2015).

    Google Scholar 

  • Landa-Jaime, V. Benthic mollusc assemblage of the Agua Dulce / El Ermitaño lagoon estuarine system, Jalisco, Mexico. Ciencias Mar. 29, 169–184 (2003).

    Google Scholar 

  • Smyth, K. & Elliott, M. Effects of changing salinity on the ecology of the marine environment. In Stressors in the Marine Environment: Physiological and Ecological Responses (eds Solan, M. & Whiteley, N.) 384 (Societal Implications. Oxford University Press, 2016).

    Google Scholar 

  • Rivera-Velázquez, G., Soto, L. A., Salgado-Ugarte, I. H. & Naranjo, E. J. Growth, mortality and migratory pattern of white shrimp (Litopenaeus vannamei, Crustacea, Penaeidae) in the Carretas-Pereyra coastal lagoon system, Mexico. Rev. Biol. Trop. 56, 523–533 (2008).

    PubMed 

    Google Scholar 

  • Gainey, L. F. & Greenberg, M. J. Physiological basis of the species abundance-salinity relationship in molluscs: A speculation*. Mar. Biol. 40, 41–49 (1977).

    CAS 

    Google Scholar 

  • Baqueiro-Cárdenas, E. R., Borabe, L. & Goldaracena-Islas, C. G. Mollusks and pollution. A review. Rev. Mex. Biodivers. 78, 1–7 (2007).

    Google Scholar 

  • Purcell, J. E., Uye, S. & Lo, W. Anthropogenic causes of jellyfish blooms and their direct consequences for humans: A review. Mar. Ecol. Prog. Ser. 350, 153–174 (2007).

    Google Scholar 

  • Nemcová, Y., Pusztai, M., Skaloudová, M. & Neustupa, J. Silica-scaled chrysophytes (Stramenopiles, Ochrophyta) along a salinity gradient: A case study from the Gulf of Bothnia western shore (northern Europe). Hydrobiologia 764, 187–197 (2016).

    Google Scholar 

  • Li, R., Jiao, N., Warren, A. & Xu, D. Changes in community structure of active protistan assemblages from the lower Pearl River to coastal Waters of the South China Sea. Eur. J. Protistol. 63, 72–82 (2018).

    PubMed 

    Google Scholar 

  • Kataoka, T. & Kondo, R. Estuarine, coastal and shelf science protistan community composition in anoxic sediments from three salinity-disparate Japanese lakes . Estuar. Coast. Shelf Sci. 224, 34–42 (2019).

    CAS 

    Google Scholar 

  • Sun, P. et al. Marked seasonality and high spatial variation in estuarine ciliates are driven by exchanges between the ‘abundant’ and ‘intermediate’ biospheres. Sci. Rep. https://doi.org/10.1038/s41598-017-10308-y (2017).

    Article 
    PubMed 
    PubMed Central 

    Google Scholar 

  • Contreras, E. F. O., Castañeda, L. R., Torres, A. & Pérez, M. A. H. Problemática sobre las lagunas costeras mexicanas V, Pesquerías. ContactoSS 25, 36–46 (1998).

    Google Scholar 

  • Reizopoulou, S. & Nicolaidou, A. Benthic diversity of coastal brackish-water lagoons in western Greece. Aquat. Conserv. Mar. Freshw. Ecosyst. 14, 93–102 (2004).

    Google Scholar 

  • Zamorano, P., Barrientos-luján, N. A. & Ramírez-luna, S. Malacofauna del infralitoral rocoso de Agua Blanca, Santa Elena Cozoaltepec, Oaxaca. Cienc. y Mar. 12, 19–33 (2008).

    Google Scholar 

  • Chávez-lópez, Y. & Cruz-gómez, C. New records of polychaetes (Annelida: Polychaeta) from three locations of Oaxaca. Mexico. 67, 157–168 (2019).

    Google Scholar 

  • Thomsen, P. F. et al. Monitoring endangered freshwater biodiversity using environmental DNA. Mol. Ecol. 21, 2565–2573 (2012).

    CAS 
    PubMed 

    Google Scholar 

  • Thomsen, P. F. & Willerslev, E. Environmental DNA—An emerging tool in conservation for monitoring past and present biodiversity. Biol. Conserv. 183, 4–18 (2015).

    Google Scholar 

  • Miller, S. E., Hausmann, A., Hallwachs, W. & Janzen, D. H. Advancing taxonomy and bioinventories with DNA barcodes. Philos. Trans. R. Soc. B Biol. Sci. 371, 20150339 (2016).

    Google Scholar 


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