The incorporation of graphene nanoplatelets (GnPs) within a polymer matrix can play an important role in the physical properties and the functionality of the composite material. Composites consisting of low-density polyethylene (LDPE) and GnPs of different concentrations were developed by mixing GnPs with a molten form of the polymeric matrix. The effect of the GnPs content on the morphological, structural, and electrical properties of the composites were investigated. As shown, graphene presence and its concentration significantly modified the polymer matrix properties, a behavior that can be employed for tailoring its applicability in electrical applications. It was found that the increase of the graphene platelets concentration seems to promote the formation of graphene agglomerates, air gaps, and inhomogeneities, while higher dielectric constant/lower dielectric losses can be achieved. Polymers have been proved to be suitable matrices for the development of composite materials due to their ease of production and processing, resistance to corrosion, good mechanical properties, and light weight. In polymer-based nanocomposites, the type and size of the filler, the morphology of the filler network, the thermal properties, and the conductivity have been considered to be important variables influencing its final properties ,2. PolymPresently, graphene, has become one of the most exciting materials , becauseSubstantial amount of research has been carried out in the field of nanocomposites incorporating graphene, some of them regarding electrical applications. In general, the increasing demand of energy is a technical challenge for the generation, transmission, storage, and distribution energy systems. This challenge often requires contradictory features such as increasing voltage levels in combination with more compact designs in urban environments. This leads to an increased electric stress on the insulation systems, which can be addressed by using insulating materials with tunable properties, as well as high dielectric constant and low losses, especially for electric field grading applications. In this particular research field, the reports in the literature on graphene nanocomposites concern, improvement of conductivity in polyethylene (PE) matrices , advanceThis work concerns nanocomposite materials made using commercial widely available ingredients using methods that can easily undergo direct further scale up, to become compatible with the industrial needs. In particular, in this work, low-density polyethylene (LDPE) was chosen as the basic matrix material for the fabrication of graphene nanocomposites, due to its significant importance in various applications such as squeeze bottles, toys, carrier bags, high frequency insulation, chemical tank linings, heavy duty sacks, general packaging, gas and water pipes . In addi3.Nanocomposite specimens were fabricated by employing commercially available LDPE from Sigma Aldrich GmbH, Darmstadt, Germany with average molecular weight—MW of 35,000 determined by gel permeation chromatography (GPC) and average numerical mass—Mn 7.700 GPC. Graphene nanoplatelets were provided by EMFUTUR Technologies Ltd., Hillsborough, Spain, with ~5 μm lateral size, 5 nm thickness, and a bulk density of 0.03 to 0.1 g/cmThe melting method was used since this is rather simple for fabricating polymer nanocomposites consisting of low molecular weight polymer matrices. This method involves melting of the polymer to its viscous form, followed by the addition and the mechanical mixing of the filler within the polymer matrix. Graphene concentration in the composite material was varied in the range 0% (%wt.) up to 5% (%wt.) at a step of 0.5%. For each composition 5 replicas were made and characterized. All the obtained formulations were characterized as follows:2 flow and a temperature increment of 20 °C/min. Finally, the electrical characterization of the nanocomposite samples was conducted by means of Broadband Dielectric Spectroscopy (BDS), using a TH2829C LCR precision bridge  capable of taking measurements in the frequency range of 20 Hz to 1 MHz.SEM characterization was performed using a (FE-SEM) Nova NanoSEM 630 , equipped with an energy-dispersive X-ray spectroscopy—EDX detector , in order to investigate and understand the formation and the structuring of the obtained nanocomposite materials. All samples were examined in the high vacuum mode, low landing energies and fast dwelling time, without any conductive coating in order to preserve the samples in their natural state for further analyses. XRD was also performed using a Rigaku Ultra high-resolution triple axis multiple reflection SmartLab X-ray Diffraction System . Moreover, Raman analysis was done using a WiTec alpha 300S GmbH Germany system , employing an Nd-YAG laser at 532 nm and confocal Raman microscopy . Raman spectra were recorded under ambient conditions, using a 532 nm diode-pumped solid-state laser  with a power of 10% (10 mW) to avoid local drying of the samples. A 20× objective and a 25 μm slit aperture were used in order to obtain quite detailed spectra, while the total acquisition time 0.5, 10 and 20 s (1 s exposure × 20 exposures) and 600 grooves/mm grating was chosen for each Raman spectrum. The only notable difference observed in spectra was a higher overall spectral intensity when the acquisition time increases. Severally spectra, in different points were recorded per sample and a representative typical spectrum for each sample was chosen. The instrument calibrations were performed on a silicon probe prior to measurements to ensure reproducible results. The spectrometer scanning data collection and processing were carried out using the WiTec Project Five software  and appropriate background corrections were performed on all spectra in Origin software . The thermal response characterization was studied using a Diamond  differential scanning calorimeter (DSC) operated at a scan rate of 10 °C/min in order to characterize the thermal transitions of employed polymer nanocomposites. Samples were prepared inside aluminum shielded pans while an empty aluminum shielded pan was used as a reference. Experimental temperature range was set from ambient to 200 °C. For thermogravimetric analysis (TGA), a Diamond  TGA was used under 200 mL/min N2 areas of the sample in order to form a capacitor. Two metallic pins were attached to the silver paste plates, and these pins were connected to an HIOKI 3532-50 LCR HITESTER , through its specialized cables. Impedance Z and corresponding loss angle δ are simultaneously measured, as a function of frequency, in the frequency range 10 Hz–1 MHz. The presented results are representative for the specific kind of samples.For the low energy impedance spectroscopy measurements, samples of a rectangular shape were used, with dimensions 10 mm × 10 mm × 0.5 mm. Conductive silver paste was smeared onto both the 10 × 10 mmSEM characterization of samples was performed to investigate the formation and structuring of the composite materials. Nanocomposite materials with higher than 2% graphene content were found to consist of a quite inhomogeneous bulk, formed by different polymerization domains as well as regions with concentrated graphene. In addition, at high graphene concentrations, large graphene agglomerations were observed in some cases, suggesting that a limit of concentration might exist, above which graphene cannot be fully dispersed in the LDPE molten material.At graphene concentrations higher than 2.5%, the structural inhomogeneity is accentuated and a mix of compact and granular regions as well as “graphene bubbles” were observed, as can be seen in Following the SEM characterization, one can understand the effect of the graphene concentration on the composites structure and morphology. The increase of concentration seems to promote the formation of graphene agglomerates, air gaps, and inhomogeneities being present at the same time. These structural SEM observations, related to the effect of the graphene concentration on the evolution of the bulk morphology, seem to correlate with the dielectric constant measurements, as it will be shown later. Finally, it should be mentioned here that to the best of our knowledge, such detailed morphology study of LDPE-graphene composite and its evolution with graphene concentration has not yet been presented in the literature before.Structural features for the LDPE composites of different graphene concentrations were further investigated using grazing incidence X-ray diffraction (GI-XRD), where the incidence angle was kept to 0.5°. In order to assess the influence of graphene, the attention was focused in the range of 5°–40°. The results can be seen in With increasing graphene concentration, the occurrence of the characteristic diffraction peak for carbon around 26° appeared for graphene concentrations higher than 2% graphene. To get the mean crystallite size τ for LDPE and graphene composites, the Scherrer equation  was empl002 = 3.37 Å, at the highest graphene concentration.The obtained results strongly indicate the embedding of the graphene in the LDPE lattice and suggest an enhancement of its crystallinity at high graphene concentration. At the same time, the interlayer distance of the graphene employed becomes closer to the hexagonal graphene one (2H-polytype), for which dRaman spectra were also recorded, using a 532 nm diode-pumped solid-state laser  with a power of 10 mW. A 20× objective and a 25 μm slit aperture were used in order to obtain quite detailed spectra, while a total acquisition time of 10 s (1 s exposure × 10 exposures) and 600 grooves/mm grating was chosen for each Raman spectrum. The spectrometer scanning data collection and processing were carried out using the WiTec Project Five software.−1, the 2D band is at about 2786 cm−1, while there is also evidence of the so-called disorder-induced D bands around 1345 cm−1. Literature reports similar peaks for graphene, in particular, in pristine-single layer graphene, the G band appearing at 1582 cm−1 (graphite) and a 2D band at about 2786 cm−1 [−1 and 1578 cm−1 respectively, denoted by the D’, while the I2D/IG ratio increases [−1, while peaks near 1420 cm−1 (the CH2 bending mode) are also present and are indicators of crystalline and amorphous PE phases. Literature reports similar results for polyethylene, both low and high density, with Sato et al. reporting four (4) peaks at 1465, 1374, 1174 and 897 cm−1 [−1 respectively [−1 and 2848 cm−1.Examples of Raman spectra collected on melt-fabricated samples corresponding to various concentrations of graphene are presented in 786 cm−1 ,20. Morencreases . Regardi897 cm−1 . Moreoveectively . It can Regarding local homogeneity of the composite material, an example is presented in Detailed characterization of the obtained nanocomposites showed that all different batches, fabricated under similar conditions, resulted in more or less similar inhomogeneous materials exhibiting similar properties. These results indicated that the mechanical mix of GnPs in melted LDPE may not be the best method for the preparation of homogeneous GnPs/LDPE nanocomposites, at least for the case of small quantities, as those used in our work. Another approach considered for obtaining GnPs/LDPE nanocomposites formulations in small quantities could be dry mixing of the LDPE polymer pellets with graphene but, as tested, it was almost not possible, since they did not really mix. Similarly, we found out that extruding the two phases resulted in even worst composite material. For proper mixing of the specific materials hot roll milling would be more proper method but it is not easily available for laboratory small scale trials. Since GnPs are not a monodisperse material, functionalization of GnPs would not be a realistic approach to solve the dispersion problems at the mixing in the mold. In any case, more homogeneous materials are expected in larger scale production, since, in this cases mixing can be done more effectively.TGA was performed to study the mass loss if a thermal event involves loss of a volatile component. To study the phase transitions of the GnPs/LDPE nanocomposites DSC analysis was performed. Two typical DSC analyses of the 2.5% and 5% graphene containing samples compared with pure LDPE are presented in The melting temperature Tm (°C) for pure LDPE was 80 °C while the crystallization temperature Tc—98 °C. All the composite materials show Tm smaller than the pure material and Tc larger. Tm increases as the GnPs increase and Tc slightly increase when GnPs concentration increased.Finally, the dielectric behavior of all fabricated samples was investigated using BDS and its dependence on the graphene content within the LDPE matrix was analyzed. As expected, the dielectric constant significantly depends on the graphene concentration and the structural characteristics of the material. In particular, the dielectric constant can be strongly influenced by the morphology of the composite, especially if this contains air gaps and agglomerations, as was found out in the SEM results. However, at this point, a quantitative correlation between the structural/morphologic parameters and the dielectric parameters variations is not possible due to the non-quantifiable structural evolution of the composite bulk structuring with increased graphene concentration.Compared to existing literature results, Gaska et al. ,24 studiGnP/LDPE composite materials with various graphene concentrations were fabricated using the melting method and were characterized using SEM, XRD and Raman spectroscopy and microscopy in order to understand the formation of the composites and the evolution of structural and morphological characteristics with increasing graphene content. SEM characterization showed that graphene concentrations up to 2.5% lead to more compact structuring, while larger concentrations promote the random formation of a kind of “graphene bubbles”. XRD characterization showed that mean crystallite size increases as the graphene concentration increases, with a value of 26.1 nm at a concentration of 2%, this becoming 40.4 nm at 5%. Moreover, the increasing of the graphene concentration within the polymer matrix indicate an expansion of crystal interplanar distance of the embedded graphene. Regarding Raman characteristic signals of the composite regions, these appeared to increase in intensity as the graphene concentration increases, revealing the formation of a composite material and not a simple component mixing. The dielectric constants of the nanocomposites presented an increased trend, proportional with graphene increased concentration; however, it was strongly affected by the morphology evolution of the material. The random formation of “graphene bubbles” could induce the formation of conductive tunnels within the samples, this phenomenon being encountered at specimens with concentration of 4.5% and 5%, a behavior requiring further studies.


Hypostomus was studied to shed light on the processes that shaped species diversity. We inferred a calibrated phylogeny, ancestral habitat preference, ancestral areas distribution, and the history of dispersal and vicariance events of this genus. The phylogenetic and distribution analyses indicate that Hypostomus species inhabiting La Plata Basin do not form a monophyletic clade, suggesting that several unrelated ancestral species colonized this basin in the Miocene. Dispersal to other rivers of La Plata Basin started about 8 Mya, followed by habitat shifts and an increased rate of cladogenesis. Amazonian Hypostomus species colonized La Plata Basin several times in the Middle Miocene, probably via the Upper Paraná and the Paraguay rivers that acted as dispersal corridors. During the Miocene, La Plata Basin experienced marine incursions, and geomorphological and climatic changes that reconfigured its drainage pattern, driving dispersal and diversification of Hypostomus. The Miocene marine incursion was a strong barrier and its retraction triggered Hypostomus dispersal, increased speciation rate and ecological diversification. The timing of hydrogeological changes in La Plata Basin coincides well with Hypostomus cladogenetic events, indicating that the history of this basin has acted on the diversification of its biota.Distribution history of the widespread Neotropical genus  However, considerable uncertainty remains about the precise links between the region’s geomorphological history and the evolutionary origin, geographical distribution and diversification pattern of modern Neotropical fish fauna. Among Neotropical freshwater fishes, armored catfishes of the family Loricariidae represent an excellent model to study the effects of landscape evolution on fish lineage diversification. This family is the most species-rich among Siluriformes and has members in all Neotropical basins. Over more than a quarter-century, investigations of the molecular phylogeny of loricariids have resulted in important discoveries and changes in their classification10. However, there are still several unresolved phylogenetic relationships in this family, such as in lineages with fast diversification, or due to the under-representation of species coming from particular river basins. These remaining challenges limit the investigation of important questions regarding the evolution of these Neotropical freshwater fishes.During the Neogene (23 to 2.6 Mya), Neotropical river networks underwent a complex evolution due to marked changes in geomorphology and environmental conditions, most likely driving great biological diversification in freshwater fishesHypostomus is one of the most diverse and widespread members of the family Loricariidae, and inhabits most of the Neotropical Realm11. The phylogeny and systematics of this group has attracted much interest17. Particularly, in La Plata Basin  this genus is represented by more than 50 species, becoming one of the most species-rich genera of this large basin19. This feature is very relevant since La Plata Basin is the third most speciose basin in South America, after the Amazon and the Orinoco basins20. Thus, Hypostomus is an excellent model to comprehend the events that have given rise to the current diversity of fish species in La Plata Basin. However, understanding the causes of this species richness requires detailed species-level knowledge of geographic distribution, ecologically relevant traits and phylogenetic relationships.The genus 21.Understanding when and/or how members of a clade have dispersed to some places and not to others is essential to recognize large-scale patterns of clade distribution. Historical biogeography studies have become focused on using phylogenies to discover the history of connections among small regions or related species. The geographical distribution of a clade will be determined by (i) the ancestral ecological niche and the opportunities for ecological niche shifts that are afforded to species by their biogeographical ranges; (ii) the ancestral distribution area and the limitations  to dispersal; and (iii) the amount of time during which niche shifts and dispersal could have occurred20. Unlike vagile terrestrial organisms, the ability of strictly freshwater fish to move between drainages is limited by the hydrological connectivity. To understand how the complex geomorphological history of La Plata Basin may have affected the geographical distribution of fish species, it is crucial to infer the phylogenies of widely distributed fish lineages. These phylogenies contain information to trace past physical connections and disconnections within and between freshwater habitats22. Paleogeographically structured biogeographic patterns may inform about species divergence times especially when no fossils have been assigned to the clade23.For freshwater fishes, distribution patterns and dispersal dynamics must be interpreted in the context of the geological and geographic histories of river basins24. However, the boundary between both basins has been locally remodeled since then, providing dispersal opportunities between basins for freshwater organisms. For instance, 25proposed a notable river capture event between the Pilcomayo River (La Plata Basin) and the Upper Rio Grande Basin  3.5 Mya, which shifted the divide between the two basins farther north. Also, 26suggested that the Amazon-Paraguay divide is a complex semipermeable geomorphological structure shaped by the Andean orogeny, the megafan environment, and climatic factors, which allowed the migration of several taxa from tributaries of the Amazon to La Plata Basin.Approximately 30 Mya, the Michicola Arc uplifted generating the division between the proto Amazonas-Orinoco and La Plata Basin28 and the second between 10 and 6.8 Mya32. The two marine transgressions covered most of the current Lower Paraná River Basin28, with a considerable impact on the distribution of freshwater organisms .Marine transgressions occurred at least twice in La Plata Basin during the Miocene 15–5 Mya. The first maximum flooding event occurred between 15 and 13 Mya34proposed that in its initial configuration, the Upper Paraná River was pouring its waters directly into the Uruguay River. Later, about 7 Mya, the lower section of the Upper Paraná changed its course towards the west and connected to the Paraguay river (forming the Lower Paraná), resulting in its current configuration. However, according to35, the Upper Paraná River was initially turning eastward and headed towards the Patos lagoon (Brazil). Then it took its current configuration about 2.5 Mya. The current hydrographical pattern of the Paraná and Uruguay rivers indicate that they are exclusively connected via the Río de la Plata estuary. However, several authors38 have proposed recent water pathways between the Middle Paraná and Middle Uruguay rivers during flood periods.The pattern of tributaries of La Plata Basin has undergone numerous modifications through time. Although there is no consensus on the different positions taken by the course of the Paraná River over time, 39. According to40, these capture events have been very numerous between 28 and 15 Mya and may explain the origin of the fish diversity of the coastal rivers of southern Brazil through dispersal processes41.On the east side of the La Plata Basin, multiple river headwater capture events between the Upper Paraná and the coastal rivers of southern Brazil have inferredHypostomus (Loricariidae) as a model group to determine from where and when Amazonian species colonized La Plata Basin, and how they diversified in this basin that has undergone intense orogenic changes. We addressed these questions by inferring a time-calibrated phylogeny for Hypostomus with an almost complete representation of species inhabiting La Plata Basin. Using our chronogram together with dated changes in the hydrological pattern of the basin such as the marine incursions , representing 64% of the species described to date in La Plata Basin and covering all the rivers of this basin. Specimens were euthanized immediately after collection in a solution containing a lethal dose of Eugenol (clove oil). Tissue samples for genetic studies were preserved in 96% ethanol and frozen at − 20 °C. The voucher specimens were fixed in formalin 4% and deposited at MHNG, IPLA, and MACN  and at Fundación de Historia Natural Félix de Azara, Buenos Aires, Argentina (CFA-IC). We included five species of closely related genera as outgroups.We analysed 52 species of https://www.conicet.gov.ar/wp-content/uploads/OCR-RD-20050701-1047.pdf) and its biological sampling guide (https://proyectosinv.conicet.gov.ar/solicitud-scientific-collection/). Also, fish handling during sampling was performed following guidelines of the UFAW Handbook on the Care and Management of Laboratory Animals (http://www.ufaw.org.uk). Fish were anesthetized and killed using water containing a lethal dose of eugenol (clove oil). Other used specimens and tissues are deposited in Museu de Ciências e Tecnologia da PUCRS; Muséum d'histoire naturelle—Ville de Genève; and Laboratório de Ictiologia e Pesca of the Universidade Federal de Rondonia . A full list of analysed specimens and their respective sequence GenBank accession numbers is given in In Argentina, fish were collected with the permission of the local authorities: Ministerio de Ecología y Recursos Naturales Renovables de Misiones (Disp. 2015 and 2016); Ministerio de Producción y Ambiente de Formosa (No. 040/2015); Secretaría de Medio Ambiente y Desarrollo Sustentable de Santa Fe (Res. 081/2015); Ministerio de Asuntos Agrarios de Buenos Aires (Res. 355/10); Dirección de Fauna y Areas Naturales Protegidas de Chaco (Cons. Aut. 2011); Dirección de Flora, Fauna Silvestre y Suelos de Tucumán (Res. 223/2015); Secretaría de Medio Ambiente y Desarrollo Sustentable de Salta (Res. 091/2015) and Dirección General de Bosques y Fauna de Santiago del Estero (Ref. 17461/2015). Our Institutions do not provide the statements on ethical approval of the study. Notwithstanding, this study was approved by the National Council of Scientific and Technical Research of Argentina (exp. 7879/14) and it is a requirement of this institution to follow the guidelines of its "Comité de Etica" (42. Polymerase chain reaction (PCR) was used to obtain two mitochondrial DNA (mtDNA) markers: 592 bp of Control Region (D-loop) and 826 bp of Cytochrome Oxidase Subunit I (COI), and partial sections of three nuclear markers43: 1345 bp of Recombinase Activating gene-1 (RAG1), 1059 bp of Hodz3 gene and 1144 bp of HAMzbtb10-3 gene. Hodz3 (Hypostomus ortholog of the Odz3 gene in vertebrates) encodes a transmembrane protein with a conserved role in eye development in vertebrates44. The anonymous marker HAMzbtb10-3 is most probably a fragment of intron number 3 of the gene zbtb10 (zinc finger and BTB domain containing 10), as identified in the genome of the catfish Ictalurus punctatus . According to SINE Base (http://sines.eimb.ru/), it includes a sequence of 313 bp that is a putative SINE of the Ras1 type45.Genomic DNA was extracted using the salt-extraction protocolEach PCR amplification was performed in a total volume of 50 μl, containing 5 μl of 10 × Taq DNA Polymerase reaction buffer, 1 μl of deoxyribonucleoside triphosphate (dNTP) mix at 10 mM each, 1 μl of each primer at 10 μM, 0.2 μl of Taq DNA Polymerase equivalent to 1 unit of Polymerase per tube, and 1 μl of genomic DNA. Cycles of amplification were programmed as follows: (1) 3 min. at 94 °C , (2) 30 s. at 94 °C, (3) 30 s. at 52–56 °C, (4) 1 min. at 72 °C, and (5) 5 min. at 72 °C . Steps 2 to 4 were repeated 40 times. All used primers are listed in 46. For each marker, we implemented JModelTest2 v.2.1.1047 to estimate appropriate substitution models with the Akaike information criterion (AIC).The editing and manually alignment of the one sequence for each species were performed using BioEdit v.7.0.148 implemented in the R library ape49. A Kendall's coefficient of concordance W value of 0 signifies complete incongruence among loci, while a value of 1 indicates complete concordance. The null hypothesis of complete incongruence among DNA sequence distance matrices was tested with 999 permutations. We also assessed congruence among gene phylogenies by running individual gene tree analyses and calculating gene (gCF) and site (sCF) concordance factors in IQtree v.1.7-beta650 and following51. In these analyses the gCF for each branch is the fraction of decisive gene trees concordant with this branch and the sCF for each branch is the fraction of decisive alignment sites supporting that branch.To assess whether the five used loci could be combined in a joint analysis, we used the Congruence Among Distance Matrices (CADM) test52. Confidence values for internal edges of the ML tree were computed via bootstrapping53, based on 1000 replicates. Second, a Bayesian Inference (BI) analysis was conducted in MrBayes 3.2.7a55. Four chains were run simultaneously  for 10 million generations, with tree space sampled every 100th generation. After a graphical analysis of the evolution of the likelihood scores, the first 300,000 generations were discarded as burn-in. The remaining trees were used to calculate the consensus tree. Both reconstruction (ML and BI) methods were run under the best fit model for the five markers on the CIPRES Science Gateway computing cluster56.We used the concatenated matrix, partitioned by gene, to perform two phylogenetic reconstruction methods. First, maximum likelihood (ML) reconstructions were performed using RAxML v. 8.2.1258. We used a different substitution model for each marker as selected through Jmodeltest2, with the Yule speciation process and a log normal-relaxed molecular clock. The analysis was run with four independent runs for 20 million generations and sampled every 1000th generation. The convergence of the runs and ESS values were investigated using Tracer v.1.7.559 and the standard deviation of split frequencies was checked (below 0.01) to confirm chain convergence. A conservative burn-in of 25% was applied after checking the log-likelihood curves. Finally, a maximum credibility tree with median ages and the 95% highest posterior density (HPD) was subsequently generated using TreeAnnotator v.1.7.558. We used two calibration time points based on dated hydrogeological changes. The first point was proposed by3 and consists in attributing 8 Mya to the speciation between Hypostomus hondae and its closest relative, H. plecostomoides. Hypostomus hondae is distributed only in the Lago Maracaibo and Magdalena basins, while H. plecostomoides is known only from the Orinoco basin and some localities in the upper Amazon. These distribution patterns match the vicariant episode that separated the Lago Maracaibo system from the Amazon and Orinoco basins 8 Mya24. We incorporated a second and new calibration point. Our phylogenetic analyses indicate that H. formosae, distributed in the Pilcomayo River (draining into La Plata Basin), is the closest relative to Hypostomus aff. plecostomus, which comes from the Rio Grande, a tributary of the Madeira River Basin (draining into the Amazon Basin). Since these distribution patterns match the headwater capture proposed by25 3.5 Mya  is the oldest known loricarioid fossil, and was used as a calibration point in recent Loricariidea phylogenetic reconstructions61. The age of this fossil is considered to correspond to the age of the split between the Corydoras lineage and its sister lineage including Hoplosternum and Callichthys genera, among others. As in previous studies, we used as prior 55 Mya and a standard deviation of 1 for the origin of the Corydoras lineage. To do so, we added to our sequence dataset a representative of the Corydoras lineage, Corydoras aff. trineatus, and a representative of its sister lineage, Callichthys callichthys .To verify the divergence dates inferred when using the hydrogeological events as calibration points, we performed new analysis using a fossil belonging to a closely related family  as calibration point. With an estimated age of 55 Mya, the fossil of Corydoras  fossil data and; (iii) using both hydrogeological and fossil data. We then assessed if important disparities in the divergence time estimates were observed among the calibration strategies.In addition, we performed a calibration analysis combining the hydrogeological events and the fossil calibration together. Thus, we used three calibration strategies: (i) using dated hydrological events, (ii) using the Hypostomus in La Plata Basin, we used the Dispersal–Extinction–Cladogenesis model (DEC)62 in the software RevBayes63.To infer the biogeographic history of . (2008): Middle and Lower Paraná Basin and the Endorreic Andean basins  and the collected specimens of CFA, MCP, MACN, MHNG.As our interest focuses on La Plata Basin, we have decided to simplify the hydrological ecoregions of the Amazon, Orinoco, Guiana Shield, and Pacific Coastal drainages in only one region,  during the time window of this marine transgression [time-stratified constraint (15–7 Mya)]. The tables of pairwise area connectivity constraints are given in 23. Both models were run based on default values as described in the RevBayes tutorial , with a total of 10,000 generations. Since the range size of the most widespread species in our phylogeny covers three ecoregions, we constrained the ancestral range reconstruction to include a maximum of three discrete ecoregions.In our analyses, we constrained rates of dispersal between ecoregions that are separated (non-adjacent) by one intercalated region to 1/10 of the dispersal rate between adjacent ecoregions. Moreover, due to Miocene marine transgression  that flooded the Middle and Lower Paraná section with saltwater, and because 64. Habitat preference was taken from the original species description, catalogues, checklists and the authors’ personal field notes:Habitat preference for each species was classified according to one or more of the following five freshwater habitat categories, taken from1.Upland streams and small rivers in Andean piedmont: swiftly flowing, well oxygenated and rich in dissolved minerals. The substrate comprises rocks and stones; usually between 800 and 1200 m above sea level.2.Upland streams and small rivers in shield escarpments: also fast flowing, well oxygenated, but carrying little suspended sediments and dissolved minerals; generally between 200 and 1000 m above sea level.terra-firme streams and medium rivers lying above the extent of seasonal flooding and over Tertiary formations; usually below 200 m contour. Water with suspended sediments and nutrients and tea-like blackwater coloration. Flow rates are typically low; the substrate is comprised of sand and clay.3.Lowland café au lait coloration.4.Deep river channels: deep, swiftly flowing rivers with a seasonal flood pattern; typically exceeding around 3–5 m maximum depth during a typical low water. Turbid water, rich in suspended sediments and nutrients, which imparts a 5. Tidal estuarine systems with freshwater.Hypostomus tree, we ran the rayDISC function in the R package corrHMM65 which is a maximum-likelihood method that allows multi-state characters and polymorphic taxa. Species showing polymorphic habitat states were assigned a priori to occur in distinct habitats. We ran three different models: (i) one-parameter equal rates (ER) model, in which a single rate is estimated for all possible transitions; (ii) symmetric (SYM) model, in which forwards and reverse transitions between states are constrained to be equal; and (iii) all rates different matrix (ARD) model, in which all possible transitions between states receive distinct values. The corrected-Akaike information criterion (AICc) was assessed to choose the best model representing the evolution of habitat preference in Hypostomus phylogeny  across time in the Hypostomus within La Plata Basin. Firstly, using our time-calibrated tree we created lineages through-time (LTT) plot implemented in the R package ape49 based on only Hypostomus from La Plata Basin. To do this we pruned from the Hypostomus phylogenetic tree the specimens not included in the La Plata Basin since the total accumulation of species would thus reflect the capacity of this basin to host new species (new for the basin because they speciated in situ or because they come from other basins).Second, to quantify historical rates of species accumulations within La Plata Basin , we compared our LTT plot with 1000 simulated trees under a constant-rate birth–death model, with identical root age and number of terminals. The simulations were performed using the function ‘ltt.plot’ , and values of speciation and extinction rates to feed this function were estimated using the R package ‘diversitree’ v. 0.9.1066. Louca and Pennell67 revealed that an infinite number of pairs of speciation and extinction rates could give rise to any given LTT plot, and it is thus unclear how to determine the correct diversification rates. Therefore, phylogenies based only on extant species may not provide reliable information about past dynamics of diversification. Then, to circumvent the potential problem that underlies LTT plots, we followed Louca and Pennell’s67 recommendation by calculating the ‘pulled diversification rate’ (PDR). PDR is a variable that is based on a set of congruent diversification scenarios that are compatible with a single LTT, and describes the relative change in diversification rate through time67. To do this analysis, we used the function ‘fit_hbd_pdr_on_grid’ of the R package ‘castor’68. Also, we graphically represented the rate of speciation of the Hypostomus genus only within La Plata Basin. This analysis shows the number of speciation for every 1 Mya time window. We plotted each principal clade separately and the total cladogenetic events for the entire basin. Unlike most of the LTT plots used, our graph is not cumulative.Analyses based on the time-calibrated phylogenetic tree were performed to quantify the evolutionary tempo and mode of diversification in Hypostomus species were represented in the alignment, 32 of which are distributed in La Plata Basin. The concatenated data set has only 1.7% of missing character data. Kendall's coefficient of concordance (W) rejected the incongruence among the matrices of the five loci, globally . Thus, the results of the CADM test indicated that all loci could be combined in a single phylogenetic analysis. The calculated gCf and sCF for each branch of the concatenated tree are shown in The final concatenated alignment comprised 4966 bp, including two mitochondrial (D-loop and COI) and three nuclear markers . A total of 52 Hypostomus , as initially proposed by Montoya–Burgos3, retrieved by Cardoso et al.16 and recently by Jardim de Queiroz et al.43. However, our analyses showed a different relationship among these clades (D1(D2(D3 + D4))) with high BS and PP values. Clade D1, forming the H. cochliodon group, comprised only one species inhabiting La Plata Basin (H. cochliodon). Clade D2, comprising the H. plecostomus group, contained 12 species from La Plata Basin. Clades D3 and D4, forming the H. regani group, were comprised of 19 species from La Plata Basin.Phylogenetic analyses based on the five loci provided substantial resolution of relationships among species of mus Fig. . Both maHypostmus phylogeny based on two dated hydrogeological events as calibration points. We also used the fossil of Corydoras revelatus60 which belongs to a distinct but closely related family, to assess whether the inferred divergence times were congruent between these two calibration strategies  from the rest of the genus. The diversifications of the Clade D1 started 15.56 Mya (10.85–20.6 Mya 95% HPD) and occurred mostly in the Amazon–Orinoco–Guyane  and a large clade including 12 species inhabiting the La Plata Basin . The first split in clade D2 generated on one side Clade D4 originated in the Paraguay ecoregion (B) after a dispersal event from the Amazon basin, while Clade D3 appeared in the initial ecoregion C Fig. . MembersClade D4 contains ten species from La Plata Basin. Its first diversification event started in the Paraguay River  in the species diversification in this basin , and geographically outside from the current La Plata Basin. The polyphyletic origin of Hypostomus species from La Plata Basin, combined with the middle Miocene age (~ 17 Mya) of the three independent colonization events, imply that Hypostomus is not part of the ‘Old Species of La Plata Basin’. However, this younger age of first colonisation agrees with others studies on Hypostomus14 and also on other subfamilies of Siluriformes .Our phylogenetic results show that the Hypostomus lineages from La Plata Basin colonized this basin via the Upper Paraná and Paraguay rivers and their descendants remained confined in this region until the end of the marine incursion which operated as a barrier in the Lower Paraná River (~ 7 Mya).Interestingly, our results suggest that the ancestors of the 74. Conversely, the disappearance of a barrier does not imply the opportunity to disperse for all taxa. Certain geomorphological and climatic processes operating on continental platforms displace barriers to organismal dispersal and probably affect all three processes of macroevolutionary diversification: speciation (species lineage splitting), dispersal (species geographic range expansion), and extinction (species termination)75. However, the dispersal also depends on the species ancestral ecological niche and its opportunities for ecological niche shifts to live in new geographical locations.The predictions of the principle of vicariance assume that multiple lineages will be affected by the raising of the putative barrier, giving origin to several sister-species pairs with similar ages76. Therefore, the dispersal of the Hypostomus lineages, after this marine incursion, depended on the ancestral ecological niche. The ancestral habitat and the geographical starting point for dispersal of all Hypostomus ancestors of La Plata Basin were estimated by our analyses of area and ancestral habitat inferences as follows. The ancestors of Clades D2, D3 and D4 occupied upland streams and small rivers in shield escarpments (habitat 2). The adaptation to lowland terra firme streams and deep river channels (habitats 3 and 4) and the colonization of other ecoregions started ~ 6.5 Mya, short after the regression of the sea. This result indicates that the ancestral species that crossed the lower parts of La Plata Basin after marine water receded had the correct habitat preference for dispersal through this basin, and thus were adapted to live in larger rivers like Lower Paraná. In contrast, the environmental effect of the marine incursion and the ancestral habitat preference of the Clade D1 could explain why the H. cochliodon group showed a very low rate of diversification and dispersion in La Plata Basin. Clade D1 gave rise to a single species that occupies highland streams and small rivers in escarpments in this basin, which it colonized later (~ 8 Mya) than the other clades. In fact, the H. cochliodon group (formerly the genus Cochliodon Kner) and the genus Panaque Eigenmann are capable of eating wood, an exceptional diet among fishes77 and have large, spoon-shaped teeth. In contrast, the other Hypostomus species inhabiting in La Plata Basin have the typical diet of loricariids, they are algivorous or detritivorous. The habitat preference and the diet differentiation reflects a particular ecological niche of La Plata Basin representatives of Clade D1 that could have played a key role in delaying the dispersion of this group in La Plata Basin.The marine incursion was an obvious dispersal barrier during late Miocene; however, its impact on freshwater fish dispersal may have persisted, at least partially, after the marine water receded, because such events can permanently alter soil properties and river water characteristicsHypostomus ancestral species of Clades D2 and D3, their descendants could have disperse throughout the La Plata Basin after the period of sea level regression, a process opening speciation opportunities. Our analysis of LLT plots rejected a model of constant net speciation and extinction and showed an upshift of speciation rate after 5 Mya and other lines of evidence10 suggest that the Paraná River acted as “biogeographical corridor” for the genus Hypostomus to colonize and speciate in other rivers belonging to La Plata Basin today . Even though the LTT plots were strongly criticized recently78, our LTT plot agrees with the PDR and the increase of the number of speciation events in our tree after 7 Mya. Consequently, the Hypostomus species of these rivers, which have a tropical origin, contrast with the xerophilous forests and temperate steppes (grassland and savannas) through which most of the rivers run. The effect of the Paraná River as biogeographical corridor of tropical freshwater-related biota towards temperate latitudes is a pattern described by numerous authors in relation to a number of diverse plant and animal groups81. The existence of a relatively mesic microclimate on the riverbanks and the development of riparian humid forests and wetlands facilitate the dispersion of tropical floristic and faunistic species in temperate latitudes of the Plata Basin, in this way promoting new diversification.Once the Upper Paraná was occupied by Hypostomus, subsequent to the multiple independent colonization events from the Amazon, diversified in La Plata Basin, showing both ecological and morphological diversification. The rate of species diversification within La Plata Basin markedly increased between 5 and 3 Mya. The current species diversity of Hypostomus in La Plata Basin is lower, but species are more phenotypically diverse than their congeners inhabiting other basins in the Neotropical Realm  could interconnect during the rainy season, functioning as portals for the dispersal of fishes possessing the appropriate headwater ecology. However, this suggestion is less supported nowadays, as many of the cases mentioned by Carvalho & Albert26 and others82 have recently been recognized as distinct species by their genetics and/or morphology. For example, Hoplias malabaricus, considered widespread in South America, has been divided into 16 vicariant species83. Inter-basin dispersal of fishes via exceptional portals followed by allopatric speciation has been documented in other regions of South America85, but these flood events would have been older than thought. Because Neotropical ichthyology remains a dynamic field, with many new species described every year86, the hypothesis of contemporary inter-basin dispersal portals opening during the rainy season will probably be challenged more time.The colonization of La Plata Basin has not been carried out exclusively through the biogeographic corridor of Upper Paraná River. In the D1 clade, the ies Fig. , and ourHypostomus highlights the key role played by river captures. Here, we used a well-documented river capture between the Amazon and Paraguay River25 to add a calibration point to the Hypostomus tree. Moreover, in our analysis of ancestral distribution range, we observed two signatures of river capture involving La Plata Basin. First, in Clade D2, we observed a vicariance event at ~ 5 Mya between La Plata Basin and the coastal rivers of southern Brazil (ecoregion G). This split was very likely due to the past capture of the Upper Paraná headwaters by coastal drainages87. Our results and previous ones10 support the invasion by ancestral lineages of Hypostomus from La Plata Basin into the coastal rivers of southern Brazil. Furthermore, different lineages of Neotropical fish show similar patterns of dispersion and colonization. For instance, the time-trees of Hoplosternum and Callichthys show that both Callichthyidae catfish genera reached the coastal rivers of southern Brazil from the Upper Paraná, and that dispersal occurred from the Amazon to the Upper Paraná but apparently never vice-versa73. In contrast, other Loricariidae (Neoplecostominae) and other freshwater fishes (Cynolebiini) had an ancestral origin in the coastal rivers of southern Brazil and dispersed later to La Plata Basin88. According to40, all these river capture events were very numerous between 28 and 15 Mya; however, our results and others73 give evidence that more recent river captures have affected fish dispersal and diversity. Our results indicate a second river capture in Clade D3, in which a very well supported lineage shows a São Francisco species nested within La Plata Basin lineages. This result suggests that the Upper Paraná River was not only a “corridor” permitting the entrance of Amazonian species into La Plata Basin, but acted also as a passageway from the La Plata Basin to the São Francisco and to the coastal rivers of southern Brazil, at least for the Hypostomus genus.Our study of species diversification in the genus Hypostomus species in this basin.Phylogenetic and biogeographical knowledge continues to be essential for the elucidation of species evolution, diversification, and the understanding of distribution patterns. However, it is necessary to note that the reconstruction of area cladograms do not depend solely on the history of geological connections among areas, but also on the history of connections among suitable habitats. The inference of a robust phylogenetic hypothesis further enhances the value of a given group of organisms as a model system for understanding Neotropical biodiversity. Our approach, incorporating different data sets  to analyse the diversification history of a densely sampled fish lineage in the La Plata Basin Fig. , has yieSupplementary Information


Ruditapes philippinarum, a major cultured shellfish species, is threatened by infection with the microparasite Perkinsus olseni, whose prevalence increases with high water temperatures. Under the current trend of climate change, the already severe effects of this parasitic infection might rapidly increase the frequency of mass mortality events. Treating infectious diseases in bivalves is notoriously problematic, therefore selective breeding for resistance represents a key strategy for mitigating the negative impact of pathogens. A crucial step in initiating selective breeding is the estimation of genetic parameters for traits of interest, which relies on the ability to record parentage and accurate phenotypes in a large number of individuals. Here, to estimate the heritability of resistance against P. olseni, a field experiment mirroring conditions in industrial clam production was set up, a genomic tool was developed for parentage assignment, and parasite load was determined through quantitative PCR. A mixed-family cohort of potentially 1,479 clam families was produced in a hatchery by mass spawning of 53 dams and 57 sires. The progenies were seeded in a commercial clam production area in the Venice lagoon, Italy, where high prevalence of P. olseni had previously been reported. Growth and parasite load were monitored every month and, after 1 year, more than 1,000 individuals were collected for DNA samples and phenotype recording. A pooled sequencing approach was carried out using DNA samples from the hatchery broodstock and from a Venice lagoon clam population, providing candidate markers used to develop a 245-SNP panel. Parentage assignment for 246 F1 individuals showed sire and dam representation were high , indicating a very limited risk of inbreeding. Moderate heritability (0.23 ± 0.11–0.35 ± 0.13) was estimated for growth traits , while parasite load showed high heritability, estimated at 0.51 ± 0.20. No significant genetic correlations were found between growth-associated traits and parasite load. Overall, the preliminary results provided by this study show high potential for selecting clams resistant to parasite load. Breeding for resistance may help limit the negative effects of climate change on clam production, as the prevalence of the parasite is predicted to increase under a future scenario of higher temperatures. Finally, the limited genetic correlation between resistance and growth suggests that breeding programs could incorporate dual selection without negative interactions.The Manila clam  Infectious diseases continue to be a major limit in fish and shellfish aquaculture, with production losses estimated to cost over 6 billion USD per annum, and far more in treatments such as vaccinations, cleaning, and antibiotics , 2. The Over the past 20 years, a number of commercial and experimental breeding programs have been introduced for mollusks –18, manyVibrio tapetis and shell repairing has also been reported after three generations of mass selection by SATMAR hatchery . In addition, an important genotype-by-environment (GxE) interaction appeared to have been induced by highly variable ranges of salinity and water temperature between sites.The Manila clam is a bivalve species of particular commercial interest as it represents 25% of global mollusk production worldwide, and although it was introduced to Europe in the early seventies, European production currently accounts for only about 2% of global production –33. To dhatchery . In anotr growth  and resiathogens . In M. polyticus , and difolyticus . Howeverelection –44. As iP. olseni has been identified as a major pathogen of the Manila clam, responsible for mass mortality of up to 70% in experimental infections, and affects clam production worldwide, including in the clam production zones of northern Italy , Legnaro (PD)].An experimental population of clams was produced by mass spawning in May 2016 at the French hatchery SATMAR  from 109 adult clams of hatchery broodstock, according to a two-factorial mating design: 31 dams × 25 sires (775 putative families) and 22 dams × 32 sires (704 putative families), representing potentially 1,479 F1 families. Soon after fertilization, the larvae from the two pools were mixed together and reared at the SATMAR hatchery in a common tank in order to avoid confounding between genetic and common environment effects. After spawning, the parents were sacrificed and a piece of mantle from each of the 109 parent clams was kept in 99% ethanol for future DNA extraction and genotyping. In September 2017 a batch of 10,000 progenies was seeded in Chioggia at 1.1 g , a commercial grow-out site in the Venice lagoon, Italy, where there is a high prevalence of the pathogen P. olseni in the gill tissues of the F1 clams was evaluated monthly since the time of seeding. Each month, about 30 clams were sampled for whole weight, shell weight, and shell length, and the gills were dissected and frozen in liquid nitrogen for subsequent DNA extraction. Biometric measurements allowed us to follow growth parameters of the clam experimental population [Over the course of 1 year, the prevalence and intensity of P. olseni infestation and, having spent 1 year in grow-out conditions, had reached commercial size. This threshold of infestation was chosen according to Chapuis et al., allowing better settings with which to maximize differences between families and thus increase the precision of the genetic parameter estimates  to stimates . The numr parent . This isr parent  demonstrP. olseni, and thus it was possible to carry out total DNA extraction from the same sample for both parasite quantification and host genotyping (see below: P. olseni quantification). All of the individual phenotype data was recorded in the INFAQUA database system dedicated to data storage and provided by SYSAAF. A description of all the traits measured can be found in In our study, 1,000 clams were individually weighed whole, then, meat and shell were separated and the shell was weighed after draining. Sex was determined, where possible, by visualization under a microscope of a gonad smear. Both gills were collected from each individual clam and stored in 1.5 ml 70% ethanol. Gill tissue was selected for the DNA extraction as it is the first tissue to become infected with P. olseni load in individual clams was estimated by real-time PCR using specific primers for P. olseni to amplify 1μl total DNA extracted from one whole clam gill . PCR cycles were performed using a 7,300 Real Time PCR system from Applied Biosystems as follows: 95°C for 10 min; 40 cycles at 95°C for 30 s and 60°C for 1 min followed by 72°C for 5 min and a melting curve at 95°C for 15 s; 60°C for 30 s and 95°C for 15 s  of the 109 Manila clams used as broodstock by the SATMAR hatchery were sent to the BCA laboratory for DNA extraction in March 2018.Fifty individuals of Extractions were carried out individually using Invisorb® DNA Tissue Kit . Total DNA was quantified by Qubit® 2.0 fluorometer and quality was evaluated on a 1% agarose gel. For the Venice lagoon samples, two equimolar pools of 25 individuals/pool were prepared, and the DNA was fragmented using Covaris and prepared for sequencing in 2017. For the broodstock individuals, samples were allocated to equimolar “lower quality” (LQ) and “higher quality” (HQ) pools based on molecular weight as visualized by electrophoresis.R. philippinarum draft reference genome  were prepared in triplicate using TruSeq® DNA Library Prep (Illumina). After Qubit 2.0 quantification and quality evaluation using Agilent 2100 Bioanalyzer, the triplicate libraries were pooled (equimolar) and sent for Illumina HiSeq 4000 for 150-bp paired-end sequencing at the Genome Center of the University of California—Davis. Reads were trimmed to remove low-quality reads using TRIMMOMATIC, and CLC Genomics Workbench was used for aligning raw reads to the e genome . SNP idee genome .SNPs detected by the PoPoolation pipeline in both Venice lagoon clams and hatchery broodstock DNA were selected based on the criteria listed in The 100 bp flanking sequences for the SNPs that matched all of the selection criteria were shared with Labogena Genotyping Company, which designed probe sequences using Illumina DesignStudio® Sequencing Assay Designer, following supplier recommendations. From the initial 250 probes designed, single probes for 245 SNPs were placed on an Illumina Infinium® genotyping chip able to genotype 96 samples/chip.−1) using the Infinium chip and analyzed on a fully automated Illumina iScan® platform, following supplier recommendations. Genotype quality control was carried out using GenomeStudio® software, according to the following standard Labogena clustering procedure: run of Illumina automated clustering algorithm  was included in the software parameters in order to correct assignments lying outside of the mating plan .Ne) were used to estimate the number of parents necessary to maintain genetic diversity, or allele frequencies, within a population. Ne can be calculated assuming random mating and equal parent representation among the F1 population, according to Kimura and Crow  and female (dam) parents, respectively.where Ne can give an indication of the risk of inbreeding, and in order to increase the accuracy of inbreeding estimations, the variability of family representation can be considered when calculating Ne, according to Harney et al.  can be calculated for a population and a number of generations (t) based on the Ne, according to Falconer  was included in the model. Genetic parameters were estimated using pedigree information with the following mixed animal model . a is a vector of additive animal genetic effects distributed following a normal distribution NA is the pedigree relationship matrix and p × p) genetic additive variance-covariance matrix. X and Z are incidence matrices for fixed and genetic additive effects, respectively, and ε is the residual component distributed following a normal distribution NI is the identity matrix and p × p) residual variance-covariance matrix.where h2) was calculated according the following formula:Heritability (p = 1), genetic additive covariance was estimated and genetic correlations (Rg) between two traits were calculated as follows:where Cova is the genetic additive covariance between trait 1 and trait 2, a(trait1)σ and a(trait2)σ are the standard deviations of additive genetic effect for trait 1 and trait 2, respectively.where Sampling occurred monthly over the course of 1 year, from the time of seeding (T0) to the final sampling event. Time-points and days post-seeding (dps) are detailed in At the time of seeding in the grow-out area, average shell length was 1.75 cm (SD = 0.33) and average whole weight was 1.115 g (SD = 0.626). At the final sampling date, 355 days post seeding (dps), average shell length was 3.48 cm (SD = 0.37) and average whole weight was 11.918 g (SD = 3.577). Growth, represented as average shell length over time, is reported in P. olseni (>10 copies.ng−1). Prevalence continued to increase, reaching 36.7% at T10 (322 dps), 1 month prior to the final sampling event (355 dps). The rise in P. olseni prevalence follows a trend similar to that of the average monthly water temperature, as it is well-known that the parasite is more virulent at higher temperatures. Final sampling was carried out when clams reached the commercial size for harvesting and at the end of the warmest summer months, when it was expected the prevalence would rise considerably. After diagnosis of all the sampled F1 individuals, the final prevalence was 51.5%.At each time-point over the course of the year-long monitoring, gill samples were collected from each of the clams and used for DNA extraction. Real-time PCR was carried out at the IZSve to quantify the parasite DNA for all samples from T4 (130 dps) to the final sampling (355 dps). Diagnosis was not carried out on samples from T0 to T3 (0 to 93 dps) as parasite levels were below the limit of detection (LOD). Prevalence (considered as the percent of samples above LOD) began to increase at T5 (187 dps), where 4.5% of the samples tested positive for Perkinsus-positive individuals above the limit of quantification (Perkinsus-free (negative).During the mass sampling event in Chioggia, clams were measured for total weight, shell weight, and shell length. Sex was recorded whenever it was possible to unambiguously assess gonadal sex, and final male to female sex ratio was 47:53 . Parasitfication . PositivP. olseni DNA, 268 showed detectable parasite DNA, but below the limit of quantification (LOQ: > 30 threshold cycle (Ct); equivalent to 100 copies of P. olseni DNA), and 544 samples contained a quantifiable amount of P. olseni DNA . Parentage assignment to a single parent pair was successful for 246 (41%) of the genotyped samples.n = 246), 163 families (or parent-pairs) containing 42 sires and 45 dams were observed out of the 1,479 putative full-sib families from the two-factorial mating design.Of the 56 sires and 53 dams used to produce the F1 clams, parentage assignment showed that most were represented in the F1 clams . DespiteNe), based on number of parents that were used in the mass spawning, was 108.9. After parentage assignment of the F1 clams, the observed Ne  was 86.9. When considering the variability in representation of the parents, i.e. the variance of the number of offspring per sire and dam,  is 1.28%.The initial expected effective population size (h2) was estimated for each trait, and genetic correlations (Rgtrait) between traits were calculated pairwise  and 0.97 ± 0.03 (SW vs. TW). The average measured values for biometric traits between negative, detectably (<LOQ), and quantifiably infected clams  tended tShifts in global temperature levels associated with climate change and other anthropogenic pressures are having increasingly negative impacts on marine ecosystems, namely with regard to pathogens. The geographical ranges of hosts and pathogens are being modified and their respective resistance capacity and virulence altered, leading to devastating mortality events due to both emerging and previously described pathogens. The viability of bivalve production will largely depend on the ability of the species to adapt to the future environmental conditions, through either natural or artificial selection of those individuals that are genetically more resistant to pathogens and stressors associated with global warming . To asseP. olseni under commercial conditions. Analysis of the growth trajectory confirmed previous findings for on-growing sites in Adriatic Sea lagoons, including the Venice lagoon (P. olseni was first detected in 4.5% of the F1 clams in March (T5), increasing steadily with the seasonal rise in seawater temperature. Optimal temperatures for proliferation of P. olseni have been shown to range between 20°C and 34°C , excluding reproductive success variance, was close to the expected Ne of 108.9), indicating that high genetic variability may be conserved when using DNA-parentage assignment to manage broodstock and limit over-representation of certain parents. When considering variance in reproductive success  Ne dropped to 39.0. When the observed Ne values were used to estimate inbreeding rate after one generation (ΔF = 1/(2*Ne), estimated ΔF was lower (0.006) or slightly higher (0.013) than the internationally recommend threshold (ΔF < 0.01) to avoid excessive increase of inbreeding rate in farmed animals  appeared to have contributed to the offspring at the time of analysis, over 2 years after the reproductive event. Comparable values of parent representation were reported in four hatchery populations of Pacific oyster, ranging between 64 and 100% for sires and 68–96% for dams. The clam progeny used here were obtained using mass spawning, a technique where all sires and dams are mixed together and release sperms and eggs in the same tank. In aquatic species such as bivalves, where a large number of animals participate in the spawning event, often only a limited number of sires and dams significantly contribute to the offspring, strongly reducing the effective population size , 77. The animals . In otheneration , while ineration . Even coiability , 82. Maiiability .h2 = 0.26 in the Manila clam . These heritability estimates fall within the range of realized heritability previously reported by mass selection for shell length of ila clam . While tTotal weight is highly correlated with shell weight (0.97) and shell length (0.9). This indicates that rapid assessment of growth can be done on the field by selecting only shell length. Similarly, as shell weight is highly correlated to total weight, it means that measurement of growth on sibs could be delayed and assessed by considering shell weight. However, as shell weight and length are less genetically correlated (0.78), and genetic correlation is lower than 0.8, these two last traits have to be considered as separate traits.When considering genetic correlations between traits, the correlated response to selection of one trait on another trait highlights the possible benefit the 52% higher heritability of SW compared to TW (0.35 vs. 0.23). Based on the Falconer (1960) formula  to estimP. olseni was estimated at 0.51 ± 0.20, indicating high potential for genetic improvement of this trait, though results should be interpreted with caution due to the low number of assigned individuals. Infections due to Perkinsus, with time and given the right environmental conditions, are likely to affect up to 100% of a Manila clam population. In the present study parasite load was recorded after 1 year on the field. In the past, natural spat was routinely used to seed on-growing areas, therefore clams were exposed to potential infection with P. olseni for a longer period (1.5–2 years) as the entire life-cycle occurred in the field. In recent years a dramatic drop in natural recruitment has forced producers to use hatchery-produced spat of larger size (12–15 mm), with a reduced potential exposure to P. olseni, much more closely resembling the experimental protocol that was followed in this study. It is important to consider that individual parasite load 1 year post-seeding was used a as a measure of resistance, which, in the case of chronic infections, may be an indirect assessment for resistance. In Penaeus monodon a limited genetic correlation was reported between gill-associated virus (GAV) load and survival (C. virginica regarding infection with P. marinus (see Introduction), evaluations for resistance to disease should integrate potential GxE effects brought on by variations in salinity or temperature between sites. Our results would thus greatly benefit from validation through future evaluations of response to realized selection experiments and larger data sets.Regarding disease resistance, heritability for parasite load with survival . MoreoveInterestingly, there was no genetic correlation between parasite load and growth-associated traits. Because of the limited number of analyzed individuals, however, the standard error for these estimates is large and caution should be exerted when considering genetic correlation. If the evidence of no genetic correlation between growth and parasite load will be confirmed using a large set of animals, then selection for one of these two traits may have no direct effect on the performance of the other, indicating that dual genetic selection could be implemented without running the risk of inducing a negative effect.P. olseni indicate there is a significant possibility of limiting the impact of this disease through genetic selection, not only in European clam aquaculture but also more broadly, as mass infestation and mortality due to this pathogen has also been frequently reported in Asia (P. olseni infection intensity and prevalence, it is likely that selected families that perform best in this specific environment may not perform as well for any of the selected traits in another clam production are such as along the Asian coast.Understanding the genetic correlations between various phenotypes is essential, as focusing selection on certain traits of interest can provoke devastating effects on other health-related traits, as demonstrated extensively in terrestrial livestock selection , 86. Ove in Asia –90. ThatP. olseni. Being a chronic parasitic disease, directly challenging selection candidates might lead to introducing the parasite in the broodstock population, while sib testing would allow to maintain a Perkinsus-free broodstock. In turn, this should guarantee the production of parasite-free spat.While the Manila clam is one of the most produced bivalve species in the world, the availability of genetic tools necessary to initiate selective breeding lag far behind those developed for most other cultured fish and shellfish. Here we showed that it is possible to design and carry out field challenges for one of the major clam pathogens, simulating commercial growing conditions. Likewise, we demonstrated that using mass spawning, the routine method for reproduction of clams in hatcheries, yields a sufficiently high number of full-sib families to reliably estimate genetic parameters and to provide a sufficient number of selection candidates. The development of a SNP panel allowed for partial parentage assignment, albeit further improvement in successful genotyping and assignment rate is required for greater efficiency and more accurate estimation of genetic parameters. The possibility of reconstructing pedigrees is important not only for estimating genetic parameters and managing inbreeding, but it will be required to use sib testing for measuring phenotypes that either cannot be recorded in selection candidates or could be risky to measure on them, as in the case of resistance to Finally, these preliminary results indicate that the high heritability for resistance against perkinsosis and the apparent lack of genetic correlation with growth-traits hold the promise to mitigate the effects of this climate-related parasitic disease without affecting growth performance.https://doi.org/10.5061/dryad.rr4xgxd6d.The dataset can be found at: MS, CP, LB, and FE designed the experiment. EV and J-FA produced the experimental population. GA and GD carried out the Perkinsus analysis. SF and MS contributed to the bioinformatic analysis. LG and RM carried out the genotyping. FE, MS, and PH carried the quantitive genetic analysis. MS and LB drafted the manuscript. All authors contributed comments to the draft.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The initial concentrations of surfactants were tested within the range of 3–17 g·L−1. The pH was also modified from 7 to >12. The uptake of nonionic surfactants into the organic phase was higher than the anionic surfactants. Solubilization of COCs with the nonionic surfactants showed similar molar solubilization ratios (MSR = 4.33 mmolCOCs·g−1surf), higher than SDS (MSR = 0.70 mmolCOCs·g−1SDS). Furthermore, under strong alkaline conditions, the MSR value of the nonionic surfactants was unchanged, and the MSR of SDS value increased (MSR = 1.32 mmolCOCs·g−1SDS). The nonionic surfactants did not produce preferential solubilization of COCs; meanwhile, SDS preferentially dissolved the more polar compounds in DNAPL. The time required to reach phase equilibrium was between 24 and 48 h, and this contact time should be assured to optimize the effect of the surfactant injected on COC solubilization.Application of surfactants in the remediation of polluted sites with dense nonaqueous phase liquid (DNAPL) still requires knowledge of partitioning between surfactants and pollutants in the organic and aqueous phases and the time necessary to reach this balance. Two real DNAPLs, generated as wastes in the lindane production and taken from the polluted sites from Sabiñanigo (Spain), were used for investigating the solubilization of 28 chlorinated organic compounds (COCs) applying aqueous surfactant solutions of three nonionic surfactants (E-Mulse Contamination of soil and groundwater by organic compounds from industrial activities has become a major environmental problem . This coDNAPLs include common industrial solvents  or other hazardous substances such as creosote and coal tar. Other chlorinated organic pollutants forming DNAPLs include pesticides and chlorinated compounds used for their synthesis ,6,7. MosThe remediation of sites polluted by DNAPLs poses essential technical and economic challenges. Conventional treatment technologies such as pump and treat have potentially high life cycle costs ,13, and Surfactants are amphiphilic compounds, and the hydrophilic group is an ionic  or highly polar group (nonionic) . When a j·mol−1surf [j·mol−1surf depending on the volume ratio of organic and aqueous phases using tetrachloroethylene and Tergitol NP15 as surfactants [j·mol−1surf [j·mol−1surf for the Tween 40/trichloroethylene system and around 5 molj·mol−1surf for Tween 80/tetrachloroethylene [Therefore, the equilibrium of the compounds presented in the DNAPL between the aqueous and the organic phase must be known for a reliable design of the Soil Flushing remediation treatment. This equilibrium is modeled using the mass solubilization ratio (WSR) or molar solubilization ratio (MSR). WSR is the ratio of the mass of solubilized organic compounds per unit of surfactant mass in micellar solution. MSR is defined as the moles of solubilized organic compounds per mole of surfactant in micellar solution ,27. Sharol−1surf . Kibbey factants . Zimmermol−1surf . Kang etethylene . The extent of micellar solubilization depends on many factors , ionic strength, temperature, and the size and chemistry of the solute) . The HLBAdditionally, due to the amphiphilic nature of the surfactant molecule, when a surfactant solution is in contact with an organic phase (such as the DNAPL), the partition of the surfactant between aqueous and organic phases has to be considered to determine the remaining amount of surfactant in solution ,31. The Correlations between the partition of a surfactant in both phases and the properties of that surfactant have been investigated in the literature. Catanoiu et al.  used thrThe pH effect on the partitioning equilibria has been scarcely studied, although it is proved that alkaline conditions can produce dehydrochlorination reactions of pesticides as lindane ,40 or alIn this research, the partitioning equilibrium of surfactants and organic compounds between the organic and the aqueous phases has been studied using two different DNAPL samples. These DNAPLs were extracted from Sardas (S) and Bailin (B) landfills, caused by the dumping of liquid wastes from lindane production  containiTo the best of our knowledge, the works available in literature studying these partitioning equilibria use pure organic compounds ,31,36 orIn addition, the effect of alkaline pH on partition equilibria (scarcely studied in literature) is analyzed since strong alkaline conditions can be used in a real treatment to promote the dehydrochlorination reactions of the most toxic COCs in DNAPLs.® 3, used in the remediation of sites with DNAPLs at full scale [Biodegradable nonionic and ionic surfactants have been selected, while good biodegradability is required for the application of a surfactant in aquifer remediation. Tween 80 and Span 80 or their mixtures have been proved to be readily biodegradable ,44 and wll scale  has alsoll scale , has bee® 3 (E3), Tween®80 (T80), and a mixture of Tween®80 at 35% and Span®80 at 65% (TS80). This mixture was tested in a previous laboratory study carried out by the Aragon Government using a DNAPL from the Sardas landfill  [Three nonionic surfactants and an ionic surfactant were used. The nonionic surfactants tested were E-Mulse, Spain) . MoreoveThe anionic surfactant selected was sodium dodecyl sulfate (SDS), which is also typically used in soil washing. The identification of surfactants and their main chemical properties are shown in the DNAPL for both DNAPLs is also included . As can be seen, both DNAPLs contain up to 28 chlorinated organic compounds (COCs): chlorobenzene (CB) and different isomers of dichlorobenzene (DCBs), trichlorobemzenes (TCBs), tetrachlorobenzene (TetraCBs), pentachlorocyclohexene (PentaCXs), hexachlorocyclohexanes (HCHs), and heptachlorocyclohexanes (HeptaCHs). Compound distribution is quite similar to that mentioned in previous research [Two different DNAPL samples obtained from the Sabiñánigo landfills and provided by the company EMGRISA and the Aragon Government were used. One DNAPL sample was obtained from the Sardas landfill (S) and the other sample from the Bailin landfill (B). The composition (molar fraction) of DNAPLs used in this research is summarized in research . These C−1). These experiments were carried out on both neutral and alkaline pH (pH > 12). An alkaline pH was achieved by adding NaOH up to 7 g L−1 in the aqueous phase containing the surfactant. Solubility experiments were conducted in sealed GC 20 mL glass vials without headspace, closed with PTFE caps. A mass of 0.4 g of each organic phase, DNAPL: B, and DNAPL: S, was added to 19.6 g of the aqueous phase containing the surfactant (surfactant concentration ranging from 0 to 17 g LThe biphasic mixture (organic and aqueous) was agitated using a magnetic stirrer under room conditions for 5 h at room-controlled temperature (T = 22 ± 2 °C). Then, the agitation was stopped and different vials were sacrificed at 0.5, 1, 3, 24, 48, and 75 h to analyze the aqueous emulsion. The experiments were carried in triplicate. The differences were lower than 10%. The qualitative identification of dissolved COCs in the aqueous phase was carried out using a gas chromatograph (GC)  along with a mass selective detector . The quantification of COCs in the emulsion was carried out using a GC  with both a flame ionization detector (FID)  and an electron capture detector (ECD) , simultaneously. The GC methods are described elsewhere [The total organic carbon (TOC) of the supernatant (aqueous phase with the surfactant and solubilized COCs) was measured once the equilibrium was reached between phases (COC concentration in emulsion did not change over time). A Shimadzu TOC-V  analyzer was used. The concentration of nonionic surfactant under equilibrium conditions was determined from the TOC value and sum of COCs in the emulsion.2CO3 (3.2 mmol·L−1) and NaHCO3 (1 mmol·L−1).Ionic surfactant (SDS) concentration in aqueous phases was quantified by measuring sulfate anions in an aqueous solution using IC . The column used as a stationary phase was Metrosep A SUPP5 5-250  , and the mobile phase used was an aqueous solution of NaTo ensure that pH conditions were maintained throughout the entire experiment, the pH was also analyzed in the samples using a 914 pH/Conductometer .surf,ORG)eqeqEQ under equilibrium conditions is plotted at pH = 7 in surf·g−1ORG, when surfactant concentration in the aqueous phase is higher than 4 g·L−1. Lower partition ratios between the organic and aqueous phases were obtained using T80, TS80, and SDS. The partition ratios of T80 and TS80 were similar. Meanwhile, the lowest values were found with SDS. Moreover, asymptotic values are not achieved with T80, TS80, and SDS for concentrations lower than 15 g·L−1. Differences in the partition behavior of the surfactant tested at neutral pH indicate a higher affinity of E3 for organic phase B.As can be seen in By comparing the results in −1. On the contrary, the higher the pH, the higher the affinity of SDS for the organic phase. These effects can be explained, taking into account that the presence of electrolytes in the aqueous phase can modify the partition of the surfactant [Results obtained at pH > 12 are shown in rfactant . Moreoves,ORGC is the saturation concentration of surfactant in the organic phase, in gsurf·g−1ORG, and sK the constant affinity of the surfactant for the organic phase, in L·g−1surf.To model the partition of surfactant between organic and aqueous phases, some authors ,31,37 haLK is the linear partitioning parameter [surf,ORG·L·g−1surf·g−1ORG. Equation (1) can be simplified using Equation (2) for small values of LK. The latter parameter is the product of s,ORGC by sK in Equation (2).The experimental values shown in arameter  in gsurfLK parameter was estimated. Since the four surfactants had a similar partitioning behavior under alkaline conditions, the experimental data was fitted together. A unique value of LK was obtained for each DNAPL, which is also shown in LK were obtained with DNAPL: S, indicating that a lower affinity of this organic phase S for the surfactant is also found at an alkaline pH.The experimental data obtained in alkaline conditions was fitted to Equation (2) using linear regression and a LK at pH = 7 are also shown in LK values for nonionic surfactants are obtained at a neutral pH, for both DANPLs B and S. On the contrary, higher LK values are obtained using the anionic surfactant SDS under alkaline conditions. In all cases, it is confirmed that pH has a strong influence on the affinity of the surfactant for the organic phases.The corresponding values of The solubilization experiments carried out are summarized in As explained in the experimental part, for each experiment in j,AQC is the concentration of j in the aqueous phase, mg·L−1 and jMW are the molecular weight of j. In mg·mmol−1, COC isomers have been lumped together, so j refers to CB, the sum of DCBs, TCBs, TetraCBs, PCB, PentaCXs, HexaCXs, HCHs, and HeptaCHs isomers.As the DNAPL samples used in this research are a complex mixture of COCs, a study has been conducted to see if the surfactants selectively dissolve some compounds in the mixture. The molar distribution of COCs in the DNAPL sample is summarized in −1 and 15 g·L−1) at neutral pH are shown in The molar percentages of solubilized COCs ) under equilibrium conditions at two different initial surfactant concentrations  parameters defined in Equation (4).−1, vs. the concentration of surfactant in the aqueous phase under equilibrium conditions, calculated by −1, has been plotted for both pHs and DNAPLs studied. As can be seen in In Moreover, the higher the surfactant concentration in solution, the higher the solubilized COC concentration, meaning that an almost linear trend is found between both variables in the concentration range studied. On the other hand, lower solubilization is achieved with SDS in comparison with that obtained with nonionic surfactants, following that reported by Zhou et al. in previous research .The slope of the plots in The SMR values obtained were in line with those reported by Pei et al. for the solubilization of DCBs using different surfactants .−1 and 1.0 mmol·L−1 for B and S, respectively, following previous results [In the linear regressions, the intercept corresponds to the solubilized DNAPL in the absence of the surfactant: 0.9 mmol·L results .The surfactants studied here  and one anionic surfactant (SDS)) significantly improved the solubilization of a complex mixture of chlorinated organic compounds contained in real DNAPL present in two landfills polluted with liquid organic wastes from lindane production. This improvement was remarkably greater for all nonionic surfactants tested, finding similar values of molar solubilization ratios (MSR) regardless of the pH used. Moreover, a significant partition of the surfactants was found between the organic and the aqueous phases. The nonionic surfactant presented a higher affinity for the organic phase at neutral pH. For each DNAPL, the composition of COCs in the micellar core was similar to the initial composition of the organic phase for the three nonionic surfactants tested. In contrast, the anionic surfactant selectively solubilized the most polar compounds in DNAPL. Moreover, under strong alkaline conditions, dehydrochlorination of some COCs trapped in the micelles was noticed obtaining an emulsion with less toxic COC. These findings are relevant in the design of the surfactant-enhanced remediation process of the site.


Erosive tooth wear has been a highly prevalent and emerging phenomenon related to eating habits of the population. Aim: This study sought to investigate industrialized fruit juices exposure in enamel and dentine substrates in terms of erosive effect. Human enamel and dentine specimens were randomized into 8 groups (n=8): Grape juice – Ades®, Grape juice – Del Valle Kapo®, Grape juice – Aurora®, Orange juice – Del Valle Kapo®, Orange juice – Ades®, Strawberry juice – Mais Vita®, Strawberry juice – Ades®, Citrus fruit juice – Tampico®. Specimens were submitted to an in vitro erosive challenge and to a microhardness test to evaluate the percentage of surface microhardness loss. The pH, titratable acidity, buffering capacity, degree of saturation and critical pH concerning hydroxyapatite and fluorapatite of the juices were measured as well as their composition of calcium, phosphate, fluoride, and total protein. Data were submitted to the analysis of variance and multivariate linear regression (α=0.05). All test agents were undersaturated concerning hydroxyapatite and fluorapatite. A significant interaction between the type of juice and substrate was found . However, Orange juice – Del Valle Kapo®, Orange juice – Ades®, and Strawberry juice – Mais Vitta® demonstrated no difference between substrates. Grape juice – Ades® promoted less mineral than other juices in enamel and dentine. The calcium concentration in juices was a protective variable for microhardness loss in both substrates. The erosive effect of industrialized fruit juices affects enamel differently from dentine, and this effect differed between some, but not all, tested juices.  Key words:Tooth erosion, dental enamel, dentine. beverages, food habits. Lifestyle changes in the past few decades, mainly those related to the patters of food consumption (high intake of acidic drinks and food), call the attention of the global scientific community for erosive tooth wear (ETW) as highly prevalent and emergent phenomena -3. EpideThe erosive lesions caused by acidic foods and beverages were related to their physicochemical properties as well as the concentration of acids, power of hydrogen, titratable acidity, buffering capacity, inorganic concentration of ions calcium, phosphate, fluoride, and mineral chelating properties. Therefore, a potentially erosive agent should have reduced ionic activities of Ca2+, PO43–, and F- and negative saturation degree concerning hydroxyapatite (HAp) and fluorapatite (FAp) crystals . BesidesThe erosive process in the enamel and dentine substrates is different due to their different histopathological aspects. While enamel erosion was expressed as superficial demineralization followed by softening and substance loss, dentine erosion beginning with a mineral loss between peritubular and intertubular dentine with subsequent peritubular dentine loss and tubule orifice enlargement. Besides, differences in organic and water components play an important role in the diffusion of H+ ions, which could determine a different behavior when exposed to erosive agents ,11.in vitro study was to evaluate the erosive effect of commonly available industrialized fruit juices in enamel and dentine substrates.Dentine erosion was particularly evident in adults with physiological or pathological root exposure, and the more relevant clinical consequence could be dentine hypersensitivity and an increase in biofilm stagnation. Prevalence data are scarce in adults individuals, even though data from epidemiological studies evidenced that erosive lesions tend to increase and became more severe during the aging process . Given t-Ethical ConsiderationConsidering the use of human teeth, a Research Ethics Committee in Brazil approved this study (CAAE: 17581413.4.0000.5188). The teeth donors were explained about the aims and relevance of this research and signed the Informed Consent Form according to the Helsinki Declaration and the Brazilian resolution (CNS/MS 466/12).-Experimental DesignThis research was conducted with human enamel and dentine samples in three stages:Stage 1: The selected juices were evaluated in terms of pH, titraTable acidity (TA), buffering capacity (β) and the amount of calcium [Ca2+], inorganic phosphate [Pi], fluoride [F-], total protein composition, and degree of saturation (pK − pI) and critical pH concerning hydroxyapatite (HAp) and fluorapatite (FAp).Stage 2: Microhardness test to evaluate the percentage of surface microhardness loss (%SMHL) was performed in human enamel N=64) and dentine (N=64) blocks. These specimens were randomly assigned into the following eight  fruit ju and dentStage 3: Erosive challenge and subsequent microhardness test to evaluate the %SMHL.-BeveragesReady-to-drink fruit juices, potentially erosive and frequently consumed, identified in Brazilian supermarkets were selected according to the flavor and manufacturing. They were kept closed at room temperature until the day of analysis and experiment. The composition, brand, and manufacturer of then are present in Box 1.-Physicochemical Characterization of the BeveragesInitially, the selected beverages had their pH measured shortly afterward the opening by means an electrode connected to a pH meter . In 50 ml of beverages was added 0.5 ml of 1 M NaOH until the pH of 5.5 and 7.0, under constant agitation, to calculate the titratable acidity (TA). It was used the Equation: β=∆C/∆pH to calculate the buffering capacity (β), where ΔC is the quantity of base used and ΔpH is the alteration in pH caused by the addition of the base  for measurements of calcium and phosphate concentrations. Briefly, it was pipetted 25 µL of each beverage in a 96-well plate to react with a calcium-sensitive reagent (Arsenazo III) and reducing phosphate agents . The reader was pre-calibrated with a standard curve of calcium carbonate (0- 100 mg/L) and phosphate (0-8.27 mg/L). The readings were performed using the absorbance of λ 650 nm for calcium and the absorbance of λ 660 nm for phosphate . Using aThe fluoride analysis was performed using the microdiffusion method . A fluorCalcium, phosphate, and fluoride concentrations were shown as mmol/L.The Bradford colorimetric method  using BrDegree of Saturation and Critical pH of BeveragesPi, and F- values of each juice and assuming a solubility product of 10-58•5 and 10-59•6 for HAp and FAp, respectively. Alternatively, the critical pH was assessed through the simulation of the degree of saturation concerning HAp and FAp at several pH values. Thus, the pH value at which the solution is exactly saturated concerning HAp and FAp  and Orange (0.7) juices, f is a function of α (0.05) and β (0.05), sd is the standard deviation of the Grape Juice (0.1). In each group, the final sample size was 6.5 enamel blocks and it was raised by 20% for offset possible losses during the experiment. Thus, 8 samples in each experimental group were necessaries.One hundred twenty-eight human third molars were collected and these teeth were equally divided into enamel and dentine experiments. Initially, they were cleaned through scraping with periodontal curettes and stored in buffered 10% formalin (pH 7.0) solution. After this, each teeth surface was examined through a magnifying glass with an increase of 5x to ensure that the selected tooth did not have caries, cracks, intrinsic staining, and none malformation in the enamel and dentine tissue.After the teeth selection, a double-sided disk of the precision diamond cutter , under slow-speed and constant irrigation, was used. Enamel and dentine blocks (3x3x2 mm) were obtained from the dental crown . The specimens were embedded in self-cured acrylic resin circular using molds of 16mm diameter and 3mm deep. Later, a metallographic polisher was used to ground flat the outer enamel  and dentine  surfaces, under constant irrigation. The polishing enamel and dentine surfaces were performed with wet felts and 1 µm diamond suspension  in a rotating polishing Machine PSK- 2V . Finally, all samples were included in a sonication with water for 5 min and stored under -20ºC until the day of the experiment.-Erosive ChallengeThe enamel and dentine samples were immersed into 50 ml (5.56 ml of the fruit juice/ mm2 exposed enamel or dentine specimens), at room temperature (22-25ºC), for 120 minutes , under g-Analyses of Microhardness A microhardness machine  determined the surface hardness of the enamel and dentine specimens before and after the erosive challenge. For this, a Vickers diamond with 100g for 15 seconds was used for the enamel specimens and 50g for 10 seconds for dentine specimens. Initially, in the dentine and enamel samples sound surface, three indentations were performed with an interval of 70 µm from one indentation to another. The dimension of each indentation was used for WIN-HCU software to calculate the hardness of the tissues. After the erosive challenge, the same procedure was performed in the eroded area. Finally, the %SMHL was calculated using the following equation: Fig. .Where the SMH0 is the average microhardness of the sound tissues and SMH1 is the average microhardness of the eroded tissues.-Statistical Approach Data were statistically analyzed using the SPSS package for Windows, version 21.0 . Data normality and homogeneity of variances were tested using the Shapiro-Wilk test and Levine test, respectively. Since data demonstrated homogeneity of variances and Gaussian distribution, no data transformation was needed.p-value of Levene’s test of equality of error variances was equal to 0.128). Alpha (α), beta (β), and partial eta squared (ηp²) represent, respectively, the type I error, effect size, and the strength of each the effect. Simple effects were used to assess the difference between the substrate within each juice. Multiple comparisons between juices within each substrate were performed by Fisher’s Least. Significant difference test F followed by Bonferroni correction as a post hoc analysis of ANOVA.Assumptions of analysis of variance were checked before the use of two-way factorial analysis of variance (ANOVA) model to investigate simple and interaction effects of two factors (Juice and Substrate) among the dependent variable (%SMHL). Levine’s test was used to prove the equality of multiple variance-covariance matrices considering the level of significance of 0.05  and Dentine (Model 2). Collinearity tests considered tolerance above 0.1 and the variance inflation factor (VIF) lower than 10. Variables as TA, critical pH and degree of saturation were not included in the model due to their high interdependence with the other variables. The supposition of error independence was tested using the Durbin-Watson statistic.The type of juice affects the %SMHL in enamel differently from dentine (significant interaction effect); however, the %SMHL after challenge with Orange juice – Del Valle Kapo®, Orange juice – Ades®, and Strawberry juice – Mais Vitta® did not differ between substrates . The proSimple effects  analysis demonstrated that the influence of the juices was significant in enamel and dentine substrates. The high effect size (Type II error) and partial eta squared of these influences evidence that these effects and their interaction are strong and with minimal likelihood of error. Post hoc analysis between juices within each substrate demonstrated that Grape juice – Ades® promoted less mineral loss than other juices in both substrates. A higher percentage of SMHL was found in Grape juice – Aurora®, Orange juice – Del Valle Kapo®, and Citrus fruit juice – Tampico® in enamel, and Grape juice – Aurora® in dentine when compared with other juices .It is noticeable the importance of screening the erosive potential of drinks and foods to obtain evidence-based information for a more comprehensive diet counseling for patients with risk of dental erosion. The consumption of carbonated drinks and fruits juice has increased erosion occurrence, while whole milk and yogurt have presented a protective effect . This isReady-to-drink juices that mimic fresh juices contain citric acid and other low-pH preservatives that could be an extrinsic source for ETW. The chemical behavior of citric acid has been dependent on two modes of actions: one related to their acid dissociation constant (pKa) and another to their chelating ability. Thus, formulations with this acid are very damaging to the tooth surface. Double chemical action is also found in phosphoric acid (a component of Coca-Cola), which has higher ionic strength and calcium affinity than citric acid . For thiin vitro single-exposure erosion simulation, this study does not provide information about what happens in the oral cavity since behavioral and biological host-related factors have a strong influence on the ETW pathogenesis  , and higet al.  could bein vitro studies , as reported by Lussi et al. . In addiet al.  could bePi for enamel and Ca2+ and F- for dentin) could explain their different erosive behavior effect on both substrates.Therefore, the erosive effect of industrialized fruit juices affects enamel differently from dentine, and this effect differed between some, but not all, tested juices. Besides, differences in the physicochemical properties (pH for dentin) and inorganic composition of juices (Ca2+ and