Taxonomic Review of South American Butter Frogs: Phylogeny, Geographic Patterns, and Species Delimitation in the Leptodactylus latrans Species Group (Anura: Leptodactylidae)

The Leptodactylus latrans species group currently comprises eight medium- to large-sized frog species with a convoluted taxonomic history, particularly related to the specific limits of the L. latrans complex, and the species pair Leptodactylus chaquensis–Leptodactylus macrosternum. Their homogeneous external morphology and continental geographic distribution in South America have posed severe limitations to a comprehensive review, such that taxonomic consensus and species limits remain uncertain. This is further worsened by the presence of chromatic polymorphism among coexisting species that can hardly be distinguished by external morphology. Based on a large-scale geographic sampling including multilocus DNA analyses, and acoustic and morphological data, we provide a comprehensive evaluation of the taxonomic status and species limits of the L. latrans group, focusing on the resolution of the L. latranscomplex and the species pair L. chaquensis–L. macrosternum. We gathered 728 mitochondrial sequences from 429 localities, encompassing the entire geographic distribution of the group. Both generalized mixed Yule coalescent and automatic barcode gap discovery species delimitation methods recovered four major mitochondrial evolutionary lineages within the L. latrans complex, also supported by distribution patterns, multilocus molecular, morphological and/or bioacoustic data. One lineage is linked to nominal L. latrans,one revalidated as Leptodactylus luctator, and the other two are formally named and described. Another lineage encompasses all specimens previously assigned to the species pair L. chaquensis–L. macrosternum, clustered as a single evolutionary entity and is now regarded as L. macrosternum. We provide a revised diagnosis for these species based on acoustic data, morphological/chromatic variation, and phylogenetic relationships of all species currently included in the L. latrans group. Our findings reinforce the view that Neotropical diversity is highly underestimated and stress that appropriate geographic sampling in an integrative framework is crucial for the establishment of specific limits among broadly distributed and morphologically cryptic Neotropical frogs

Evaluation of Stage-Dependent Genotoxic Effect of Roundup® (Glyphosate) on Caiman latirostris Embryos

The agricultural expansion over the past decades, along with the associated increase in the use of pesticides, represents a high risk for many wild species. Caiman latirostris is a South American caiman with many features that make it highly vulnerable to pesticide exposure. Considering previous finding on the genotoxicity of the glyphosate-based formulation Roundup®in this species, the aim of this study was to evaluate the possible stage-dependent effect of this compound on C. latirostris embryos through the Comet assay (CA), micronuclei (MN), and nuclear abnormalities (NA) tests. Caiman eggs were exposed to three effective concentrations of Roundup® (750, 1250, 1750 µg/egg) in three different stages of the incubation period (total duration 70 ± 3 days at 31 ± 2 °C) of approximately 23 days each. A statistically significant difference in DNA damage determined by the CA was found between groups exposed to different concentrations of RU (p 0.05). There was no differences in the MN or NA frequencies between the different groups and the negative control (p > 0.05), nor among the different stages within each treatment. The results obtained in this study indicate that RU produce DNA damage on C. latirostris embryos independently of the developmental stage where the exposure occurs, implying an important risk for the species during all its period of development, when pesticide application is at maximum rate.Fil: Burella, Pamela Mariana. Universidad Nacional del Litoral. Facultad de Humanidades y Ciencias. Departamento de Ciencias Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; ArgentinaFil: Simoniello, Maria Fernanda. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Cátedra de Toxicología y Bioquímica Legal; ArgentinaFil: Poletta, Gisela Laura. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Cátedra de Toxicología y Bioquímica Legal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentin

Oxidative damage and antioxidant defense in Caiman latirostris (Broad-snouted caiman) exposed in ovo to pesticide formulations

The surface used for agricultural production in Argentina significantly increased in recent years, mainly due to the expansion of soybean crops. As a result, the use of agrochemicals increased too. Many natural populations of Caiman latirostris (broad-snouted caiman) are affected by habitat fragmentation and the constant exposure to pesticides. This exposure could produce Reactive Oxygen Species. The negative imbalance between ROS generation and the capacity of the biological systems to eliminate the reactive intermediaries or avoid the damage is called Oxidative Stress. The aim of this study was to evaluate oxidative damage and antioxidant defense in C. latirostris hatchlings after in ovo exposure to widely used pesticide formulations. Embryos were exposed by topical exposure on the eggshell, from the beginning of incubation period, to sub-lethal concentrations of two glyphosate formulations: PanzerGold® (PANZ) and Roundup® Full II (RU): 500, 750, 1000 μg/egg; to the endosulfan (END) formulation Galgofan® and the cypermethrin (CYP) formulation Atanor®: 1, 10, 100, and 1000 μg/ egg. Blood samples were taken to all animals immediately after hatching for the application and comparison of the following oxidative stress biomarkers between the exposed groups and their respective controls: lipoperoxidation through thiobarbituric acid reactive substances (TBARS), DNA base oxidation through the modified comet assay, and the activities of Catalase (CAT) and Superoxide dismutase (SOD) in erythrocytes. Our results showed lipoperoxidation in caiman exposed to END (10, 100, 1000 µg/egg), CYP (1, 10, 1000 µg/egg), RU (500, 1000 µg/egg) and PANZ (500, 1000 µg/egg), DNA base oxidation in those exposed to END (10, 100, 1000 µg/egg), CYP (1, 10 µg/egg) and PANZ (500, 750 µg/egg) as well as alteration in the activity of SOD in END 1 µg/egg and CYP (10, 1000 µg/egg). This study demonstrated the incidence of oxidative stress in animals exposed to pesticide formulations widely used in agricultural activity associated mainly with soybean crops, and add further information to that previously reported about pesticide effects in this native reptile species.Fil: Burella, Pamela Mariana. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Cátedra de Toxicología y Bioquímica Legal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; ArgentinaFil: Odetti, Lucia Magdalena. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Cátedra de Toxicología y Bioquímica Legal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; ArgentinaFil: Simoniello, Maria Fernanda. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Cátedra de Toxicología y Bioquímica Legal; ArgentinaFil: Poletta, Gisela Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Cátedra de Toxicología y Bioquímica Legal; Argentin

Biomarkers of Environmental Contamination in Reptile Species: The Effect of Pesticide Formulations on Broadsnouted Caiman Caiman latirostris (Crocodilia, Alligatoridae)

Pesticide formulations are currently considered as one of the main factors responsible for environmental contamination worldwide. Consequently, several wild species can be affected by the overuse of pesticides and fertilizers in agricultural activities. In recent years, we have been evaluating the effects of different pesticide formulations (glyphosate-, endosulfan-, cypermethrin- and chlorpyrifos-based formulations) and mixtures widely used in Argentina and other neighboring countries on broad snouted caiman (Caiman latirostris) as a sentinel reptile species for pesticide contamination. During this period, several instances of evaluation have been conducted in embryos and hatchlings, under controlled and semi-natural conditions. We used biomarkers of genotoxicity (micronucleus, other nuclear abnormalities, and the Comet assay) and oxidative stress (oxidation of lipids and DNA, and activity of the antioxidant enzymes catalase and superoxide dismutase), previously adapted by our group to be applied on blood, in order to avoid damage to the animals, as well as growth. All pesticides tested as well as the complex mixtures induced genotoxicity and oxidative stress through several biomarkers of different endpoints, in independent studies, but under these conditions, no synergistic effects were observed for the mixtures. The final aim is to continue with the integral in situ evaluation of environmentally exposed wild populations.Fil: Poletta, Gisela Laura. Universidad Nacional del Litoral. Facultad de Humanidades y Ciencias. Departamento de Ciencias Naturales. Laboratorio de Zoología Aplicada: Anexo Vertebrados (FHUC-UNL/MASPyMA); Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Cátedra de Toxicología y Bioquímica Legal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Ecología, Genética y Evolución de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Ecología, Genética y Evolución de Buenos Aires; ArgentinaFil: López González, Evelyn Cecilia. Universidad Nacional del Litoral. Facultad de Humanidades y Ciencias. Departamento de Ciencias Naturales. Laboratorio de Zoología Aplicada: Anexo Vertebrados (FHUC-UNL/MASPyMA); Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Burella, Pamela Mariana. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Cátedra de Toxicología y Bioquímica Legal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Romito, María Laura. Universidad Nacional del Litoral. Facultad de Humanidades y Ciencias. Departamento de Ciencias Naturales. Laboratorio de Zoología Aplicada: Anexo Vertebrados (FHUC-UNL/MASPyMA); ArgentinaFil: Siroski, Pablo Ariel. Universidad Nacional del Litoral. Facultad de Humanidades y Ciencias. Departamento de Ciencias Naturales. Laboratorio de Zoología Aplicada: Anexo Vertebrados (FHUC-UNL/MASPyMA); Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; ArgentinaFil: Mudry, Marta Dolores. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Ecología, Genética y Evolución de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Ecología, Genética y Evolución de Buenos Aires; Argentin