Body size, age and growth in males populations of Boana pulchella (Anura, Hylidae).

Age determination in amphibians is crucial to the investigation of life-history traits. In this context, we studied, for the first time, the life-history traits of Boana pulchella from a sample (63 adult males) of three populations (Las Acequias, Río Cuarto, Alejandro Roca) in central Argentina using the skeletochronological method. All adults of B. pulchella studied showed recognizable bone structures that allowed age determination. The average snout-vent length of sexually mature males was 43.39 mm. The maximum observed longevity was 5 years (5 Las Acequias, 4 Río Cuarto, 3 Alejandro Roca) and minimum age at sexual maturity was 2 years (same in the three populations), with mean of 2.96 years. Body size and age were positively correlated (except in Alejandro Roca populations). The growth patterns, estimated by the von Bertalanffy growth equation, do not show differences between populations, and the growth rate decrease after the attainment of sexual maturity. In conclusion, the determination of the individual age of the different populations of B. pulchella allowed us for the first time to establish the characteristics of the life history of the species, important for future comparisons whit others populations and future conservation biology studies.

Morphological abnormalities in natural populations of the common South American toad Rhinella arenarum inhabiting fluoride-rich environments.

Morphological abnormalities in amphibians may be attributed to contaminants, ultraviolet radiation and trematode parasites, or a synergistic effect between them. In the present study, morphological abnormalities in Rhinella arenarum adults from natural and artificial fluoride-rich environments were identified and evaluated. Three sites were sampled in central Argentina: Los Vallecitos stream (LF-LV), Los Cerros Negros stream (MF-CN), and Decantation ponds (HF-DP), with low (0.33 mg/L), middle (2.03 mg/L) and high (14.0 mg/L) fluoride levels respectively; the latter site is associated with a fluoride mine. Abnormal individuals were photographed and then standard radiographs were taken. Abnormality frequencies and relative percentage of abnormal individuals were calculated for each site. In addition, skeletochronology was used to estimate toad's age. Five abnormality types were identified: syndactyly, ectrodactyly, polydactyly, microphthalmia and ectromelia. Percentages of abnormal individuals per site were: LF-LV = 4%, MF-CN = 21.2% and HF-DP = 6.4%. The MF-CN and HF-DP populations had morphological abnormality frequencies that exceeded the reference value (5%) reported in the literature. The average age did not differ between sites. The results of this study indicate that there is an association between frequency of morphological abnormalities and high fluoride levels.

Reproductive endpoints of Rhinella arenarum (Anura, Bufonidae): Populations that persist in agroecosystems and their use for the environmental health assessment.

Degradation of the environment by agriculture affects the persistence and health of the amphibian populations. Characteristics related to reproduction of anuran can be used to evaluate the status of populations and as endpoints in environmental health assessment. In this in situ study the aspects related to the amplexus and ovipositions of the bioindicator species Rhinella arenarum that inhabits agroecosystems were analyzed. The hypothesis of this study is that perturbations of agroecosystems have a negative impact on the size of reproductive adults, on the size of ovipositions and eggs, and on the survival of eggs and embryos. Study area is located in the rural landscape of central Argentina. Four sampling sites were selected: C1, C2 and C3 are ponds on agroecosystems; and SM is a reference site that is not affected by agriculture or livestock. Abundance of amplexus pairs, oviposition and tadpoles per site was recorded. Individuals´ snout-vent length (SVL) in amplexus was measured. The fecundity was calculated like number of eggs per oviposition. The eggs' Gosner stage, the diameter eggs and the frequency of dead and abnormal eggs were recorded by oviposition. Killing-power between egg-embryo and egg-tadpole was calculated. The higher phosphate concentration was detected in all agroecosystems and nitrate was detected in C1 and C2. Conductivity, salinity and SDT were higher in C1 site Male SVL from the SM site was lower than the other sites while the largest SVL was of female from the C3 site. The higher frequencies of sprouted eggs and of dead eggs were recorded in the C2 site. Egg diameter was associated with SM and correlated negatively to SVL of the male and female. No correlation between female SVL and oviposition size was recorded. Killing-power in the passage from egg to tadpole classes was higher in the three agroecosystems. The hypothesis of this study was corroborated in part. Reproductive adults in agroecosystems did not have smaller body size. However, in the agroecosystem ponds, the eggs with smaller diameter were registered, the oviposition had higher frequency of abnormal eggs and the higher mortality was registered. This confirms the high sensitivity of the early stages to environmental disturbances and sustains their use as endpoints for the environmental health assessment.

Assessment in situ of genotoxicity in tadpoles and adults of frog Hypsiboas cordobae (Barrio 1965) inhabiting aquatic ecosystems associated to fluorite mine.

Non-lethal biological techniques such as blood biomarkers have gained attention due to their value as early signals of anthropic effects of contamination representing significant tools to evaluate ecosystems health. We evaluate and characterize in situ genotoxicity of water samples collected from aquatic ecosystems around a fluorite mine using amphibian frogs Hypsiboas cordobae as bioindicator species complemented with 16 physicochemical parameters. Four stations associated with fluorite mine sampling were sampled: a stream running on granitic rock with natural high fluorite content; two streams both running on metamorphic rock with low fluorite content; and an artificial decantation pond containing sediments produced by fluorite flotation process with high variation in physicochemical parameters. We analyses the blood of tadpoles and adults of H. Cordobae, calculated frequencies of micronuclei, erythrocyte nuclear abnormalities, mitosis, immature and enucleated erythrocytes. Individuals were measured and weighed and body condition was calculated. The results of this study indicate that individuals of decantation pond are exposed to compounds or mixtures which are causing cell damage when compared to those that were collected of stream. Larval stage was more vulnerable than the adult phase and it could be related mainly to the higher exposure time to xenobiotics, which can penetrate easily by skin, mouth and gills; additionally this site offers a reduced availability of food than other sites. Therefore, chronic exposure to pollutants could derive in degenerative and neoplastic diseases in target organs. Moreover these individuals may experience reproductive and behavioral disturbances which could lead to population decline in the long term.

Adverse effect of agroecosystem pond water on biological endpoints of common toad (Rhinella arenarum) tadpoles.

Chemical prroducts used in farming and wastes from livestock can contaminate pond water in agroecosystems due to runoff. Amphibians using these ponds for breeding are probably exposed to pollutants, and serious consequences might be observed afterward at the population level. Assessment biological endpoints of anuran to water quality give a realistic estimate of the probability of occurrence of adverse effects and provide an early warning signal. In this study, the ecotoxicity of agroecosystem ponds from the south of Córdoba province, Argentina, was investigated. Ponds in four sites with different degrees of human disturbance were selected: three agroecosystems (A1, A2, A3) and a site without crops or livestock (SM). The effect of pond water quality on the biological endpoint of Rhinella arenarum tadpoles was examined using microcosms with pond water from sites. Biological endpoints assessed were as follows: mortality, growth, development, morphological abnormalities (in body shape, gut, and labial tooth row formula), behavior, and blood cell parameters (micronucleus and nuclear abnormalities). Results indicated that water from agroecosystems has adverse effect on early life stage of R. arenarum. High mortality and fewer metamorphs were recorded in the A1 and A3 treatments. Tadpoles and metamorphs from A1 and A2 treatments had lower body condition. Tadpoles from A1 and A3 showed the highest prevalence of morphological abnormalities. The lowest amount of tadpoles feeding and the highest percentage of tadpoles swimming on the surface were observed in treatments with agroecosystem pond water. The higher frequencies of micronuclei and nuclear abnormalities were recorded in tadpoles from A1, A2, and A3 treatments. We check the sensitivity of the biological endpoints of R. arenarum tadpoles like early warning indicators of water quality. We found that the poor water quality of agroecosystem ponds has impact on the health of the tadpoles, and this could affect the persistence of populations. We recommend implementation of management actions before the harmful effects of agroecosystem pond water on early life stage of anuran become evident in higher ecological levels.

Health status of tadpoles and metamorphs of Rhinella arenarum (Anura, Bufonidae) that inhabit agroecosystems and its implications for land use.

Perturbations of water bodies near agricultural and livestock systems can affect embryonic and larval stages of anurans and negatively impact adult populations and structure of amphibian communities. This study is focused on early development of Rhinella arenarum, for which body growth, abnormalities in the oral disc and genetic damage on erythrocytes were analyzed to establish the impact of agroecosystems on local populations of amphibians. Tadpoles and metamorphs of R. arenarum were collected in three agroecosystems (namely, C1, C2, and C3) and in a site without agricultural and livestock activities (SM) from central Argentina. Egg masses of C1 were extracted for breeding tadpoles under laboratory conditions (Lab). Tadpoles were in small size and lighter in weight in C1 and C2. Metamorphs were shorter and lighter in weight in C1 and C3. In SM and Lab samples, no tadpoles with abnormal LTRF (labial tooth row formula) or without labial teeth were observed. In C1, the highest frequency of abnormal LTRF was recorded and was the only site in which tadpoles without labial teeth were found. In C1 and C2 the tadpoles had highest micronucleus frequencies and nuclear abnormalities. C1 can be considered as the site with the highest anthropogenic perturbation and with less healthy tadpoles. Livestock practices such as alternating cattle between parcel and keeping a buffer between crops and water bodies, would allow a better development of the first aquatic stages that are essential for the conservation of the anuran populations.

Common toad Rhinella arenarum (Hensel, 1867) and its importance in assessing environmental health: test of micronuclei and nuclear abnormalities in erythrocytes.

Anthropogenic activities may generate significant changes in the integrity of aquatic ecosystems, so long-term monitoring of populations that inhabit them is crucial. Counting micronucleated erythrocytes (MN) and erythrocytic nuclear abnormalities (ENA) in peripheral blood is a widely used method for detecting chromosomal damage due to chemical agents in the water. We analyzed MN and ENA frequency in blood obtained from the common toad Rhinella arenarum populations in sites with different degrees of environmental degradation. The results of this study indicate that there is an association between the frequency of micronuclei and nuclear abnormalities and the degree of environmental alteration recorded for the sites studied.

Reproductive biology of Pleurodema guayapae (Anura: Leptodactylidae: Leiuperinae).

Pleurodema guayapae is a species that inhabits saline environments and semidesert zones from central Argentina. To date, the knowledge about the reproductive biology of this species is very poor, and our aim is to contribute to its knowledge with the description of some important reproductive aspects. For this, field work was undertaken in an area near to Patquia, La Rioja province. Sampling was undertaken during three summer periods (2006-2007; 2007-2008; 2008-2009) in Chamical-Patquia area, where we could find reproductively active populations. We observed and described breeding sites, type of clutch, process of foam nest construction, clutch and egg number and sizes, and hatching time and stage. Behaviour observations were performed from the time that males began to call until the pairs ended up the foam nests building, and layed the eggs. Additionally, one amplected pair was observed and filmed in the process of foam nest construction, and four amplectant pairs were collected and separatelly placed in plastic containers, for nests observations in the laboratory. Hatching time was based on three different foam nests of known age. We found that P. guayapae populations were acoustically active only after a rainfall. Its breeding sites were represented by ephemeral ponds of fresh water, product of rains. The males emitted their calls inside or outside these ponds. A detailed description of the foam nest construction process by both females and males was made. The clutches were in dome-shaped foam nest type of 6-9cm in diameter and 1-3cm in height, some of which were in communal nests. The nests had an average of 1 137 pigmented eggs. This species showed a short hatching time. Our results allow us to conclude that this species should be considered an extreme explosive breeder. Our results are discussed with others obtained for related species.

Impacts of horticultural environments on Rhinella arenarum (Anura, Bufonidae) populations: exploring genocytotoxic damage and demographic life history traits.

Horticulture poses a significant ecological risk, as agrochemicals are applied more frequently and in larger quantities per unit of production compared to extensive crop fields. The native amphibian Rhinella arenarum serves as a reliable bioindicator of environmental health. This study aimed to assess genocytotoxic damage and demographic life history traits of R. arenarum inhabiting horticultural environments. Sampling was conducted in suburban sites in central Argentina: H1 and H2 (sites associated with horticultural activity) and a reference site, RS. Environmental parameters were recorded, and the frequency of micronuclei (Mn), nuclear abnormalities (ENA), and indicators of cytotoxic damage were determined in tadpoles and adults. Demographic variables (age at maturity, longevity, potential reproductive lifespan, size at maturity, modal lifespan) were calculated. The highest nitrate and phosphate values, along with low dissolved oxygen values, were recorded at sites H1 and H2. Organisms inhabiting horticultural environments exhibited higher frequencies of Mn and ENA, surpassing those recorded in previous studies on tadpoles from sites with extensive crop production. Size at maturity and age at maturity of females, as well as size at maturity, longevity, mean age, and mean adult SVL of males, were lower in horticultural sites. The results support the hypothesis that anuran populations inhabiting horticultural environments demonstrate a diminished health status attributed to subpar environmental quality. Monitoring endpoints at different biological levels provides information on the ecotoxicological risk for amphibians and human populations inhabiting nearby areas.

Erythrocyte nuclear size as a better diagnostic character than cell size in the identification of live cryptic polyploid species.

It is well documented in anurans the cryptic condition of many species complexes involving polyploids. In these complexes the character that clearly differentiates them is the number of chromosome complements. The blood cells of amphibians conserve their nucleus, and so the erythrocyte size is correlated with the DNA content. We analyzed two cryptic-polyploid complexes occurring in the center of Argentina: Odontophrynus cordobae (2n)/O. americanus (4n) and Pleurodema kriegi (4n)/P. cordobae (8n). Our aim was evaluate the efficiency in the utilization of nuclear area with respect to cellular area of the erythrocytes to define the limits values for the identification of cryptic-polyploid species. We studied 110 individuals of Pleurodema and 116 individuals of Odontoprhynus. For each individual, we measured the cellular and nuclear length (L) and width (A) of 40 erythrocytes (Area = L*A*π/4) and boundary values were calculated using distribution curves. In both complexes studied, the erythrometric parameters showed significant differences between related species. Moreover, in both complexes the nuclear area was more efficient for identifying the species (Pleurodema: 34.39 µm2 (probability = 99.96%) and Odontophrynus: 24.02 µm2 (99.075%)) than the cell area (Pleurodema: 273.08 µm2 (97.55%) y Odontophrynus: 197.69 µm2 (97.94%)). Greater efficiency found using nuclear area is novel and significant because most studies use only the cell area to differentiate polyploid complexes.

[Bioacoustic of the advertisement call of Ceratophrys cranwelli (Anura: Ceratophryidae)]. / Bioacústica del canto de advertencia de Ceratophrys cranwelli (Anura: Ceratophrydae).

The advertisement call plays an important role in the life history of anuran amphibians, mainly during the breeding season. Call features represent an important character to discriminate species, and sound emissions are very effective to assure or reinforce genetic incompatibility, especially in the case of sibling species. Since frogs are ectotherms, acoustic properties of their calls will vary with temperature. In this study, we described the advertisement call of C. cranwelli, quantifying the temperature effect on its components. The acoustic emissions were recorded during 2007 using a DAT record Sony TCD-100 with stereo microphone ECM-MS907 Sony and tape TDK DAT-RGX 60. As males emit their calls floating in temporary ponds, water temperatures were registered after recording the advertisement calls with a digital thermometer TES 1300+/-0.1 degreeC. Altogether, 54 calls from 18 males were analyzed. The temporal variables of each advertisement call were measured using oscillograms and sonograms and the analyses of dominant frequency were performed using a spectrogram. Multiple correlation analysis was used to identify the temperature-dependent acoustic variables and the temperature effect on these variables was quantified using linear regression models. The advertisement call of C. cranwelli consists of a single pulse group. Call duration, Pulse duration and Pulse interval decreased with the temperature, whereas the Pulse rate increased with temperature. The temperature-dependent variables were standardized at 25 degreeC according to the linear regression model obtained. The acoustic variables that were correlated with the temperature are the variables which emissions depend on laryngeal muscles and the temperature constraints the contractile properties of muscles. Our results indicated that temperature explains an important fraction of the variability in some acoustic variables (79% in the Pulse rate), and demonstrated the importance of considering the effect of temperature in acoustic components. The results suggest that acoustic variables show geographic variation to compare data with previous works.

[Population demography in Rhinella arenarum (Anura: Bufonidae) and Physalaemus biligonigerus (Anura: Leiuperidae) in agroecosystems in the province of Córdoba, Argentina]. / Demografía poblacional de Rhinella arenarum (Anura: Bufonidae) y Physalaemus biligonigerus (Anura: Leiuperidae) en agroecosistemas de la provincia de Córdoba, Argentina.

The advancing agricultural frontier has led to an important loss of natural habitats, with significant consequences for biodiversity. The demography for two species of anurans, Physalaemus biligonigerus and Rhinella arenarum, both associated with agricultural systems in the central region of the C6rdoba Province, were analyzed and compared in this study. Four sites were sampled: three agroecosystems with different alteration degrees (C1, C2 and SM1) and a fourth site not cultivated (SM2). The sampling was conducted during two reproductive periods, from September 2008 to April 2009 and September 2009 to April 2010. Individuals were captured using live pitfall traps for the metamorphic, juveniles and adults; and visual encounter survey, for the capture of eggs and larvae. With the abundance data, the survival for each age class was estimated using the KNM method (Kiritani Nakasuki Manly). With survival rates and fertility population, Leslie matrices were elaborated to obtain a quantitative projection of the population size. Altered environments showed lower eggs and larvae survival. Population projections were favorable in the site SM2 and were less favorable and a tendency to extinction, in sites dominated by crops. This study showed that the agroecosystems of this region are possibly inhospitable environments for reproduction and survival of the species studied. The aquatic stages in the life cycle of both species would be the more affected, since water bodies deterioration is present or may occur in those areas. We can recognize species-specific effects of agricultural ecosystems; P. biligonigerus was the most affected species, possibly because of their life histories and habitat requirements. We suggested that environmental degradation caused by the cropland in the central region of Argentina would impact on the demographics of the anuran populations in the area.

Effect on growth and development of common toad (Rhinella arenarum) tadpoles in environment related to fluorite mine.

Fluorite mining activities have been scarcely evaluated so far and the potential effect of this activity on larval stages is poorly known. Thus, studies addressing the effect of contaminants present in water bodies on the health of amphibian larvae are crucial information for their conservation and constitute a warning sign of environmental modification. This study aims to evaluate the effects of natural and artificial surface waters associated with a fluorite mine on the early life stages of Rhinella arenarum. Using microcosms, we assessed four sites with different degrees of disturbance by the fluorine mine: Vallecitos stream (undisturbed); Cerros Negros upstream of mining camp (low disturbance); Cerros Negro downstream of mining camp (medium disturbance); Decantation Ponds (high disturbance). For 65 days we measured different endpoints at different periods of tadpole development. The highest mortality of tadpoles was observed in the Decantation Ponds. In the same way, larval body condition was lower at Decantation Ponds throughout the study, due mainly to the lower weight. A significant decrease in growth and development was observed in Cerros Negro downstream of the mining camp and Decantation Ponds. No significant differences in growth, development and mortality were observed for the other two sites. At the metamorphic climax we observe a lower body condition and a reduced recruitment of individuals in Decantation Ponds. This study allowed us to assess the effect on larvae of R. arenarum of fluorite mining wastewater compared with other near-natural sites. Given the potentially negative synergic effects of mixed water pollutants on tadpoles, this study suggests that chronic exposure to fluorite mining water may significantly impact the adult amphibian population structure, thus altering population viability. Therefore, we propose to monitor the correct functioning of the mine and especially of Decantation Ponds to avoid discharges into the natural streams.

Hemoparasites Do Not Affect Life-History Traits and Cellular Immune Response in Treefrog Hosts Boana cordobae.

We provide the first evidence for hemoparasites in the endemic Cordoba treefrog Boana cordobae. We collected 37 adult frogs at 1200 m a.s.l. in the Comechingones Mountains in the Córdoba province (Argentina). Each individual was sexed, then snout-vent length and body mass were recorded, a toe was collected for skeletochronological age determination, and a slide with a blood smear was prepared for hemoparasite screening, before releasing the frogs in situ. A total of 81% (n = 30) of the frogs were infected by hemogregarines and trypanosomes with a high intensity of infections. Dactylosoma was found for the first time in Argentina. Hemoparasites had no significant effect on the leukocyte profile, which we assessed from the May-Grünwald-Giemsa-stained blood smears. The neutrophils/lymphocytes ratio, indicative of stress, was insignificantly higher (0.06) in parasitized frogs than in parasite-free individuals (0.04). Infected frogs were larger than the controls, but this effect vanished when correcting size data for age. Young frogs (first-breeders) dominated the age distribution of parasite-free individuals, suggesting that infection of frogs takes usually place after sexual maturation. Vectors transmitting hemoparasites to B. cordobae remain to be identified. We demonstrate that moderate to high intensities of hemoparasites do not significantly affect the cellular immune response of B. cordobae, or any of the life-history traits studied, nor did they show any external sign of disease.

[Intra- and inter-specific morphometric variation between Odontophrynus populations (Anura: Cycloramphidae) of central Argentina]. / Variación morfométrica intra e interespecífica entre poblaciones de Odontophrynus (Anura: Cycloramphidae) del área central de Argentina.

Intra- and inter-specific morphometric variation between Odontophrynus populations (Anura: Cycloramphidae) of central Argentina. Morphometric analyses are particularly important, and for many years they have supported evolutionary and ecological phenomena, and have been useful for the classification of new species, mainly to the lowest taxonomic levels. In order to assess the degree of sexual dimorphism, the intra-specific morphometric variation and the inter-specific morphological differences, we performed morphometric analyses of two morphologically cryptic species, Odontophrynus cordobae (diploid) and O. americanus (tetraploid). For this, we measured 15 morphometric variables on 211 individuals from 18 localities of Córdoba province. We found sexual dimorphism in six and three parameters in O. cordobae and O. americanus, respectively. Diploid and tetraploid males significantly differed in six morphometric variables. Discriminant Function Analysis (DFA) including all populations correctly classified a 76.37% of individuals within their respective species. DFA based on four groups (1- allopatric O. cordobae, 2-syntopic O. cordobae; 3- allopatric O. americanus; 4- syntopic O. americanus) accurately classified a 73.45% of individuals within their respective groups. Allopatric individuals of each species and, allopatric and syntopic individuals within each species were clearly segregated. Syntopic individuals of O. cordobae and O. americanus showed the lowest percentages of correct classification. Morphometric differences between sympatric diploids and tetraploids were not greater than those observed in allopatry. These findings deviate from the expected results under the hypothesis of character displacement, and they suggest that external morphological characters would not have a major influence on the recognition and choice of conspecific males by females.

[Trophic ecology in tadpoles of Rhinella arenarum (Anura: Bufonidae) in agroecosystems and their possible implications for conservation]. / Ecología trófica en larvas de Rhinella arenarum (Anura: Bufonidae) en agroecosistemas y sus posibles implicaciones para la conservación.

The progress of the agriculture border has led an important loss of natural habitats, with significant consequences for biodiversity. In this sense, the studies in anuran amphibian tadpoles inhabiting these environments are relevant, because the larval stage is a phase of population regulation. The aim of this study was to analyze the body condition and diet in Rhinella arenarum, tadpoles, an anuran species widely distributed in South America and that inhabit agroecosystems. Three sites were sampled, two agroecosystems with different alteration degrees (C1 and C2) and an uncultured (SM) third place. The captured tadpoles were anesthetized, fixed and preserved in formaldehyde (10%). Subsequently, body measurements were made and the complete intestine was removed and analyzed for food items under a binocular microscope. The diet in R. arenarum tadpoles has a dominance of algae Bacillariophyceae, followed by Cyanophyceae. In particular, the class Bacillariophyceae, due to the presence of the genus Navicula, Nitzschia, Gomphonema and Hantzschia, was important in the diet of the anurans in those agroecosystems. Class Cyanophyceae, mainly represented by genus Oscillatoria and Euglenophyceae represented by Euglena and Strombomonas, were predominant in the diet of the anurans in SM. Some differences in the total items consumed by tadpole were observed between the studied sites. Tadpoles that inhabit the modified sites (C1 and C2) recorded a significantly smaller amount of food. Moreover, the tadpoles that inhabit these sites showed a lower body condition. The presence of certain algae associated with eutrophic environments, could indicate some pollution in agroecosystems (C1 and C2). Food resources would be lesser in places with strong agricultural activity, possibly with a greater degree of eutrophication. A smaller food amount could have consequences at population level for the short and long time terms, because of its impact on individual growth. Larval diet is suggested as a potential bioindicator of environmental health for these areas.

Asymmetric Male Mating Success in Lek-Breeding Rhinella arenarum.

Mate choice is the attempt of an individual to gain higher reproductive fitness by preferring to mate with some individuals and not with others. We studied the role of mate choice in the mating system of the neotropical toad Rhinella arenarum by assessing male reproductive tactics for mate acquisition and the contribution of female choice for pair formation. In a shallow pond in central Argentina, we estimated male mating success and the corresponding reproductive tactics by focal observation. The variation of phenotypic and genotypic traits (size and shape, longevity, vocalization features, heterozygosity) was related to the observed mating success in 110 males. The phonotactic response of 21 reproductive females to conspecific advertisement call features was tested in arena experiments. Mating success was limited to 32 males, pair formation was size-assortative. The dominant reproductive tactics were advertising from call positions near suitable breeding sites and pre-mating fights with intruding males, whereas the interception of amplectant pairs and the displacement of mated males were never observed. Female phonotaxis was directed to conspecific choruses but complex and simple call structures were not distinguished. We conclude that the mating system is a lek combining pre-mating fights among males and female choice of slightly smaller males. Fights interfere with female choice, undermining size-assortative mating. This is a unique system in the R. marina species group, in which interception behavior dominates reproduction.

Integrative taxonomic reassessment of Odontophrynus populations in Argentina and phylogenetic relationships within Odontophrynidae (Anura).

Amphibians are the most vulnerable vertebrates to biodiversity loss mediated by habitat destruction, climate change and diseases. Informed conservation management requires improving the taxonomy of anurans to assess reliably the species' geographic range. The genus Odontophrynus that is geographically refined to Argentina, Bolivia, Brazil, Uruguay and Paraguay includes currently 12 nominal species with many populations of uncertain taxonomic assignment and subsequently unclear geographic ranges. In this study, we applied integrative taxonomic methods combining molecular (mitochondrial 16S gene), allozyme, morphological and bioacoustic data to delimit species of the genus Odontophrynus sampled from throughout Argentina where most species occur. The combined evidence demonstrates one case of cryptic diversity and another of overestimation of species richness. The populations referred to as O. americanus comprise at least three species. In contrast, O. achalensis and O. barrioi represent junior synonyms of the phenotypically plastic species O. occidentalis. We conclude that each of the four species occurring in Argentina inhabits medium to large areas. The Red List classification is currently "Least Concern". We also propose a phylogenetic hypothesis for the genus and associated genera Macrogenioglottus and Proceratophrys (Odontophrynidae).

Anuran tadpoles inhabiting a fluoride-rich stream: diets and morphological indicators.

We in situ assessed the influence of natural fluoride concentrations in lotic freshwater ecosystems on diet and morphology of Boana cordobae tadpoles. Two streams were sampled in Argentina: Los Vallecitos stream (LF-LV) and Los Cerros Negros stream (HF-CN) with low and high fluoride levels, respectively. We captured tadpoles of B. cordobae in each stream using nets. Body weight (BW), total length (TL) and body condition (BC) of tadpoles was registered. Food items were identified to genus level and assigned to functional traits. Tadpoles showed significant differences in TL between streams, with smaller individuals in HF-CN, while did not show differences in BW and BC. The diet of tadpoles consisted mostly of microalgae. In tadpoles from HF-CN stream the proportion of cyanobacteria was lower than tadpoles from LF-LV. In relation to functional traits, small algae, high profile and colonial algae were more abundant in HF-CN. Algae attached by pads showed a higher proportion in HF-CN diets and stalked algae were more abundant in LF-LV. The differences in TL and diet of tadpoles can be attributed to differences in algal community composition, with genera that are affected by high concentrations of natural fluoride; for example cyanobacteria. The low algal richness registered in HF-CN stream does not affect the physiological state of the tadpoles, possibly because of a higher algal density in HF-CN diets. However, in further studies it would be important to evaluate the population status of B. cordobae from the HF-CN, because a small body length of tadpoles could have consequences at the population level.

Evaluation in situ of genotoxic and cytotoxic response in the diploid/polyploid complex Odontophrynus (Anura: Odontophrynidae) inhabiting agroecosystems.

Polyploidization has been documented across a wide range of vertebrates. Gene duplication could promote better adaptation to environmental changes and to chronic injury or stress. We investigated if genotoxic and cytotoxic responses to agricultural impact are affected by ploidy. We evaluate syntopic populations of the cryptic diploid/polyploid complex Odontophrynus cordobae/O. americanus breeding in an agroecosystem from Central Argentina. The blood of 72 adult anurans was analysed. We used erythrometry to distinguish Odontophrynus individuals with different ploidy levels. We calculated micronucleus frequencies (Mn) and erythrocytic nuclear abnormalities (ENAs) as genotoxic effects and enucleated, mitotic, pyknotic and immature erythrocytes as cytotoxic endpoints (CYT). Mn, ENAs and CYT frequencies were significantly different between diploid and polyploid organisms. The higher frequencies of Mn and CYT were recorded in polyploid organisms, and the higher frequency of ENAs was recorded in diploids. These results indicate that stress response, as indicated by most genotoxic and cytotoxic endpoints, was higher in polyploids respect to diploids. Polyploidy could provide greater genetic flexibility increasing buffering against exogenous DNA-damaging agents and thus confer an advantage over diploids under certain environmental conditions.