Adolfo Ludovico Martino

Speciation by polyploidy in Odontophrynus americanus

Diploid and tetraploid populations of the leptodactylid frog Odontophrynus americanus were studied in the Cordoba province (central Argentine) to evaluate whether they represent a cryptic species pair rather than forms of the same species as they are considered at present. We examined three independent character complexes (external morphology, advertisement calls, allozymes) and quantified the character-specific state of differentiation between diploids and tetraploids in comparison to that of the congeneric and widely sympatric O. occidentalis. Multivariate analysis of 15 morphometric characters indicated a remarkable degree of differention between both forms, but did not permit an unequivocal identification of individuals (rate of erroneous classification: 25%), whereas all O. occidentalis were classified correctly. In contrast, the temporal structure of the advertisement call permitted a distinction of diploids, tetraploids and O. occidentalis even based on a single call. The latter differed from the other by producing a call consisting of several pulse trains. The pulse rate of the O. americanus call, which consists of a single pulse train, was significantly lower in tetraploids than in diploids. The allozyme pattern and the corresponding allele frequencies indicated a close relationship among the three Odontophrynus taxa, but genetic distances obtained suggested a differentiation of gene pools at the level of species. Considering the degree of differentiation demonstrated in this study, the ploidy and the different geographical distribution, we conclude that diploids and tetraploids represent two species, similar to its North American counterpart Hyla chrysoscelis/H. versicolor. As the name O. americanus refers to tetraploids collected near Buenos Aires (Argentina), we describe the diploids as the new species Odontophrynus cordobae sp. nov.

Longevity, Demography and Sex-Specific Growth of the Pampa de Achala Toad, Bufo achalensis CEI, 1972

The demography and life history parameters of the neotropical Bufo achalensis, an endemic toad of the Pampa de Achala, Sierras Grandes de Cordoba, Argentina, were studied in 13 activity periods between 1970/71 and 1998/99. We used phalange bones for skeletochronological age estimation and to assess annual growth rates in 243 individuals. Maximum longevity was 11 years, and sexual maturity was attained at an age of 2–4 years. Reproductive females were on average 4–8 mm smaller than males of the same age. There is no indication that the population of B. achalensis is declining as observed in several neotropical amphibian species.

Reproductive Ecology of the Common South American Toad Rhinella arenarum (Anura: Bufonidae): Reproductive Effort, Clutch Size, Fecundity, and Mate Selection

Abstract Understanding the causes of population declines is difficult when the ecology of the organisms themselves is insufficiently known. The common South American toad, Rhinella arenarum, inhabits a wide variety of habitats. Their populations are abundant; however, life-history characteristics of the adult, including reproductive traits, are poorly known. We studied the reproductive ecology of R. arenarum for several populations in central Argentina during two breeding seasons (2007–08). Breeding activity was compared for the two seasons with respect to variation in habitat variables. Females deposited a mean of 23,226.67 eggs per clutch (N  =  10), and snout–vent length did not show any significant effect on the number of eggs. Spawning females lost 27% of their mass when spawning occurred, and the heavier females had greater reproductive output. We found a significantly positive relationship between amplexed male and female snout–vent lengths, indicating size-assortative mating. Females in good body and somatic condition produced larger clutches independent of snout–vent length. Because clutch hydration in ponds occurs, reproductive effort as a function of body loss mass of spawning females is a more appropriate estimate of reproductive output for R. arenarum. Trends in the number of egg strings over several breeding periods can provide information about changes in population size. Long-term studies designed to monitor populations are needed to determine the impacts and consequences of environmental changes.

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.

Variation in erythrocyte size between juveniles and adults of Odontophrynus americanus

Differences in erythrocyte size between tadpoles and adults have been found in numerous anuran species but there are no references about erythrocyte size of juveniles. We measured the size of red blood cells and nuclei of juvenile and adult individuals of Odontophrynus americanus. Erythrocyte and nuclear size of juvenile were significantly smaller than the corresponding sizes of adults. The erythrocytes of juvenile O. americanus were comparable in size to the erythrocytes of adult O. cordobae. We discuss about the possibility of correctly distinguishing juvenile O. americanus (tetraploid) from adult O. cordobae (cryptic diploid counterpart) specimens using erythrocytes. We also discuss possible causes of variation.

Intra-specific variation in advertisement call of Odontophrynus cordobae (Anura, Cycloramphidae): a multilevel and multifactor analysis

Advertisement call is a complex signal that can vary at different scales and the characterization of this variation is crucial to understand its adaptive function. In our study, we examined different factors that could influence intra and inter-population call variation in Odontophrynus cordobae. We analyzed calls of individuals from different breeding sites across the species distribution, including a syntopic site with their related species O. americanus. Dominant frequency, pulse rate, call duration and pulses/call were the acoustic parameters that showed the lowest intra-individual variation (CV ≤ 3%). The discriminant function analysis (DFA) assigned 92% of calls to the correct individual, indicating that the advertisement calls of O. cordobae are individually distinctive. Univariate and multivariate statistical analysis showed a significant contribution of pulse rate in differentiation of populations. Interpulse interval and pulse rate showed a moderate clinal variation, even when syntopic populations were excluded from the analysis. We found no association between acoustic and linear geographical or altitudinal distances and observed an evident divergence between the syntopic population and the other populations analyzed. This result is reinforced by DFA, which showed a differentiation among syntopic and allopatric populations of O. cordobae. We discuss our results from the hypothesis of a possible character displacement influencing call properties in this species in syntopy with their related species O. americanus. However, new syntopic areas and tests of female preferences would be needed to strengthen our results.

Longevity and sexual size dimorphism of the Pampa de Achala copper lizard Pristidactylus achalensis (Gallardo, 1964)

Longevity and sex-specific growth of the neotropical lizard Pristidactylus achalensis was studied from 1993 to 1998 in a population inhabiting the Pampa de Achala, Sierras Grandes de Cordoba, Argentina, at an elevation of 2,000 m. Capture effort concentrated on the territorial adults which were detected while basking. Each of the 149 individuals studied was sexed, morphometrically described and toe-clipped. The second phalanx was used for skeletochronological age estimation and retrospective growth reconstruction, in some voucher specimens additionally the humerus to validate LAG counts in the phalanx cross sections. Longevity was about the same in males (9 LAGs) and females (10 LAGs) but may be even larger in non-territorial individuals. Sexual maturity was usually attained at an age of 3 LAGs, only occasionally one year earlier. With respect to snout-cloaca length, females were always significantly smaller than males of the same age. Bone growth (increase of LAG diameter) revealed that the sexual size dimorphism was related to a significantly lower growth rate of females following hatching. Age and size were correlated ( r s = 0.564, n = 149, P << 0.001), but still most of the total size variance remained unexplained by age. Maximum size was approached asymptotically with more than 90% of size increase occuring during the first three years of life: 46.0 ± 1.7 mm (1 LAG), 81.3 ± 5.6 mm (2 LAG), 100.1 ± 0.7 mm (3 LAGs). Territorial individuals rarely were successful to defend a territory more than one activity period, as evidenced by a low recapture probability and a stable between-years age structure of this group.

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.

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.

Evaluation in situ of genotoxicity and stress in South American common toad Rhinella arenarum in environments related to fluorite mine

Little attention has been paid to the impact of wastewater generated by mining activities on fluoride. In this study, we evaluated the hematology responses of common South American toad Rhinella arenarum inhabiting natural and artificial environments associated with a fluorite mine from central Argentina. We analyzed three sampling stations associated with the fluorite mine: (I) Los Cerros Negros stream (CN), which runs on granitic rock with a high fluorite content; (II) Los Vallecitos stream (LV), which runs on metamorphic rock with low fluorite content; and (III) artificial decantation ponds (DP) containing sediments produced by fluorite flotation process. We calculated frequencies of micronuclei, erythrocyte nuclear abnormalities, mitosis, and immature erythrocytes. In addition, we performed a differential leukocyte count and determined neutrophils/lymphocyte ratio as a stress response estimator. We found high micronucleus (MN) and erythrocyte nuclear abnormality (ENA) frequencies in DP and CN but low frequencies in LV. The neutrophil/lymphocyte ratio was different among sites, with a significant increase in individuals from DP. Values registered in DP could be caused by exposure to mixture of compounds registered in dams that hold wastewater, while high values registered in CN stream might be due to natural concentrations of fluoride. Our results suggest that blood is an effective and non-destructive sensitive indicator for monitoring genotoxic agents in freshwater ecosystems.