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.

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.

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.

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.

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.

Effect to age on the geographic variation in morphometric traits among populations of Boana cordobae (Anura: Hylidae) / Efecto de la edad en la variación geográfica de características morfométricas entre poblaciones de Boana cordobae (Anura: Hylidae)

Abstract The morphometric variation of body size is an important topic of the natural history of the species which has been received particular interest. In this study, we estimated differences in body size and age structure of six populations of Boana cordobae living at different altitudes, 808-2 310 m.a.s.l. in Córdoba and San Luis provinces (Argentina). We measured 15 morphometric variables and used skeletochronology to age determination of 79 individuals. Morphometric variables showed significant differences between sexes, being females larger than males, even when the effect of age was taken into account. We found a significant relationship between age and most of the morphometric variables. When removing the effects of age, we found significant inter-population differences in body size. Males from the high-elevation populations were larger than individuals from low-elevation populations. These results suggest that a difference in age structure between populations is a main factor for the geographic variation in body size of this species.(AU)

Resumen La variación morfométrica del tamaño corporal es un aspecto importante de la historia natural de las especies, que ha sido de particular interés. En este estudio se estimaron diferencias en el tamaño corporal y la estructura de edad de seis poblaciones de Boana cordobae que viven a diferentes altitudes, 808-2 310 m.a.s.l. en las provincias de Córdoba y San Luis (Argentina). La técnica de esqueletocronología se utilizó para la determinación de la edad, mediante la medición de 15 variables morfométricas en 79 individuos. Las variables morfométricas mostraron diferencias significativas entre sexos, siendo las hembras de mayor tamaño que los machos, incluso cuando el efecto de la edad se tuvo en cuenta; y se encontró una relación significativa entre la edad y la mayoría de las variables morfométricas. Cuando el efecto de la edad fue removido, se encontraron diferencias significativas en el tamaño del cuerpo entre las poblaciones estudiadas. Machos de poblaciones de mayor altitud son más grandes que los de altitudes menores. Estos resultados sugieren que las diferencias en la estructura de edad entre las poblaciones es un factor de importancia a tener en cuenta para analizar la variación en el tamaño corporal de esta especie según el área geográfica.(AU)

Morphology and size of blood cells of Rhinella arenarum (Hensel, 1867) as environmental health assessment in disturbed aquatic ecosystem from central Argentina.

Four populations of Rhinella arenarum from aquatic environments with different degrees of disturbance in central Argentina were compared to assess the ability of cytomorphology and cytomorphometry of blood cells as a hematological biomarker. A total of 93 specimens of R. arenarum (adults sexually mature) were captured during the spring. From the analysis of cell, no variations were found in terms of morphology, whereas in nuclear and cell areas and Price-Jones curves, we observed a smaller size in erythrocytes of individuals inhabiting the site most altered, "Villa Dalcar," as well as for leukocytes, lymphocytes, neutrophils, and eosinophils for the same site. This could be caused by presence of different pollutants in the lake. Furthermore, this was confirmed by the high levels of environmental variables (conductivity, total dissolved solids, and salinity) show that Villa Dalcar is the site most affected by human activities.

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.

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.

Reproductive biology of Pleurodema guayapae (Anura: Leptodactylidae: Leiuperinae) / Biología reproductiva de 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 Patquía, La Rioja province. Sampling was undertaken during three summer periods (2006-2007; 2007-2008; 2008-2009) in Chamical-Patquía 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.

Pleurodema guayapae es una especie que habita en zonas semidesérticas y salinas del área central de Argentina. Los conocimientos actuales sobre la biología reproductiva de esta especie son muy pobres, por lo que nuestro objetivo fue contribuir a su conocimiento con la descripción de algunos aspectos reproductivos importantes. Para ello se realizaron tres campañas de muestreo durante los veranos (2006-2007, 2007-2008 y 2008-2009) en el área de Chamical-Patquía, provincia de La Rioja, donde se encontraron poblaciones reproductivamente activas. Se observaron y describieron los sitios reproductivos, tipo de puesta, proceso de construcción del nido de espuma, tamaño de la puesta y de los huevos, número de huevos por puesta y tiempo y estado de eclosión de la larva. Las observaciones de comportamiento se realizaron desde el momento en que los machos comenzaron a emitir los cantos hasta que la pareja en amplexo terminó de construir el nido de espuma con los huevos en su interior. Además, se observó y filmó una pareja en amplexo, en el proceso de construcción del nido de espuma y se recolectaron cuatro parejas en amplexo y se colocaron separadamente en recipientes plásticos para que sus puestas pudieran ser observadas en el laboratorio. El tiempo de eclosión se determinó en tres nidos de espuma de edad conocida. Se encontró que las poblaciones de P. guayapae solo estuvieron acústicamente activas luego de una lluvia torrencial. Los sitios de reproducción fueron charcas efímeras de agua de lluvia. Los machos emitieron sus cantos de advertencia desde adentro o fuera de esas charcas. Se realizó una descripción detallada del proceso de construcción del nido de espuma por parte de la hembra y el macho de P. guayapae. El tipo de la puesta en esta especie es un nido de espuma flotante con forma de domo de 6 a 9cm de diámetro y de 1 a 3cm de alto, algunos de los cuales estaban unidos entre sí formando puestas comunales. Los nidos de espuma poseen en promedio 1 137 huevos pigmentados. Esta especie mostró un tiempo de eclosión corto. Nuestros resultados nos permiten concluir que P. guayapae debe ser considerada como reproductor explosivo extremo. Nuestros resultados son discutidos con los obtenidos en otras especies afines.

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.

Variación morfométrica intra e interespecífica entre poblaciones de Odontophrynus (Anura: Cycloramphidae) del área central de Argentina / Intraand 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.

El análisis morfométrico ha servido como apoyo para explicar fenómenos ecológicos y evolutivos y para la clasificación de nuevas especies. Nosotros realizamos el análisis morfométrico de dos especies morfológicamente crípticas con el objeto de evaluar el grado de diferenciación intra e interespecífica. Se midieron 15 variables sobre 211 individuos pertenecientes a 18 localidades del centro de Argentina. Se encontró dimorfismo sexual en seis variables morfométricas en Odontophrynus cordobae y en tres variables en O. americanus. Se encontraron diferencias significativas entre especies en seis variables. El análisis discriminante incluyendo los individuos de todas las poblaciones relevadas para O. cordobae y O. americanus, mostró una clasificación positiva de los individuos dentro de su respectiva especie del 76.37%. En el análisis discriminante realizado con base en cuatro grupos (O. cordobae alopátrico y sintópico y O. americanus alopátrico y sintópico) se obtuvo una reclasificación de los individuos dentro de cada grupo del 73.45%. Las diferencias morfométricas entre especies fueron mayores en sintopía. Los resultados se alejan de los resultados esperados bajo la hipótesis del desplazamiento de caracteres y podrían sugerir que los caracteres morfológicos externos no tendrían una influencia importante en el reconocimiento y elección de machos coespecíficos por parte de las hembras.

[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.