Rana Latastei Research Articles

Evaluating the risk of pesticide exposure for amphibian species listed in Annex II of the European Union Habitats Directive

Environmental contaminants like pesticides concern amphibian conservationists. For many European amphibian species, special areas of conservation were created as they are listed in Annex II of the EU Habitats Directive. Agriculture is not prohibited within these conservation areas. In the present study, a risk evaluation at the European level was conducted to identify proportions of land use with regular pesticide applications within the conservation areas and the specific risk of pesticide exposure depending on the species’ biology and ecology. The proportion of agricultural land use and the risk of habitat and individual contamination differ among species but also at national scale. Nearly all species with high risk of habitat pesticide exposure are not threatened within their entire territories and Europe. Conversely, most globally threatened and European priority species are at a lower exposure risk in their habitats – with the exceptions Rana latastei, Pelobates fuscus insubricus, Triturus dobrogicus and Discoglossus jeanneae. In the conservation areas for these species, Habitat Directive management plans need to consider monitoring of habitat contamination with pesticides. Such a monitoring should also be conducted in conservation areas for amphibians that seem to be not threatened yet but are at high exposure risk, e.g. Bombina bombina. Monitoring and conservation action should also take place site-specifically to avoid national or regional loss of amphibian biodiversity. Overall, intensive use of agrochemicals and recent land use changes have the potential to be a serious threat for amphibian species, which can be found within cultivated areas – regardless of their current IUCN status.

New data on the distribution of the Italian agile frog Rana latastei Boulenger, 1879 in Slovenian Istra

After the first confirmed record of the Italian agile frog in the Dragonja River Valley, a herpetological field survey was organized in 2012 to estimate the species range in this area. We confirmed its reproduction in the Slovenian part of the watersheds of the rivers Dragonja, Mirna and upper part of Rižana. Here we present a preliminary report on the presence of this amphibian in the Slovenian part of Istra.

On the occurrence of the Italian agile frog (Rana latastei Boulenger, 1879) in the Slovenian part of Istria

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Rapid selection against inbreeding in a wild population of a rare frog.

Populations that are small and isolated can be threatened through loss of fitness due to inbreeding. Nevertheless, an increased frequency of recessive homozygotes could increase the efficiency of selection against deleterious mutants, thus reducing inbreeding depression. In wild populations, observations of evolutionary changes determined by selection against inbreeding are few. We used microsatellite DNA markers to compare the genetic features of tadpoles immediately after hatch with those of metamorphosing froglets belonging to the same cohort in a small, isolated population of the threatened frog Rana latastei. Within a generation, the inbreeding coefficient (FIS) decreased: at hatch, FIS was significantly >0, whereas FIS was <0 after metamorphosis. Furthermore, heterozygosity increased and allelic frequencies changed over time, resulting in the loss of genotypes at metamorphosis that were present in hatchlings. One microsatellite locus exhibited atypically large FST values, suggesting it might be linked to a locus under selection. These results support the hypothesis that strong selection against the most inbred genotypes occurred among early life-history stages in our population. Selective forces can promote changes that can affect population dynamics and should be considered in conservation planning.

Starting size and tadpole performance in the frogRana latastei

Egg provisioning is a major maternal effect in amphibians. We evaluated the relationship between starting body size (a proxy of egg provisioning) and multiple measures of larval performance in the Italian agile frog Rana latastei; we analysed within-clutch variation, to remove co-variation between provisioning and genetic maternal effect. We reared tadpoles from multiple clutches in a common environment under two food treatments (high- and low-protein content), and measured the mortality, tadpole size during development and development rate. We used a Bayesian framework (Markov chain Monte Carlo generalized linear mixed models) for the multivariate analysis of performance measures. Tadpoles with a large starting size remained the largest ones through the entire larval development, and attained metamorphosis earlier. Food with a high-protein content reduced mortality and increased the growth and development rate; the choice of food may be important in captive-breeding/headstarting programmes. We did not detect effects of the interaction between provisioning and type of food on tadpole performance. Our study confirms the importance of egg provisioning in amphibians, showing that it can affect multiple traits, and that their effects can last through the entire larval development.

Polygyny, census and effective population size in the threatened frog, Rana latastei

AbstractEffective population size (Ne) is a key determinant of genetic diversity of populations. In amphibians, the ratio effective population size/census size (Ne/N) is often very low, raising concerns for the long‐term persistence of genetic diversity in isolated populations. It has been proposed that the phenomenon of ‘genetic compensation’ increases the ratio Ne/N in small populations, but the underlying mechanisms are poorly understood. Polygyny can decrease Ne/N because of the negative relationship between polygyny and Ne. We used genetic information (microsatellites) to evaluate the relationship between census size, polygyny and Ne in populations of the threatened Italian agile frog Rana latastei. We reconstructed parentage in tadpoles from nine populations with eight to 32 breeding females, using a likelihood‐based method; we analysed simulated datasets with known properties to confirm the reliability of this approach in reconstructing polygyny. Furthermore, we estimated Ne using approximate Bayesian computation. The level of polygyny differed strongly among populations (average number of mates per breeding male: 2–6.4). Polygyny was greater in populations with larger census sizes. Moreover, variance in male mating success was larger in large populations. Effective population size increased with population size, but was negatively related to polygyny; as polygyny increased in large populations, this was associated with reduced Ne/N. In polygynous species, increasing levels of polygyny in large populations may explain the low Ne/N values, with important implications for the conservation of genetic diversity and for long‐term population persistence.

Factors affecting repatriation success of the endangered Italian agile frog (Rana latastei)

AbstractRepatriations and translocations are among the most frequent actions deployed for conservation of amphibians all over the world. However, very few studies have analysed the effectiveness of these actions. The aims of this study were i) to measure the success of repatriations of Rana latastei carried out within a conservation project in Lombardy (Northern Italy) from 1999 to 2001, and ii) to analyse how the environmental factors affected repatriation success. Eighteen ponds were surveyed from February to April 2006, for an overall of 45 field surveys. Each pond was carefully checked for frogs and egg masses. We classified each pond as newly excavated or pre-existing according to habitat management actions and we measured 16 variables concerning micro- and macro-habitat characteristics, human disturbance and presence of predators. Rana latastei was found in 33.3% of monitored sites and the repatriation outcome of tadpole release did not differ between newly excavated and non managed ponds. Habitat variables influenced the success of repatriations, which increased in sites with higher percentage of woodlands. Finally, repatriation outcome was also negatively influenced by human disturbance and predator occurrence.

Genetic diversity, but not hatching success, is jointly affected by postglacial colonization and isolation in the threatened frog, Rana latastei

Both postglacial colonization and habitat fragmentation can reduce the genetic diversity of populations, which in turn can affect fitness. However, since these processes occur at different spatial and temporal scales, the consequences of either process may differ. To disentangle the relative role of isolation and postglacial colonization in determining genetic diversity and fitness, we studied microsatellite diversity of 295 individuals from 10 populations and measured the hatch rate of 218 clutches from eight populations of a threatened frog, R. latastei. The populations that were affected by fragmentation to a greater extent suffered higher embryo mortality and reduced hatch rate, while no effects of distance from glacial refugium on hatch rate were detected. Altogether, distance from glacial refugium and isolation explained > 90% of variation in genetic diversity. We found that the genetic diversity was lowest in populations both isolated and far from the glacial refugium, and that distance from refugium seems to have the primary role in determining genetic diversity. The relationship between genetic diversity and hatch rate was not significant. However, the proportion of genetic diversity lost through recent isolation had a significant, negative effect on fitness. It is possible that selection at least partially purged the negative effects of the ancestral loss of genetic diversity.

Testing Experimental Results in the Field: Comment on Ficetola and De Bernardi (2005)

Ficetola & De Bernardi (2005) test the effects of reproductive interference between Rana latastei and Rana dalmatina on the former’s reproductive success under natural conditions. Their study examines predictions by Hettyey & Pearman (2003), based on natural history observations and experimental results, that reproductive failure may occur because of heterospecific matings when species relative abundance is highly skewed. Ficetola & De Bernardi (2005) measure R. latastei reproductive success, represented by mean embryo viability across clutches raised in the laboratory, using three populations that are syntopic with R. dalmatina and two other populations, where this congener is absent. Ficetola and De Bernardi find no decrease in reproductive success of R. latastei populations in syntopy. They also find no relationship between the relative abundance of heterospecifics and R. latastei reproductive success. Ficetola & De Bernardi (2005) conclude that heterospecific individuals do not interfere with R. latastei reproduction under natural conditions and they emphasize validating experimental results using natural populations. We agree with Ficetola & De Bernardi (2005) that tests of experimental results under natural conditions are needed. Determining an optimal experimental setup is difficult because different setups may yield different results (e.g. Michalak & Rafinski 1999). Further, influential factors in experimental manipulations may be unimportant under natural conditions (e.g. Skelly & Kiesecker 2001). In this sense, the aim of Ficetola & De Bernardi (2005) to test the validity of experimental results using natural populations is admirable. Nonetheless, correlative tests of experimental studies must be designed with statistical rigor and a thorough understanding of the original studies in order to provide valid tests of predictions. Ficetola & De Bernardi (2005) is flawed in this respect. The design in Ficetola & De Bernardi (2005) creates two biases toward accepting the null hypothesis that reproductive interference by R. dalmatina does not affect R. latastei in nature. Firstly, Ficetola & De Bernardi (2005) ignore the result that the relationship between species relative abundance and effectiveness of sexually isolating mechanisms is strongly non-linear (Hettyey & Pearman 2003). Abundance of R. latastei and R. dalmatina in the syntopic study populations of Ficetola and De Bernardi is estimated to be 1:1, 1:2, and 1:5. Hettyey & Pearman (2003) observe no significant decrease in fertilization success in trials, where R. latastei males are not outnumbered by R. dalmatina males by at least 1:5, probably because of the mitigating effect of mate recognition mechanisms. The results and model of Hettyey & Pearman (2003) predict significant decrease in average reproductive success in nature only when one species occurs at very low relative abundance. Decreased reproductive success where R. latastei’s relative abundance is 1:1 and 1:2 is not expected, thus grouping all syntopic populations together biases Ficetola and De Bernardi’s test. Further, absence of a bimodal distribution of embryo viability and no decrease in embryo viability in the one population where R. latastei was outnumbered by 1:5 would suggest that conspecific–heterospecific ratios be <1:5 in some natural populations to disrupt sexual isolation. However, one cannot draw such a conclusion from the single population in Ficetola & De Bernardi (2005) in which the phenomenon was expected. Secondly, Ficetola & De Bernardi (2005) suffers from small sample size, which biases the study Ethology

Testing Experimental Results in the Field: Reply to Hettyey and Pearman

In their comment to our recent paper (Ficetola & De Bernardi 2005), Hettyey and Pearman outline two main weakness of our observational study: our sample size was too small and our data did not allow us to test the possibility of non linearity of the relationship between species ratio and egg fertility. The issue of our sample size has already been addressed in our paper, but we stress that, despite our small sample size, our effect size was very small. Our power analysis shows that, given the observed effect size, a disproportionate number of populations would need to be sampled in order to obtain a significant result. For example, the estimated number of populations required to detect an effect significant at a 1⁄4 0.05 with power 0.8 was 1015 populations, a number greater than the number of known Rana latastei populations (Grossenbacher 1997). Hettyey and Pearman suggest a second weakness of our paper. The relationship between the abundance of heterospecifics and the fertility of R. latastei is expected to be nonlinear and our test is biased as the reduction of fertility could be observable only at very high abundance of heterospecifics. In this regard, we stress that there is a difference between a nonlinear relationship and a relationship that has an effect only after a given threshold (Huggett 2005). Under outdoor laboratory conditions, the effect of heterospecific abundance on fertility of R. latastei is depicted by Fig. 2 in Hettyey & Pearman (2003). It is difficult to draw conclusions about the shape of this relationship, given the small number of different heterospecific densities tested (n 1⁄4 4), but this relationship seems to be monotonic and there is no evident threshold effect, since fertility always decreases as heterospecific abundance increases. Indeed, in their paper Hettyey & Pearman (2003, p. 297) state that ‘Mean embryo viability declined with decreasing abundance of conspecific males’, and a model with a linear decline provides an adequate fit to their data (Pearson’s correlation based on the values of Fig. 2: r 1⁄4 0.959, n 1⁄4 4, p 1⁄4 0.041). The above pattern is completely different from those in Fig. 3 of our paper. When heterospecifics outnumber R. latastei, the difference in fertility between egg masses laid in plastic wading pools and in nature is pronounced and cannot be overlooked as Hettyey and Pearman do in their comment. Under skewed species ratio (1:5), the average egg fertility was 90% in the 32 egg masses we collected in nature (SE 1⁄4 2.7) and approx. 14% in the four egg masses laid during the Hettyey and Pearman’s experiment (SE @ 12). Despite similar species ratio, the overlap of fertility ranges between the two studies is close to zero and the fertility observed under natural conditions is much higher than those observed in the laboratory study (Z test: Z 1⁄4 5.1, p < 0.0001). Hettyey and Pearman suggest that raising embryos could create biases in the observed hatch rate, but we would be very surprised to observe a hatch rate of 90% from egg masses having <15% of fertility when they were laid. In their comment, Hettyey and Pearman do not discuss the main point addressed by our paper. The main question that we addressed was whether the social interaction observed under laboratory conditions is important for the reproductive outcome of natural populations. Species are not randomly distributed in nature and therefore we wonder why Hettyey and Pearman suggest a random selection of populations. Our populations were selected from a large database (Bernini et al. 2004), to be representative of the situation in an area encompassing approx. 12% of the range of R. latastei. In these populations, there are no obvious trends of fertility in relationship to the presence/abundance of heterospecifics and there is no evidence of drop of fertility under very skewed species ratio (Ficetola & De Bernardi 2005). Moreover, the difference in egg fertility Ethology

Amphibian Diversity and Nestedness in a Dynamic Floodplain River (Tagliamento, NE-Italy)

Amphibian distribution and assemblage structure were investigated along the last morphologically intact river corridor in Central Europe (Tagliamento). Thirteen taxa were identified with Rana latastei and Bufo bufo being the predominant species. In the main study reach, a 2 km2 dynamic island-braided floodplain in the middle section of the river, 130 water bodies were delineated that were situated either in the active floodplain (82 sites) or in the adjacent riparian forest (48 sites). Results demonstrated that the active floodplain increased appreciably the available habitat for amphibians, despite frequent disturbances by floods or droughts. Amphibian richness within a given habitat was significantly correlated with distance from vegetated islands, fish density, and water temperature. In the active floodplain, species distribution was highly predictable, exhibiting nearly perfect nestedness, suggesting that selective colonisation and extinction processes predominated. The degree of nestedness was much higher than in the adjacent riparian forest or in regulated floodplains in Central Europe. Results clearly emphasise that amphibians can exploit the entire hydrodynamic gradient, except the main channel. In the active floodplain, vegetated islands and large woody debris are important, directly and indirectly, in maintaining both habitat and amphibian diversity and density in this gravel-bed river.

Trade-off between larval development rate and Post-metamorphic Traits in the Frog Rana latastei

Development rate early in the ontogeny is believed to correlate positively with fitness. Geographic variation in intrinsic development rate suggests the existence of trade-offs between development rate and other fitness related traits. We investigated whether these trade-offs exist between intrinsic larval development rate and post-metamorphic traits in an organism with a complex life cycle. In laboratory, we measured if the tadpoles of the frog Rana latastei with fast intrinsic development rate have a suboptimal post-metamorphic morphology, by comparing froglets from five populations. Then, we evaluated the relationship between age at metamorphosis, hindlimb length and jumping performance for frogs grown in nature in two populations. Under laboratory conditions, froglets with fast intrinsic development had shorter absolute and shorter size-adjusted tibiofibulas. We observed a strong, positive relationship between tibiofibula length and jumping performance. In nature, froglets from the last metamorphosing population had longer absolute and size-adjusted tibiofibulas, and were able to jump further. The cost of fast development could be the shorter legs of early metamorphosing frogs, and their poor jumping performance. Thus, a fast intrinsic development rate may not always be positively related to lifetime fitness, since delayed effects of larval development persist also across life history stages.

Susceptibility of Italian agile frog populations to an emerging strain ofRanavirusparallels population genetic diversity

AbstractWestern populations of the Italian agile frog (Rana latastei) experience widespread genetic depletion. Based on population genetic theory, molecular models of immunity and previous empirical studies, population genetic depletion predicts increased susceptibility of populations to emergent pathogens. We experimentally compared susceptibility ofR. latasteipopulations upon exposure to an emerging strain ofRanavirus, frog virus 3 (FV3), using six populations spanning the geographical range and range of population genetic diversity found in nature. Our findings confirm this prediction, suggesting that the loss of genetic diversity accompanying range expansion and population isolation is coincident with increased mortality risk from an emergent pathogen. Loss of heterozygosity and escape from selection imposed by immunologically cross‐reactive pathogens may potentially generate range‐wide variation in disease resistance.

Supplementation or in situ conservation? Evidence of local adaptation in the Italian agile frog Rana latastei and consequences for the management of populations

Relocation of endangered species can be an effective conservation tool if it does not mix populations that represent significant intraspecific variation. The threatened Italian agile frog, Rana latastei, has small populations with low genetic diversity: translocation has been proposed to improve the likelihood of survival of populations. Using a common environment experiment and field surveys, we investigated whether there were differences in larval growth and developmental rate between foothill and lowland R. latastei populations, to evaluate if they are evolutionarily significant units. In nature, the colder climate of the foothills causes delayed metamorphosis. Conversely, in a common environment, larvae from foothill populations show faster growth and development. We did not find a significant egg-size related maternal effect or any differences in size at metamorphosis. We hypothesise that counter-gradient selection promoted fast growing phenotypes in a cold environment, where low temperatures slow down larval development. Foothill populations, despite being only a small geographical distance away from lowland populations, seem to be adapted to a colder climate and represent an evolutionarily significant unit. Different populations should, therefore, be managed independently, avoiding translocation. We suggest that evolutionary divergence between populations should be verified prior to planning relocation programmes, to prevent the risk of genetic homogenisation.

RESPONSE OF THE ITALIAN AGILE FROG (RANA LATASTEI) TO A RANAVIRUS, FROG VIRUS 3: A MODEL FOR VIRAL EMERGENCE IN NAÏVE POPULATIONS

Ranavirus (family Iridoviridae) is a genus of pathogens of poikilotherms, and some ranaviruses may play a role in widespread mortality of amphibians. Ecology of viral transmission in amphibians is poorly known but can be addressed through experimentation in the laboratory. In this study, we use the Ranavirus frog virus 3 (FV3) as an experimental model for pathogen emergence in naive populations of tadpoles. We simulated emerging disease by exposing tadpoles of the Italian agile frog (Rana latastei), to the North American Ranavirus FV3. We demonstrated that mortality occurred due to viral exposure, exposure of tadpoles to decreasing concentrations of FV3 in the laboratory produced dose-dependent survival rates, and cannibalism of virus-carrying carcasses increased mortality due to FV3. These experiments suggest the potential for ecological mechanisms to affect the level of exposure of tadpoles to Ranavirus and to impact transmission of viral pathogens in aquatic systems.

Distribution and status of Rana latastei in Italy (Amphibia, Ranidae)

Data regarding distribution, demographic parameters, ecological preferences and conservation status of Rana latastei in Italy are reported. By comparing bibliographic information and original field data, the authors wish to provide a first estimate concerning the possible risk of rarefaction of this species throughout the national territory. Observations were carried out in an area corresponding lo a large portion of the distribution range in Italy; in particular, the following regions were considered: Piedmont, Lombardy, Venetia and Friuli-Venezia Giulia. The populations of Rana latastei seem to have undergone a constant decline in recent decades; the species is threatened mainly from the limited extension and fragmentation of suitable habitats and should probably be considered in the category “vulnerable”.

Large golden-ringed dragonfly Cordulegaster heros Theischinger 1979, new for the fauna of Italy (Odonata: Cordulegastridae)

The species has been recorded at three localities in the extreme eastern part of the Friuli-Venezia Giulia region, north-eastern Italy, thus bringing the number of Italian dragonfly species to 89. The distribution of C. heros in Slovenia and Italy is mapped and discussed from the zoogeographical point of view. Additionally, the collected faunistic data on other dragonfly species and sympatric occurrence of endangered frog species Rana latastei Boulenger are given. Due to inclusion of C. heros and R. latastei to the Annexes II and IV of the Habitat Directive of EU, the conservation of their habitats in Italy and western Slovenia is proposed within the European network of protected areas NATURA 2000.

A skeletochronological study of growth, longevity, and age at sexual maturity in a population of Rana latastei (Amphibia, Anura).

Longevity and age at sexual maturity in an Italian population of Rana latastei were studied by skeletochronology performed on the phalanges. Frogs collected in 1998 and 1999 by drift fences and pitfall traps were marked by toe-clipping. After marking, individuals were released and the cut phalanges were processed for skeletochronological analysis. The maximum age so far recorded was 3 years in males and 4 years in females. The smallest male and female that were sexually mature on the basis of histological analysis of the gonads were 36 and 35 mm snout vent length (SVL), respectively. In both sexes, most individuals were estimated to breed shortly after emergence from their first overwintering. Among the European Brown Frogs, Rana latastei appears to be one of the shortest-lived and one of the first to reach sexual maturity.

Genetic depletion in Swiss populations of Rana latastei: conservation implications

Rana latastei, the Italian Agile Frog, is found over a restricted range in Central Europe, with populations in Switzerland (Tessin) occurring at the very margin of the species distribution. We used microsatellite DNA polymorphisms to study the genetic diversity of Swiss populations of Rana latastei and compared them with several Italian populations located in the central and western parts of the species’ range. On average, Swiss populations showed a much lower level of within-population genetic diversity relative to their Italian counterparts. Existing Swiss conservation efforts do not include genetic considerations and our results show that any attempt to increase genetic diversity within Switzerland would require a collaborative effort with Italian wildlife agencies.

Social environment and reproductive interference affect reproductive success in the frog Rana latastei

Variation in the social environment produces selection on morphological and behavioral traits. It is less clear how the social environment generates variation in demography through behavioral mechanisms. Theory suggests that one aspect of social environment, relative abundance of heterospecifics, influences the intensity of reproductive interference and its demographic effects. These effects are countered by species recognition and female preferences. We studied the effects of social environment on reproductive success in replicated, mixed breeding populations of two ranid frogs, Rana latastei and Rana dalmatina, the former being of international conservation concern. We manipulated the social environment of female R. latastei experimentally by varying the relative abundance of potential conspecific and heterospecific sexual partners. We measured amplexus frequency and recorded the reproductive success of R. latastei females. When conspecific males were relatively uncommon, (1) the absolute and relative frequencies of conspecific amplexus decreased, indicating a breakdown of sexual isolation, (2) oviposition was less frequent, and (3) the percentage of viable embryos in deposited clutches decreased. R. latastei females in an environment of low relative conspecific abundance (1:5, R. Latastei:R. dalmatina) demonstrated 6.8% the reproductive success of females in an environment exclusively with conspecifics. We present a model for the dependence of conspecific amplexus on the social environment. We discuss several mechanisms that may influence reproduction by R. latastei, and we support conservation of the species' preferred habitat to reduce opportunities for reproductive interference that occur at shared breeding sites.