Strong reproductive barriers in a narrow hybrid zone of West-Mediterranean green toads (Bufo viridis subgroup) with Plio-Pleistocene divergence

Background One key question in evolutionary biology deals with the mode and rate at which reproductive isolation accumulates during allopatric speciation. Little is known about secondary contacts of recently diverged anuran species. Here we conduct a multi-locus field study to investigate a contact zone between two lineages of green toads with an estimated divergence time of 2.7 My, and report results from preliminary experimental crosses. Results The Sicilian endemic Bufo siculus and the Italian mainland-origin B. balearicus form a narrow hybrid zone east of Mt. Etna. Despite bidirectional mtDNA introgression over a ca. 40 km North-South cline, no F1 hybrids could be found, and nuclear genomes display almost no admixture. Populations from each side of the contact zone showed depressed genetic diversity and very strong differentiation (FST = 0.52). Preliminary experimental crosses point to a slightly reduced fitness in F1 hybrids, a strong hybrid breakdown in backcrossed offspring (F1 x parental, with very few reaching metamorphosis) and a complete and early mortality in F2 (F1 x F1). Conclusion Genetic patterns at the contact zone are molded by drift and selection. Local effective sizes are reduced by the geography and history of the contact zone, B. balearicus populations being at the front wave of a recent expansion (late Pleistocene). Selection against hybrids likely results from intrinsic genomic causes (disruption of coadapted sets of genes in backcrosses and F2-hybrids), possibly reinforced by local adaptation (the ranges of the two taxa roughly coincide with the borders of semiarid and arid climates). The absence of F1 in the field might be due to premating isolation mechanisms. Our results, show that these lineages have evolved almost complete reproductive isolation after some 2.7 My of divergence, contrasting sharply with evidence from laboratory experiments that some anuran species may still produce viable F1 offspring after > 20 My of divergence.

Inferring the degree of incipient speciation in secondary contact zones of closely related lineages of Palearctic green toads (Bufo viridis subgroup).

Reproductive isolation between lineages is expected to accumulate with divergence time, but the time taken to speciate may strongly vary between different groups of organisms. In anuran amphibians, laboratory crosses can still produce viable hybrid offspring >20 My after separation, but the speed of speciation in closely related anuran lineages under natural conditions is poorly studied. Palearctic green toads (Bufo viridis subgroup) offer an excellent system to address this question, comprising several lineages that arose at different times and form secondary contact zones. Using mitochondrial and nuclear markers, we previously demonstrated that in Sicily, B. siculus and B. balearicus developed advanced reproductive isolation after Plio-Pleistocene divergence (2.6 My, 3.3-1.9), with limited historic mtDNA introgression, scarce nuclear admixture, but low, if any, current gene flow. Here, we study genetic interactions between younger lineages of early Pleistocene divergence (1.9 My, 2.5-1.3) in northeastern Italy (B. balearicus, B. viridis). We find significantly more, asymmetric nuclear and wider, differential mtDNA introgression. The population structure seems to be molded by geographic distance and barriers (rivers), much more than by intrinsic genomic incompatibilities. These differences of hybridization between zones may be partly explained by differences in the duration of previous isolation. Scattered research on other anurans suggests that wide hybrid zones with strong introgression may develop when secondary contacts occur <2 My after divergence, whereas narrower zones with restricted gene flow form when divergence exceeds 3 My. Our study strengthens support for this rule of thumb by comparing lineages with different divergence times within the same radiation.

New polymorphic microsatellites markers and development of mitotyping primers for West-Mediterranean green toad species (Bufo viridis subgroup)

We report new polymorphic microsatellites for three species of Palearctic green toads (Bufo viridis subgroup): 10 in B. balearicus and seven each in B. siculus and B. boulengeri. Diversity at these loci, measured for 27 B. balearicus, 23 B. siculus and 11 B. boulengeri, ranged from low to high (two to 10 alleles). Mitotyping primers, specific to the control region, which allow fast screening of parapatric Sicilian endemic B. siculus and Italian mainland-origin B. balearicus, were developed.

Thirteen polymorphic microsatellite markers for the European green toad Bufo viridis viridis, a declining amphibian species.

We report 13 new polymorphic microsatellite markers for the European green toad Bufo viridis viridis (B. viridis subgroup), a declining amphibian from Central, Southeastern and Eastern Europe. Diversity at these loci estimated for 19 individuals ranged from two to ten alleles. Most of these primers also cross-amplify in related West-Mediterranean green toad species (Bufo balearicus, B. siculus and B. boulengeri). These microsatellites will be useful for conservation genetics of threatened Bufo viridis viridis populations and evolutionary studies of green toad taxa in secondary contact to examine hybridization.

A genetic isolation gradient of populations of the Balearic green toad (Bufo balearicus) follows rising eastward fragmentation of the rural landscapes on the island of Menorca.

We present the first approach to the genetic diversity and structure of the Balearic toad (Bufo balearicus Boettger, 1880) for the island of Menorca. Forty-one individ- uals from 21 localities were analyzed for ten microsatellite loci. We used geo-refer- enced individual multilocus genotypes and a model-based clustering method for the inference of the number of populations and of the spatial location of genetic dis- continuities between those populations.¦Only six of the microsatellites analyzed were polymorphic. We revealed a northwest- ern area inhabited by a single population with several well-connected localities and another set of populations in the southeast that includes a few unconnected small units with genetically significant differences among them as well as with the individ- uals from the northwest of the island. The observed fragmentation may be explained by shifts from agricultural to tourism practices that have been taking place on the island of Menorca since the 1960s. The abandonment of rural activities in favor of urbanization and concomitant service areas has mostly affected the southeast of the island and is currently threatening the overall geographic connectivity between the different farming areas of the island that are inhabited by the Balearic toad.

Development and cross-amplification of thirty microsatellite loci in five diploid and polyploid Central Asian species of Palearctic green toads (Bufo viridis subgroup)

We report 30 polymorphic microsatellite markers for five species of Palearctic green toads (Bufo viridis subgroup): 23 in the diploid B. latastii, 19 in diploid B. turanensis, 20 in diploid B. shaartusiensis, 27 in tetraploid B. pewzowi, and 30 in triploid B. baturae. Genetic diversity at these loci, measured for 10-18 individuals per species, ranged from 2 to 19 alleles. These microsatellite loci will be useful for conservation plans (genetic diversity, population structure, evolutionary units), inheritance patterns, and evolution of green toads.

Estudi de les estratègies comportamentals dels gripau corredor (Bufo Calamita) a la zona semi-àrida de Mas de Melons (terme municipal de Lleida) durant l'hivern i principis de primavera del 2007

En aquest treball s’han radiomarcat, un total de 4 gripaus corredors (Bufo calamita) que posteriorment foren alliberats a la Bassa de l’Astor, per tal d’estudiar les estratègies comportamentals de l’espècie en una zona semi-àrida. Dels 4 animals alliberats, un es perdé als pocs dies, la resta foren seguits mitjançant radioseguiment uns 64 dies, entre finals d’hivern i principis de primavera del 2007. En el treball de seguiment s’anotà en cada una de les localitzacions la temperatura ambiental del moment i la freqüència a la qual emetia l’emissor. Donat que es tenien els emissors calibrats, a partir d’aquesta freqüència es podia obtenir la temperatura a la qual es trobava l’emissor, és a dir la temperatura corporal del gripau en aquest cas. Mitjançant programes estadístics s’ha pogut determinar que durant el període hivernal i principis de primavera existeix una relació estadísticament significativa entre la variació de la temperatura ambiental i la temperatura corporal dels gripaus. Aquesta relació no es dona però en el període estival. Això fa pensar que el comportament del calamita esdevé una forma de termoregulació actuant per tal d’evitar extrems crítics de temperatura i humitat. S’ha pogut determinar també que gràcies a les característiques dels refugis utilitzats en la zona d’estudi (munts de pedres o caus d’altres espècies) els gripaus aconsegueixen mantenir-se en ambients on la variació de temperatura és inferior a la variació de la temperatura exterior. Així la variació de la temperatura corporal dels gripaus és també inferior. Pel que fa als moviments entre els diferents refugis, aquests han variat en funció de cada animal. La distància mitja recorreguda en el total dels desplaçaments ha estat d’uns 185 metres lineals. Aquests desplaçaments s’han donat sempre en dies de pluja o l’endemà d’un dia de pluja.

Acción del etanol y del acetaldehido sobre la contracción isométrica en el músculo sartorio del sapo (Bufo sp.)

Tesis (Maestría en Ciencias con Especialidad en Fisiología Médica) U.A.N.L.

Genetic analysis of complex demographic scenarios: Spatially expanding populations of the cane toad, Bufo marinus

Inferring the spatial expansion dynamics of invading species from molecular data is notoriously difficult due to the complexity of the processes involved. For these demographic scenarios, genetic data obtained from highly variable markers may be profitably combined with specific sampling schemes and information from other sources using a Bayesian approach. The geographic range of the introduced toad Bufo marinus is still expanding in eastern and northern Australia, in each case from isolates established around 1960. A large amount of demographic and historical information is available on both expansion areas. In each area, samples were collected along a transect representing populations of different ages and genotyped at 10 microsatellite loci. Five demographic models of expansion, differing in the dispersal pattern for migrants and founders and in the number of founders, were considered. Because the demographic history is complex, we used an approximate Bayesian method, based on a rejection-regression algorithm. to formally test the relative likelihoods of the five models of expansion and to infer demographic parameters. A stepwise migration-foundation model with founder events was statistically better supported than other four models in both expansion areas. Posterior distributions supported different dynamics of expansion in the studied areas. Populations in the eastern expansion area have a lower stable effective population size and have been founded by a smaller number of individuals than those in the northern expansion area. Once demographically stabilized, populations exchange a substantial number of effective migrants per generation in both expansion areas, and such exchanges are larger in northern than in eastern Australia. The effective number of migrants appears to be considerably lower than that of founders in both expansion areas. We found our inferences to be relatively robust to various assumptions on marker. demographic, and historical features. The method presented here is the only robust, model-based method available so far, which allows inferring complex population dynamics over a short time scale. It also provides the basis for investigating the interplay between population dynamics, drift, and selection in invasive species.

Comparative cytogenetic studies of Bufo ictericus, B. paracnemis (Amphibia, Anura) and an intermediate form in sympatry

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effects of inhibition gastric acid secretion on arterial acid-base status during digestion in the toad Bufo marinus

Digestion affects acid-base status, because the net transfer of HCl from the blood to the stomach lumen leads to an increase in HCO3- levels in both extra- and intracellular compartments. The increase in plasma [HCO3-], the alkaline tide, is particularly pronounced in amphibians and reptiles, but is not associated with an increased arterial pH, because of a concomitant rise in arterial Pco(2) caused by a relative hypoventilation. In this study, we investigate whether the postprandial increase in Paco(2) of the toad Bufo marinus represents a compensatory response to the increased plasma [HCO3-] or a state-dependent change in the control of pulmonary ventilation. To this end, we successfully prevented the alkaline tide, by inhibiting gastric acid secretion with omeprazole, and compared the response to that of untreated toads determined in our laboratory during the same period. In addition, we used vascular infusions of bicarbonate to mimic the alkaline tide in fasting animals. Omeprazole did not affect blood gases, acid-base and haematological parameters in fasting toads, but abolished the postprandial increase in plasma [HCO3-] and the rise in arterial Pco(2) that normally peaks 48 h into the digestive period. Vascular infusion of HCO3-, that mimicked the postprandial rise in plasma [HCO3-], led to a progressive respiratory compensation of arterial pH through increased arterial Pco(2) Thus, irrespective of whether the metabolic alkalosis is caused by gastric acid secretion in response to a meal or experimental infusion of bicarbonate, arterial pH is being maintained by an increased arterial Pco(2). It seems, therefore, that the elevated Pco(2), occuring during the postprandial period, constitutes of a regulated response to maintain pH rather than a state-dependent change in ventilatory control. (C) 2003 Elsevier B.V. All rights reserved.

Cytotaxonomic considerations with the description of two new NOR locations for South American toads, genus Bufo (Anura: Bufonidae)

Cytogenetic studies were made on Brazilian Bufo: B. marinus, B. paracnemis, B. ictericus, B. rufus, B. arenarum, B. crucifer, Bufo granulosus, B. pygmaeus and B. margaritifer (= B. typhonius). All these species had a typical karyotype of 2n = 22. Species from the marinus and crucifer groups had NORs on Chromosome 7, species from the granulosus group had NORs on Chromosome 5, and B. margaritifer had NORs on Chromosome 10. The last two locations of NORs are described for the first time for the genus Bufo in South America.

Os jogos do texto ou as facetas da escrita?: gênero policial, intertextualidade e literatura francesa em Bufo & Spallanzani (1985) de Rubem Fonseca

Pós-graduação em Letras - FCLAS