José Ayarzagüena’s research while affiliated with Doñana Biological Station and other places

The success of a reintroduction program is determined by the ability of individuals to reproduce and thrive. Hence, an understanding of the mating system and breeding strategies of reintroduced species can be critical to the success, evaluation and effective management of reintroduction programs. As one of the most threatened crocodile species in the world, the Orinoco crocodile (Crocodylus intermedius) has been reduced to only a few wild populations in the Llanos of Venezuela and Colombia. One of these populations was founded by reintroduction at Caño Macanillal and La Ramera lagoon within the El Frío Biological Station, Venezuela. Twenty egg clutches of C. intermedius were collected at the El Frío Biological Station for incubation in the lab and release of juveniles after one year. Analyzing 17 polymorphic microsatellite loci from 335 hatchlings we found multiple paternity in C. intermedius, with half of the 20 clutches fathered by two or three males. Sixteen mothers and 14 fathers were inferred by reconstruction of multilocus parental genotypes. Our findings showed skewed paternal contributions to multiple-sired clutches in four of the clutches (40%), leading to an overall unequal contribution of offspring among fathers with six of the 14 inferred males fathering 90% of the total offspring, and three of those six males fathering more than 70% of the total offspring. Our results provide the first evidence of multiple paternity occurring in the Orinoco crocodile and confirm the success of reintroduction efforts of this critically endangered species in the El Frío Biological Station, Venezuela.

Additional information on 17 microsatellite loci used in Crocodylus intermedius paternity analysis. (DOC)

Parental genotypes reconstructed for 20 Crocodylus intermedius clutches across 17 polymorphic microsatellite loci. (DOC)

Basic information about the taxonomy, biology and distribution of Hyalinobatrachium glassfrogs of the Guiana Shield (GS) is scarce, ambiguous, and in many cases even contradictory. In this review we aim to clarify the current taxonomic status of this group by means of phenotypic (morphology, morphometrics and bioacoustics) and molecular (mitochondrial DNA sequences) comparisons. Eight species have previously been recognized for the GS: H. crurifasciatum, H. eccentri-cum, H. fleischmanni (initially described as Hylella cappellei in the GS), H. iaspidiense (with the putative synonym H. nouraguense), H. ignioculus, H. mesai, H. mondolfii, and H. taylori. Our data support the resurrection of H . cappellei from its synonymy with H. fleischmanni. Hyalinobatrachium crurifasciatum, H. eccentricum, and H. ignioculus are pro-posed as junior synonyms of H. cappellei. We show that none of the four paratypes of H. taylori belong to this species and we assign two to H. cappellei and two to H. mondolfii. Additional specimens previously identified as H. taylori are reas-signed to H. cappellei, and hence H. taylori is redefined. Hyalinobatrachium nouraguense is confirmed as a junior synonym of H. iaspidiense. We also describe two new species of Hyalinobatrachium from French Guiana: Hyalinobatrachium kawense sp. nov. and Hyalinobatrachium tricolor sp. nov. In addition, and in concordance with the new taxonomic rearrangements, we provide diagnostic characters for all species, known distributions and main sources of references for their biology. We also report new distribution records for H. iaspidiense and H. mondolfii, and describe the formerly unknown tadpole of the later. Consequently, we recognize seven species of Hyalinobatrachium for the Guiana Shield: H. cappellei, H. iaspidiense, H. kawense sp. nov., H. mesai, H. mondolfii, H. taylori, and H. tricolor sp. nov. We discuss the suitability of integrative taxonomy as an approach to identify taxonomic uncertainty and consider its signifi-cance for conservation purposes. We also address the implications of our results to understand phylogeographic patterns in this area.

We present data on the reproductive ecology of the Orinoco crocodile (Crocodylus intermedius) in a newly established population at the El Frío Biological Station, Venezuela, from 2003 to 2007. Nesting occurs during the dry season, and hatching of young takes place at the beginning of the rainy season. Elliptical hole-like nests are constructed in artificial sand beaches with a median nest depth of 42.6 cm. Nest depth is positively correlated with female total size, enabling us to predict the size of the female based on nest characteristics. Temperature in the egg chamber was on average 31.9 °C. The thermal amplitude of the nest was positively correlated with nest depth, and less than 1.3 °C when the nest was deeper than 30 cm. The average clutch size was 41.2 eggs, the average clutch mass was 4256.2 g, and egg viability was 75.4%. The average length, width and weight of eggs was 7.61 cm, 4.73 cm and 111.07 g, respectively. As part of the conservation programme, we also artificially incubated eggs from the species. Hatching rate in the incubator was 84.3%. Total length and mass at hatching were 28.6 cm and 66.9 g, respectively. Our data demonstrate that head-starting our population through egg incubation is a suitable conservation strategy for this endangered species.

Glassfrogs (family Centrolenidae) represent an exceptionally diverse group among Neotropical anurans, but their evolutionary relationships never have been assessed from a molecular perspective. Mitochondrial and nuclear markers were used to develop a novel hypothesis of centrolenid phylogeny. Ingroup sampling included 100 terminals, with 78 (53%) of the named species in the family, representing most of the phenotypic diversity described for the group. Thirty-five species representing taxa traditionally associated with glassfrogs were used as outgroups. Gene sampling consisted of complete or partial sequences of three mitochondrial (12S, 16S, ND1) and three nuclear markers (c-myc exon 2, RAG1, POMC) for a total of 4362 bp. Phylogenies were estimated using maximum parsimony, maximum likelihood, and Bayesian analyses for individual genes and combined datasets. The separate analysis of mitochondrial and nuclear datasets allowed us to clarify the relationships within glassfrogs; also, we corroborate the sister-group relationship between Allophryne ruthveni and glassfrogs. The new phylogeny differs significantly from all previous morphology-based hypotheses of relationships, and shows that hypotheses based on few traits are likely to misrepresent evolutionary history. Traits previously hypothesized as unambiguous synapomorphies are shown to be homoplastic, and all genera in the current taxonomy (Centrolene, Cochranella, Hyalinobatrachium, Nymphargus) are found to be poly- or paraphyletic. The new topology implies a South American origin of glassfrogs and reveals allopatric speciation as the most important speciation mechanism. The phylogeny profoundly affects the traditional interpretations of glassfrog taxonomy, character evolution, and biogeography—topics that now require more extensive evaluation in future studies.

Hyalinobatrachium orientale has a complex taxonomic history suggesting that more than one species could be under this name. In this review, we try to clarify the current taxonomic status of this species by means of morphological, bioacoustic, and mitochondrial DNA sequence comparisons of specimens from Tobago Island and the Venezuelan Cordillera de la Costa (Oriental Sector, Cordillera del Litoral and Cordillera del Interior). Our data support the resurrection of Hyalinobatrachium orocostale, restricted to the Cordillera del Interior. Additionally, specimens from Cordillera del Litoral and Oriental Sector do not form a monophyletic group; hence, we define as Hyalinobatrachium sp. the populations from Cordillera del Litoral and H. orientale sensu stricto the populations from the Oriental Sector. Preliminary bioacoustic and morphological analyses indicate that the populations from Tobago are conspecific with Hyalinobatrachium orientale sensu stricto.

Glassfrogs (family Centrolenidae) represent an exceptionally diverse group among Neotropical anurans, but their evolutionary relationships never have been assessed from a molecular perspective. Mitochondrial and nuclear markers were used to develop a novel hypothesis of centrolenid phylogeny. Ingroup sampling included 100 terminals, with 78 (53%) of the named species in the family, representing most of the phenotypic diversity described for the group. Thirty-five species representing taxa traditionally associated with glassfrogs were used as outgroups. Gene sampling consisted of complete or partial sequences of three mitochondrial (12S, 16S, ND1) and three nuclear markers (c-myc exon 2, RAG1, POMC) for a total of ∼4362 bp. Phylogenies were estimated using maximum parsimony, maximum likelihood, and Bayesian analyses for individual genes and combined datasets. The separate analysis of mitochondrial and nuclear datasets allowed us to clarify the relationships within glassfrogs; also, we corroborate the sister-group relationship between Allophryne ruthveni and glassfrogs. The new phylogeny differs significantly from all previous morphology-based hypotheses of relationships, and shows that hypotheses based on few traits are likely to misrepresent evolutionary history. Traits previously hypothesized as unambiguous synapomorphies are shown to be homoplastic, and all genera in the current taxonomy (Centrolene, Cochranella, Hyalinobatrachium, Nymphargus) are found to be poly- or paraphyletic. The new topology implies a South American origin of glassfrogs and reveals allopatric speciation as the most important speciation mechanism. The phylogeny profoundly affects the traditional interpretations of glassfrog taxonomy, character evolution, and biogeography—topics that now require more extensive evaluation in future studies.

We describe a new species of Hyalinobatrachium from Serranía de Perijá, Estado de Zulia, Venezuela. The new species is placed in the Hyalinobatrachium fleischmanni group and can be differentiated from all other species of Hyalinobatrachium by having small enamelled glands on the lower part of the upper lip and on the skin covering the jaw, iris with a horizontal dark band that connects the pupil with the lateral edges of the eye, marked ulnar and tarsal white enamelled folds, extensive webbing between fingers and between toes, white pericardium and an advertisement call formed by a high-pitched single note composed of two parts: a group of modulated pulses increasing in frequency followed by a long and modulated sound at a constant frequency.