Parotoid gland secretions of toad species are a vast reservoir of bioactive molecules with a wide range of biological properties. Herein, for the first time, it is described the isolation by preparative reversed-phase HPLC and the structure elucidation by NMR spectroscopy and/or mass spectrometry of nine major bufadienolides from parotoid gland secretions of the Cuban endemic toad Peltophryne fustiger: ψ-bufarenogin, gamabufotalin, bufarenogin, arenobufagin, 3-(N-suberoylargininyl) marinobufagin, bufotalinin, telocinobufagin, marinobufagin and bufalin. In addition, the secretion was analyzed by UPLC-MS/MS which also allowed the identification of azelayl arginine. The effect of arenobufagin, bufalin and ψ-bufarenogin on Na(+)/K(+)-ATPase activity in a human kidney preparation was evaluated. These bufadienolides fully inhibited the Na(+)/K(+)-ATPase in a concentration-dependent manner, although arenobufagin (IC50 = 28.3 nM) and bufalin (IC50 = 28.7 nM) were 100 times more potent than ψ-bufarenogin (IC50 = 3020 nM). These results provided evidence about the importance of the hydroxylation at position C-14 in the bufadienolide skeleton for the inhibitory activity on the Na(+)/K(+)-ATPase.

Habitat loss and climate change are major threats to amphibian species worldwide. We combined niche modelling under various climatic scenarios with analysis of habitat loss and the appropriateness of Cuban protected areas to identify major risk zones for the Endangered Cuban toad Peltophryne longinasus. Four subspecies with disjunct distributions associated with mountain forests are recognized. Our results suggest that the western subspecies, P. longinasus longinasus and P. longinasus cajalbanensis, are at risk from global warming, habitat degradation and potential additive effects. Peltophryne longinasus dunni, in central Cuba, has the lowest threat level related to climate change and habitat loss but could become increasingly threatened by the presence of the infectious disease chytridiomycosis. The eastern subspecies, P. longinasus ramsdeni, faces moderate impacts of climate change and habitat loss; however, low opportunity of migration to new areas and population decline justify a high threatened status for this subspecies. Our results predict minor temperature increases and precipitation decreases in the future. Nevertheless, at the biological level these changes could generate variations in species physiology, vocal behaviour and prey availability, and could probably increase the risk of predation. In Cuba protected areas have contributed to avoiding excessive forest loss but the potential impact of climate change was not considered in their original design. Our findings confirm that all subspecies of P. longinasus are threatened but management measures should be tailored according to the various predicted impacts.

1. Global change processes (including climate change, sea-level rise, and land-use change, among others) represent significant threats to biodiversity. These changes are usually studied across broad areas, and understanding their effects on narrowly distributed endangered species is challenging. Peltophryne florentinoi is a critically

Pigmentation anomalies may occur due to genetic or environmental factors and can affect restricted parts of the body or the entire surface. Eleutherodactylus zeus is frog endemic to western Cuba where it is adapted to life in caves, rock crevices, and other sheltered sites in limestone landscapes associated with forest habitats. We observed 43 frogs in Santo Tomás cave, in Viñales National Park, of which 26 % showed depigmented blotches, typical of piebaldism, along their bodies. No unusual behaviour was detected in any of these frogs. This is the first reported case of piebaldism in frogs of the West Indies and consequently of Cuba. Records of piebaldism in amphibians are very scarce in the literature, not necessarily as a consequence of its rarity in nature but possibly due to inconsistencies in the classification of pigmentation abnormalities.

We studied the variation in genetics, bioacustics, and morphology in Eleutherodactylus glamyrus, a regionally endemic frog species restricted to high elevations in the Sierra Maestra Massif, Western Cuba that was originally described as a cryptic species hidden under the name E. auriculatus. Genetic analysis of mtDNA sequences of the 16S and cob genes identify two allopatric and strongly supported mitochondrial clades (phylogroups) which also showed no haplotype sharing in the nuclear Rag-1 gene. Bioacustic, and morphological comparisons concordantly identify these two phylogroups as independent evolutionary lineages. Therefore, we herein restrict the name Eleutherodactylus glamyrus Estrada and Hedges to populations represented in our analyses as the western phylogroup (Cordillera del Turquino to Pico La Bayamesa) and consider specimens from the eastern phylogroup (Sierra del Cobre) to represent a new species described and named as Eleutherodactylus cattus. Our results add to the growing list of Eleutherodactylus species endemic to Cuba and highlight the importance of combining different sources of evidence for obtaining robust assessments of species limits in amphibians.