Amphibians stand at the forefront of a global biodiversity crisis. More than one-third of amphibian species are globally threatened, and over 120 species have likely suffered global extinction since 1980. Most alarmingly, many rapid declines and extinctions are occurring in pristine sites lacking obvious adverse effects of human activities. The causes of these ''enigmatic'' declines remain highly contested. Still, lack of long-term data on amphibian populations severely limits our understanding of the distribution of amphibian declines, and therefore the ultimate causes of these declines. Here, we identify a systematic community-wide decline in populations of terrestrial amphibians at La Selva Biological Station, a protected old-growth lowland rainforest in lower Central America. We use data collected over 35 years to show that population density of all species of terrestrial amphibians has declined by Ϸ75% since 1970, and we show identical trends for all species of common reptiles. The trends we identify are neither consistent with recent emergence of chytridiomycosis nor the climate-linked epidemic hypothesis, two leading putative causes of enigmatic amphibian declines. Instead, our data suggest that declines are due to climate-driven reductions in the quantity of standing leaf litter, a critical microhabitat for amphibians and reptiles in this assemblage. Our results raise further concerns about the global persistence of amphibian populations by identifying widespread declines in species and habitats that are not currently recognized as susceptible to such risks.conservation ͉ long-term studies ͉ tropical wet forest

We report the comparative proteomic characterization of the venoms of adult and newborn specimens of the lancehead pitviper Bothrops asper from two geographically isolated populations from the Caribbean and the Pacific versants of Costa Rica. The crude venoms were fractionated by reverse-phase HPLC, followed by analysis of each chromatographic fraction by SDS-PAGE, N-terminal sequencing, MALDI-TOF mass fingerprinting, and collision-induced dissociation tandem mass spectrometry of tryptic peptides. The two B. asper populations, separated since the late Miocene or early Pliocene (8-5 mya) by the Guanacaste Mountain Range, Central Mountain Range, and Talamanca Mountain Range, contain both identical and different (iso)enzymes from the PLA 2 , serine proteinase, and SVMP families. Using a similarity coefficient, we estimate that the similarity of venom proteins between the two B. asper populations may be around 52%. Compositional differences between venoms among different geographic regions may be due to evolutionary environmental pressure acting on isolated populations. To investigate venom variability among specimens from the two B. asper populations, the reverse-phase HPLC protein profiles of 15 venoms from Caribbean specimens and 11 venoms from snakes from Pacific regions were compared. Within each B. asper geographic populations, all major venom protein families appeared to be subjected to individual variations. The occurrence of intraspecific individual allopatric variability highlights the concept that a species, B. asper in our case, should be considered as a group of metapopulations. Analysis of pooled venoms of neonate specimens from Caribbean and Pacific regions with those of adult snakes from the same geographical habitat revealed prominent ontogenetic changes in both geographical populations. Major ontogenetic changes appear to be a shift from a PIII-SVMP-rich to a PI-SVMP-rich venom and the secretion in adults of a distinct set of PLA 2 molecules than in the neonates. In addition, the ontogenetic venom composition shift results in increasing venom complexity, indicating that the requirement for the venom to immobilize prey and initiate digestion may change with the size (age) of the snake. Besides ecological and taxonomical implications, the geographical venom variability reported here may have an impact in the treatment of bite victims and in the selection of specimens for antivenom production. The occurrence of intraspecies variability in the biochemical composition and symptomatology after envenomation by snakes from different gegraphical location and age has long been apreciated by herpetologist and toxinologists, though detailed comparative proteomic analysis are scarce. Our study represents the first detailed characterization of individual and ontogenetic venom protein profile variations in two geographical isolated B. asper populations, and highlights the necessity of using pooled venoms as a statistically representative venom for antivenom production.

Aim  We used inferences of phylogenetic relationships and divergence times for three lineages of highland pitvipers to identify broad‐scale historical events that have shaped the evolutionary history of Middle American highland taxa, and to test previous hypotheses of Neotropical speciation. Location  Middle America (Central America and Mexico). Methods  We used 2306 base pairs of mitochondrial gene sequences from 178 individuals to estimate the phylogeny and divergence times of New World pitviper lineages, focusing on three genera (Atropoides, Bothriechis and Cerrophidion) that are broadly co‐distributed across Middle American highlands. Results  We found strong correspondence across three highland lineages for temporally and geographically coincident divergences in the Miocene and Pliocene, and further identified widespread within‐species divergences across multiple lineages that occurred in the early–middle Pleistocene. Main conclusions  Available data suggest that there were at least three major historical events in Middle America that had broad impacts on species divergence and lineage diversification among highland taxa. In addition, we find widespread within‐species genetic structure that may be attributable to the climatic changes that affected gene flow among highland taxa during the middle–late Pleistocene.

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