Roberto Ibáñez

The Status of the Panama Canal Watershed and Its Biodiversity at the Beginning of the 21st Century

P is a small Central American country, but it operates a big canal and the world keeps an eye on developments there. Problems with the canal or ecological disasters in its watershed would attract a lot of attention. As Theodore Roosevelt planned, the Panama Canal is a major shipping corridor, transporting 37 ships a day and providing substantial income to the Panamanian government. Yet as Roosevelt could not have recognized—despite his interest in conservation—the canal sits in the center of one of the world’s most biologically diverse areas (Myers et al. 2000): Southern Central America has more forest bird species than any other region in the world, except Amazonia and the northern and central Andes, each of which is vastly larger than southern Central America (Stotz et al. 1996); and Panama has as many plant species per 10,000 km2 as any region in the world, more than Amazonia or the Malay Peninsula (Barthlott et al. 1996). Roosevelt may have suspected, though, that forests are crucial for protecting the water supply of the Panama Canal and for maintaining the plant and animal communities. Fortunately, the year 2000 still found extensive forests around the canal, protected largely thanks to military and shipping interests, but it also found the watershed adjacent to a large and expanding capital city. Maintaining the ecosystem integrity of the canal will pose a major challenge for conservation in the 21st century. Is urban and economic development compatible with a hydrologically functioning canal and conservation of an extremely diverse flora and fauna?

AN ECOSYSTEM REPORT ON THE PANAMA CANAL: MONITORING THE STATUS OF THE FOREST COMMUNITIES AND THE WATERSHED

In 1996, the Smithsonian Tropical Research Institute and the Republic of Panamas Environmental Authority, with support fromthe United States Agency for International Development, undertook a comprehensive program to monitor the ecosystem of the Panama Canal watershed. The goals were to establish baselineindicators for the integrity of forest communities and rivers. Based on satellite image classification and ground surveys, the2790 km2 watershed had 1570 km2 of forest in 1997, 1080 km2 of which was in national parks and nature monuments. Most of the 490 km2 of forest not currently in protected areas lies along the west bank of the Canal, and its managementstatus after the year 2000 turnover of the Canal from the U.S. to Panama remains uncertain. In forest plots designed to monitorforest diversity and change, a total of 963 woody plant specieswere identified and mapped. We estimate there are a total of 850–1000 woody species in forests of the Canal corridor. Forestsof the wetter upper reaches of the watershed are distinct in species composition from the Canal corridor, and have considerably higher diversity and many unknown species. Theseremote areas are extensively forested, poorly explored, and harbor an estimated 1400–2200 woody species. Vertebrate monitoring programs were also initiated, focusing on species threatened by hunting and forest fragmentation. Large mammals are heavily hunted in most forests of Canal corridor, and therewas clear evidence that mammal density is greatly reduced in hunted areas and that this affects seed predation and dispersal. The human population of the watershed was 113 000 in 1990, and grew by nearly 4% per year from 1980 to 1990. Much of this growth was in a small region of the watershed on the outskirts of Panama City, but even rural areas, including villages near and within national parks, grew by 2% per year. There is no sewage treatment in the watershed, and many towns have no trashcollection, thus streams near large towns are heavily polluted. Analyses of sediment loads in rivers throughout the watershed did not indicate that erosion has been increasing as a result ofdeforestation, rather, erosion seems to be driven largely by total rainfall and heavy rainfall events that cause landslides.Still, models suggest that large-scale deforestation would increase landslide frequency, and failure to detect increases inerosion could be due to the gradual deforestation rate and the short time period over which data are available. A study of runoff showed deforestation increased the amount of water fromrainfall that passed directly into streams. As a result, dry season flow was reduced in a deforested catchment relative to aforested one. Currently, the Panama Canal watershed has extensive forest areasand streams relatively unaffected by humans. But impacts of hunting and pollution near towns are clear, and the burgeoningpopulation will exacerbate these impacts in the next few decades.Changes in policies regarding forest protection and pollution control are necessary.

Antifungal isolates database of amphibian skin-associated bacteria and function against emerging fungal pathogens

Microbial symbionts of vertebrate skin have an important function in defense of the host against pathogens. In particular, the emerging chytrid fungus Batrachochytrium dendrobatidis, causes widespread disease in amphibians but can be inhibited via secondary metabolites produced by many different skin-associated bacteria. Similarly, the fungal pathogens of terrestrial salamander eggs Mariannaea elegans and Rhizomucor variabilis are also inhibited by a variety of skin-associated bacteria. Indeed, probiotic therapy against fungal diseases is a recent approach in conservation medicine with growing experimental support. We present a comprehensive Antifungal Isolates Database of amphibian skin-associated bacteria that have been cultured, isolated, and tested for antifungal properties. At the start, this database includes nearly 2000 cultured bacterial isolates from 37 amphibian host species across 18 studies on five continents: Africa, Oceania, Europe, and North and South America. As the research community gathers information on additional isolates, the database will be updated periodically. The resulting database can serve as a conservation tool for amphibians and other organisms, and provides empirical data for comparative and bioinformatic studies. The database consists of a FASTA file containing 16S rRNA gene sequences of the bacterial isolates, and a metadata file containing information on the host species, life-stage, geographic region, and antifungal capacity and taxonomic identity of the isolate.

Avian predation upon lizards and frogs in a neotropical forest understorey

Although tropical forest birds are known to prey upon small lizards and frogs, no study has documented the attributes of vertebrate-eating birds or whether birds prey opportunistically on the different elements of the herpetofauna within tropical communities. This study is based on a 14-mo investigation on avian diet, supplemented with a 3-y census of frogs and a 1-y census of lizards in a humid forest of central Panama. From 91 bird species, 1086 regurgitates were collected, in which were found 75 lizards and 53 frogs. Over 50% of the common, primarily insectivorous bird species preyed upon lizards or frogs, with a mean frequency of 0.26 prey/sample. These birds (22 species, nine families) foraged on various sub- strates from different strata of the forest, fed on invertebrates averaging from 3.3 to 17.2 mm in length, weighed from 11 to 195 g, and had bill lengths that varied from 12.2 to 49.8 mm. Based on a logistic regression analysis, intensity of foraging at army-ant swarms was the variable that best explained the likelihood that a bird species preyed upon lizards, leading to a classification that was 91% correct. In contrast, bill length and body length classified correctly 88% of the frog-eating birds, which showed a fairly constant 1:7 bill length/body length ratio (as opposed to a mean but highly variable 1:10 ratio in other species). A multiple regression analysis showed that seasonal variation in intensity of lizard predation was posit- ively related to arthropod abundance except during the breeding season when lizard intake decreased, presumably because nesting birds did not follow ant swarms. Intensity of frog predation correlated with frog abundance over time, the latter being inversely related to arthropod availability. Ninety-seven per cent of all lizards and frogs identified in the diet samples (n = 105) were from two genera, Anolis and Eleutherodactylus, respectively. Prey size distribution in the regurgitates suggested an optimal prey size of 33.5 mm snout-vent length (SVL) for lizards and 14.5 mm SVL for frogs. Birds preyed opportunistically on the different Anolis spe- cies, but almost exclusively upon juvenile individuals. Abundances of the different Eleutherodactylus species correlated with their predation rates, but these frogs rep- resented only 10% of all the frogs observed during the censuses. The two most common local anurans, Colostethus flotator and Bufo typhonius, were not taken by any bird species.

The Great American Biotic Interchange in frogs: multiple and early colonization of Central America by the South American genus Pristimantis (Anura: Craugastoridae).

The completion of the land bridge between North and South America approximately 3.5-3.1 million years ago (Ma) initiated a tremendous biogeographic event called the Great American Biotic Interchange (GABI), described principally from the mammalian fossil record. The history of biotic interchange between continents for taxonomic groups with poor fossil records, however, is not well understood. Molecular and fossil data suggest that a number of plant and animal lineages crossed the Isthmus of Panama well before 3.5 Ma, leading biologists to speculate about trans-oceanic dispersal mechanisms. Here we present a molecular phylogenetic analysis of the frog genus Pristimantis based on 189 individuals of 137 species, including 71 individuals of 31 species from Panama and Colombia. DNA sequence data were obtained from three mitochondrial (COI, 12S, 16S) and two nuclear (RAG-1 and Tyr) genes, for a total of 4074 base pairs. The resulting phylogenetic hypothesis showed statistically significant conflict with most recognized taxonomic groups within Pristimantis, supporting only the rubicundus Species Series, and the Pristimantis myersi and Pristimantis pardalis Species Groups as monophyletic. Inference of ancestral areas based on a likelihood model of geographic range evolution via dispersal, local extinction, and cladogenesis (DEC) suggested that the colonization of Central America by South American Pristimantis involved at least 11 independent events. Relaxed-clock analyses of divergence times suggested that at least eight of these invasions into Central America took place prior to 4 Ma, mainly in the Miocene. These findings contribute to a growing list of molecular-based biogeographic studies presenting apparent temporal conflicts with the traditional GABI model.

Skin bacterial diversity of Panamanian frogs is associated with host susceptibility and presence of Batrachochytrium dendrobatidis

Symbiotic bacteria on amphibian skin can inhibit growth of the fungus Batrachochytrium dendrobatidis (Bd) that has caused dramatic population declines and extinctions of amphibians in the Neotropics. It remains unclear how the amphibians’ skin microbiota is influenced by environmental bacterial reservoirs, host-associated factors such as susceptibility to pathogens, and pathogen presence in tropical amphibians. We sampled skin bacteria from five co-occurring frog species that differ in Bd susceptibility at one Bd-naive site, and sampled one of the non-susceptible species from Bd-endemic and Bd-naive sites in Panama. We hypothesized that skin bacterial communities (1) would be distinct from the surrounding environment regardless of the host habitat, (2) would differ between Bd susceptible and non-susceptible species and (3) would differ on hosts in Bd-naive and Bd-endemic sites. We found that skin bacterial communities were enriched in bacterial taxa that had low relative abundances in the environment. Non-susceptible species had very similar skin bacterial communities that were enriched in particular taxa such as the genera Pseudomonas and Acinetobacter. Bacterial communities of Craugastor fitzingeri in Bd-endemic sites were less diverse than in the naive site, and differences in community structure across sites were explained by changes in relative abundance of specific bacterial taxa. Our results indicate that skin microbial structure was associated with host susceptibility to Bd and might be associated to the history of Bd presence at different sites.

Chytridiomycosis and Amphibian Population Declines Continue to Spread Eastward in Panama

Chytridiomycosis is a globally emerging disease of amphibians and the leading cause of population declines and extirpations at species-diverse montane sites in Central America. We continued long-term monitoring efforts for the presence of the fungal pathogen Batrachochytrium dendrobatidis (Bd) and for amphibian populations at two sites in western Panama, and we began monitoring at three new sites to the east. Population declines associated with chytridiomycosis emergence were detected at Altos de Campana National Park. We also detected Bd in three species east of the Panama Canal at Soberanía National Park, and prevalence data suggests that Bd may be enzootic in the lowlands of the park. However, no infected frogs were found further east at Tortí (prevalence <7.5% with 95% confidence). Our results suggest that Panama’s diverse and not fully described amphibian communities east of the canal are at risk. Precise predictions of future disease emergence events are not possible until factors underlying disease emergence, such as dispersal, are understood. However, if the fungal pathogen spreads in a pattern consistent with previous disease events in Panama, then detection of Bd at Tortí and other areas east of the Panama Canal is imminent. Therefore, development of new management strategies and increased precautions for tourism, recreation, and biology are urgently needed.

More than skin deep: functional genomic basis for resistance to amphibian chytridiomycosis.

The amphibian-killing chytrid fungus Batrachochytrium dendrobatidis (Bd) is one of the most generalist pathogens known, capable of infecting hundreds of species globally and causing widespread population declines and extinctions. However, some host species are seemingly unaffected by Bd, tolerating or clearing infections without clinical signs of disease. Variation in host immune responses is commonly evoked for these resistant or tolerant species, yet to date, we have no direct comparison of amphibian species responses to infection at the level of gene expression. In this study, we challenged four Central American frog species that vary in Bd susceptibility, with a sympatric virulent strain of the pathogen. We compared skin and spleen orthologous gene expression using differential expression tests and coexpression gene network analyses. We found that resistant species have reduced skin inflammatory responses and increased expression of genes involved in skin integrity. In contrast, only highly susceptible species exhibited suppression of splenic T-cell genes. We conclude that resistance to chytridiomycosis may be related to a species’ ability to escape the immunosuppressive activity of the fungus. Moreover, our results indicate that within-species differences in splenic proteolytic enzyme gene expression may contribute to intraspecific variation in survival. This first comparison of amphibian functional immunogenomic architecture in response to Bd provides insights into key genetic mechanisms underlying variation in disease outcomes among amphibian species.

DNA barcoding applied to ex situ tropical amphibian conservation programme reveals cryptic diversity in captive populations

Amphibians constitute a diverse yet still incompletely characterized clade of vertebrates, in which new species are still being discovered and described at a high rate. Amphibians are also increasingly endangered, due in part to disease‐driven threats of extinctions. As an emergency response, conservationists have begun ex situ assurance colonies for priority species. The abundance of cryptic amphibian diversity, however, may cause problems for ex situ conservation. In this study we used a DNA barcoding approach to survey mitochondrial DNA (mtDNA) variation in captive populations of 10 species of Neotropical amphibians maintained in an ex situ assurance programme at El Valle Amphibian Conservation Center (EVACC) in the Republic of Panama. We combined these mtDNA sequences with genetic data from presumably conspecific wild populations sampled from across Panama, and applied genetic distance‐based and character‐based analyses to identify cryptic lineages. We found that three of ten species harboured substantial cryptic genetic diversity within EVACC, and an additional three species harboured cryptic diversity among wild populations, but not in captivity. Ex situ conservation efforts focused on amphibians are therefore vulnerable to an incomplete taxonomy leading to misidentification among cryptic species. DNA barcoding may therefore provide a simple, standardized protocol to identify cryptic diversity readily applicable to any amphibian community.

Panamanian frog species host unique skin bacterial communities

Vertebrates, including amphibians, host diverse symbiotic microbes that contribute to host disease resistance. Globally, and especially in montane tropical systems, many amphibian species are threatened by a chytrid fungus, Batrachochytrium dendrobatidis (Bd), that causes a lethal skin disease. Bd therefore may be a strong selective agent on the diversity and function of the microbial communities inhabiting amphibian skin. In Panamá, amphibian population declines and the spread of Bd have been tracked. In 2012, we completed a field survey in Panamá to examine frog skin microbiota in the context of Bd infection. We focused on three frog species and collected two skin swabs per frog from a total of 136 frogs across four sites that varied from west to east in the time since Bd arrival. One swab was used to assess bacterial community structure using 16S rRNA amplicon sequencing and to determine Bd infection status, and one was used to assess metabolite diversity, as the bacterial production of anti-fungal metabolites is an important disease resistance function. The skin microbiota of the three Panamanian frog species differed in OTU (operational taxonomic unit, ~bacterial species) community composition and metabolite profiles, although the pattern was less strong for the metabolites. Comparisons between frog skin bacterial communities from Panamá and the US suggest broad similarities at the phylum level, but key differences at lower taxonomic levels. In our field survey in Panamá, across all four sites, only 35 individuals (~26%) were Bd infected. There was no clustering of OTUs or metabolite profiles based on Bd infection status and no clear pattern of west-east changes in OTUs or metabolite profiles across the four sites. Overall, our field survey data suggest that different bacterial communities might be producing broadly similar sets of metabolites across frog hosts and sites. Community structure and function may not be as tightly coupled in these skin symbiont microbial systems as it is in many macro-systems.