Diversity patterns of amphibians in lowland Amazonian forests in southeastern Peru

Biological diversity is threatened worldwide and it is a priority to generate more information that can be used both for understanding ecological processes and determining conservation strategies. For my dissertation, I focused on amphibian diversity patterns in lowland rainforests of southwestern Amazonia to evaluate the importance of habitat heterogeneity in the region. My main purpose was to test the hypothesis that amphibian communities in different forest types differ in species richness, composition, and abundance. I used standardized visual encounter surveys to quantify the species composition and abundance of amphibians at four sites, each containing four forest types (floodplain, terra firme, bamboo, and palm swamp). I used leaf-litter plots to evaluate the effect of soil and leaf-litter characteristics on species richness and abundance of leaf-litter frogs. I intensively sampled at one site and then sampled three other sites (distance among sites varied 3.5–105 km) to evaluate whether the patterns observed at one site were similar elsewhere. I also updated the information on threatened and potentially threatened amphibians in Peru and my study region. I found that no species appears to have experienced population declines in southeastern Peru, suggesting that the region still contains the original species pool. My results support the hypothesis that amphibian communities differ across forest types and that patterns observed at the local scale (one site) are similar at the regional scale (four sites). My data also indicate that there is no correlation between species composition and geographic distance among sites. Instead, an important proportion of the gamma diversity is represented by habitat-related beta diversity. My leaf-litter plot data showed that part of the variation in the leaf-litter community structure is explained by soil and litter characteristics. I found that soil total phosphorus and, to a lesser extent, humidity, leaf-litter mass, and pH is linked to species presence/absence and abundance. My study provides the first standardized, quantitative comparison of amphibian community structure across four major forest types in southwestern Amazonia and highlights the fact that forest types are complementary and necessary for maintaining high species richness in the region.

Enigmatic faunal declines at La Selva, Costa Rica: Patterns and processes in a collapsing neotropical herpetofauna

Amphibian populations are declining even in pristine areas in many parts of the world, and in the Neotropics most such enigmatic amphibian declines have occurred in mid- to high-elevation sites. However, amphibian populations have also declined at La Selva Biological Station in the lowlands of Costa Rica, and similar declines in populations of lizards have occurred at the site as well. To set the stage for describing amphibian declines at La Selva, I thoroughly review knowledge of amphibian decline and amphibian conservation in Central America: I describe general patterns in biodiversity, evaluate major patterns in and ecological correlates of threat status, review trends in basic and applied conservation literature, and recommend directions for future research. I then synthesize data on population densities of amphibians, as well as ecologically similar reptiles, over a 35-year periods using quantitative datasets from a range of studies. This synthesis identifies assemblage-wide declines of approximately 75% for both amphibians and reptiles between 1970 and 2005. Because these declines defy patterns most commonly reported in the Neotropics, it is difficult to assess causality evoking known processes associated with enigmatic decline events. I conduct a 12-month pathogen surveillance program to evaluate infection of frogs by the amphibian chytrid fungus, an emerging pathogen linked to decline events worldwide Although lowland forests are generally believed to be too warm for presence or adverse population effects of chytridiomycosis, I present evidence for seasonal patterns in infection prevalence with highest prevalence in the coolest parts of the year. Finally, I conducted a 16-month field experiment to explore the role of changes to dynamics of leaf litter, a critical resource for both frogs and lizards. Population responses by frogs and lizards indicate that litter regulates population densities of frogs and lizards, particularly those species with the highest decline rate. My work illustrates that sites that are assumed to be pristine are likely impacted by a variety of novel stressors, and that even fauna within protected areas may be suffering unexpected declines.

Treefrogs in Forested Swamps at the La Selva Biological Station: Assemblage Variation through Space and Time

Swamp-breeding treefrogs form conspicuous components of many tropical forest sites, yet remain largely understudied. The La Selva Biological Station, a rainforest reserve in Costa Rica, harbors a rich swamp-breeding treefrog fauna that has been studied in only one of the many swamps found at the site. To understand if the species composition of treefrogs at La Selva varies over space or time, frogs were censused in 1982-83, 1994-95, 2005 and 2011 at two ponds located in the reserve. Data on treefrog habitat utilization were also collected. Species composition varied spatially only in 2011. Temporal variation was observed at both ponds for all groups tested. Habitat use varied among species and between swamps. The pattern of variation suggests that temporally dynamic systems such as temporary Neotropical forest swamps will converge and diverge in species composition over time.

Ontogenetic Scaling of Poison Glands in Dendrobates Pumilio

The relationship between granular (poison) gland size and density was examined in an ontogenetic series of the strawberry dart-poison frog, Dendrobates pumilio. Specimens used in this study were collected from the La Selva Biological Station in northeastern Costa Rica. Patches of skin from the dorsal surface of seven frogs, ranging in size from 11 to 23 mm snout-vent length (SVL), were fixed and embedded in paraffin for histological sectioning. Poison gland size and density were quantified microscopically in these sections. Poison glands are uniformly distributed across the skin and mean poison gland diameter increases at a rate faster than snout-vent length from 42.5 at SVL 11mm to 120.0 at SVL 23 mm. Conversely, gland density decreases with body size from 71.9 glands/mm2 to 33.2 glands/mm2 • Due to the positive allometric growth of the poison glands, the percentage of skin surface occupied by poison glands increases from 10.1-22.1% in small frogs (SVL<18 >mm) to 50.0-65.2% in large frogs (SVL>19MM), resulting in more toxin per mm2 in the larger animals. The largest increase in toxicity is correlated temporally with the onset of sexual maturity rather than with changes in aposematic coloring.

Diversity Patterns of Amphibians in Lowland Amazonian Forests in Southeastern Peru

Biological diversity is threatened worldwide and it is a priority to generate more information that can be used both for understanding ecological processes and determining conservation strategies. For my dissertation, I focused on amphibian diversity patterns in lowland rainforests of southwestern Amazonia to evaluate the importance of habitat heterogeneity in the region. My main purpose was to test the hypothesis that amphibian communities in different forest types differ in species richness, composition, and abundance. I used standardized visual encounter surveys to quantify the species composition and abundance of amphibians at four sites, each containing four forest types (floodplain, terra firme, bamboo, and palm swamp). I used leaf-litter plots to evaluate the effect of soil and leaf-litter characteristics on species richness and abundance of leaf-litter frogs. I intensively sampled at one site and then sampled three other sites (distance among sites varied 3.5-105 km) to evaluate whether the patterns observed at one site were similar elsewhere. I also updated the information on threatened and potentially threatened amphibians in Peru and my study region. I found that no species appears to have experienced population declines in southeastern Peru, suggesting that the region still contains the original species pool. My results support the hypothesis that amphibian communities differ across forest types and that patterns observed at the local scale (one site) are similar at the regional scale (four sites). My data also indicate that there is no correlation between species composition and geographic distance among sites. Instead, an important proportion of the gamma diversity is represented by habitat-related beta diversity. My leaf-litter plot data showed that part of the variation in the leaf-litter community structure is explained by soil and litter characteristics. I found that soil total phosphorus and, to a lesser extent, humidity, leaf-litter mass, and pH is linked to species presence/absence and abundance. My study provides the first standardized, quantitative comparison of amphibian community structure across four major forest types in southwestern Amazonia and highlights the fact that forest types are complementary and necessary for maintaining high species richness in the region.

Conservation ecology of amphibians and reptiles in Sarapiqui, Costa Rica : forest fragmentation and long term population change

In order to explore the conservation ecology of frogs and lizards in the Sarapiqui region of Costa Rica, I compared populations and communities among forest fragments and La Selva Biological Station, as well as across 35 years of sampling at La Selva. Species richness in nine fragments combined was 85% of that found in La Selva, and community composition varied among sites and by fragment size class. Although communities in fragments differed fundamentally from those in intact forest, the high diversity observed across all fragments indicates that preserving a network of small forest patches may be of great conservation value to the herpetofauna of this region. According to data from past studies at La Selva, most common species of leaf-litter frogs and lizards demonstrated significant decreases in density over the 35-year period. My findings may represent either natural population fluctuations or sweeping faunal declines at this site.

Potential impact of invasive anuran species in western Newfoundland

Successful dispersal and establishment of invasive anurans (frogs and toads) may be influenced by competitive exclusion and/or niche differentiation with competing species. I investigated the dispersal of anurans in western Newfoundland using anuran calling surveys and pond-edge visual encounter surveys. The Mink Frog, Lithobates septentrionalis, had dispersed ~50 km northeast from the original (2001) discovery location and ~34 km southwest; displaying spatial separation from Green Frogs, Lithobates clamitans, at landscape and local scales. Visual encounter surveys did not reveal any correlation between adult Mink Frogs and odonate competitors. Additionally, I assessed the impact of varying tadpole densities on removal of epilithic periphyton by providing epilithon covered substrates for American Toad, Anaxyrus americanus, tadpoles raised in laboratory or field enclosures. Higher tadpole densities resulted in smaller tadpoles that removed more periphyton from substrates. As anuran population ranges expand, there may be effects on ecological resources for vertebrate and invertebrate competitors.

Peptidomic approach identifies cruzioseptins, a new family of potent antimicrobial peptides in the splendid leaf frog, Cruziohyla calcarifer

Phyllomedusine frogs are an extraordinary source of biologically active peptides. At least 8 families of antimicrobial peptides have been reported in this frog clade, the dermaseptins being the most diverse. By a peptidomic approach, integrating molecular cloning, Edman degradation sequencing and tandem mass spectrometry, a new family of antimicrobial peptides has been identified in Cruziohyla calcarifer. These 15 novel antimicrobial peptides of 20–32 residues in length are named cruzioseptins. They are characterized by having a unique shared N-terminal sequence GFLD– and the sequence motifs –VALGAVSK– or –GKAAL(N/G/S) (V/A)V– in the middle of the peptide. Cruzioseptins have a broad spectrum of antimicrobial activity and low haemolytic effect. The most potent cruzioseptin was CZS-1 that had a MIC of 3.77 μM against the Gram positive bacterium, Staphylococcus aureus and the yeast Candida albicans. In contrast, CZS-1 was 3–fold less potent against the Gram negative bacterium, Escherichia coli (MIC 15.11 μM). CZS-1 reached 100% haemolysis at 120.87 μM. Skin secretions from unexplored species such as C. calcarifer continue to demonstrate the enormous molecular diversity hidden in the amphibian skin. Some of these novel peptides may provide lead structures for the development of a new class of antibiotics and antifungals of therapeutic use.

Identification of Miscellaneous Peptides from the Skin Secretion of the European Edible Frog, Pelophylax kl. Esculentus

The chemical compounds synthesised and secreted from the dermal glands of amphibian have diverse bioactivities that play key roles in the hosts' innate immune system and in causing diverse pharmacological effects in predators that may ingest the defensive skin secretions. As new biotechnological methods have developed, increasing numbers of novel peptides with novel activities have been discovered from this source of natural compounds. In this study, a number of defensive skin secretion peptide sequences were obtained from the European edible frog, P. kl. esculentus, using a 'shotgun' cloning technique developed previously within our laboratory. Some of these sequences have been previously reported but had either obtained from other species or were isolated using different methods. Two new skin peptides are described here for the first time. Esculentin-2c and Brevinin-2Tbe belong to the Esculentin-2 and Brevinin-2 families, respectively, and both are very similar to their respective analogues but with a few amino acid differences. Further, [Asn-3, Lys-6, Phe-13] 3-14-bombesin isolated previously from the skin of the marsh frog, Rana ridibunda, was identified here in the skin of P. kl. esculentus. Studies such as this can provide a rapid elucidation of peptide and corresponding DNA sequences from unstudied species of frogs and can rapidly provide a basis for related scientific studies such as those involved in systematic or the evolution of a large diverse gene family and usage by biomedical researchers as a source of potential novel drug leads or pharmacological agents.

Ex Vivo Smooth Muscle Pharmacological Effects of a Novel Bradykinin-Related Peptide, and Its Analogue, from Chinese Large Odorous Frog, Odorrana livida Skin Secretions

Bradykinin-related peptides (BRPs) are one of the most extensively studied frog secretions-derived peptide families identified from many amphibian species. The diverse primary structures of BRPs have been proven essential for providing valuable information in understanding basic mechanisms associated with drug modification. Here, we isolated, identified and characterized a dodeca-BRP (RAP-L1, T6-BK), with primary structure RAPLPPGFTPFR, from the skin secretions of Chinese large odorous frogs, Odorrana livida. This novel peptide exhibited a dose-dependent contractile property on rat bladder and rat ileum, and increased the contraction frequency on rat uterus ex vivo smooth muscle preparations; it also showed vasorelaxant activity on rat tail artery smooth muscle. In addition, the analogue RAP-L1, T6, L8-BK completely abolished these effects on selected rat smooth muscle tissues, whilst it showed inhibition effect on bradykinin-induced rat tail artery relaxation. By using canonical antagonist for bradykinin B1 or B2 type receptors, we found that RAP-L1, T6-BK -induced relaxation of the arterial smooth muscle was very likely to be modulated by B2 receptors. The analogue RAP-L1, T6, L8-BK further enhanced the bradykinin inhibitory activity only under the condition of co-administration with HOE140 on rat tail artery, suggesting a synergistic inhibition mechanism by which targeting B2 type receptors.

Community- and population-level effects of chytridiomycosis in a Neotropical amphibian community

As the number of fungal pathogen outbreaks become more frequent worldwide across taxa, so have the number of species extirpations and communities persisting with the pathogen. This phenomenon raises questions, such as: “what leads to host extinction during an outbreak?” and “how are hosts persisting once the pathogen establishes?.” But the data on host populations and communities across life stages before and after pathogen arrival rarely exist to answer these questions. Over the past three to four decades, the amphibian-killing fungus Batrachochytrim dendrobatidis (Bd) spread in a wave-like manner across Central America, leading to rapid species extirpations and population declines. I collected data on tadpole and adult amphibians in El Copé, Panama before, during, and after the Bd outbreak to answer these questions. I used Bayesian statistical approaches to account for imperfect host and pathogen detection of marked and unmarked individuals. In the tadpole community, within 11 months of Bds arrival, density and occupancy rapidly declined. Species losses were phylogenetically correlated, with glass frogs disappearing first, and tree frogs and poison-dart frogs remaining. I found that tadpole communities resembled one another more strongly after the outbreak than they did before Bd invasion. I found no tadpoles within 22 months of the outbreak and limited signs of recovery within 10 years. In contrast, at the same site, for a population of male glass frogs, Espadarana prosopleon, I found that 10 years post-outbreak, the population was consistently half its historic abundance, and that the lack of recruits to the population explained why the population had not rebounded, rather than high pathogen-induced mortality. And finally, examining the entire amphibian community, I found high pathogen prevalence, low infection intensities, and high survival rates of uninfected and infected hosts. Bd transmission risk, i.e., the probability a susceptible host becomes infected, did not relate to host density, pathogen prevalence, or infection intensity– Bd transmission risk was uniform across the study area. My results are especially relevant to conservation biologists aiming to predict the future impacts of Bd outbreaks, those trying to manage persisting populations, and those interested in reintroducing species back into wild amphibian communities.

Desempenho zootécnico de girinos de rã-touro com diferentes níveis de proteína e energia digestíveis

A partir dos valores de digestibilidade de alguns ingredientes foram propostas dietas, com diferentes níveis de proteína e energia digestíveis, em esquema fatorial 3 x 3, destinadas a girinos de rã-touro para avaliar o desempenho zootécnico. O experimento, com duração de 60 dias, foi conduzido com 810 girinos, oriundos da mesma desova, distribuídos em 27 caixas de polipropileno, em uma densidade de 1girino/L. O delineamento experimental foi um esquema fatorial com três níveis de proteína digestível (27; 31 e 35%) e três níveis de energia digestível (2.700; 2.800 e 2.900kcal/kg) com três réplicas. Os parâmetros avaliados aos 60 dias foram ganho de peso, consumo da dieta, consumo em proteína da dieta, conversão alimentar, taxa de eficiência proteica e índice de sobrevivência. Os resultados foram submetidos à análise de variância e ao teste de Duncan (95% de precisão). Os resultados encontrados não apresentaram interação entre proteína e energia digestível para nenhum parâmetro avaliado. em relação à proteína digestível, foram verificadas diferenças nos parâmetros consumo em proteína da dieta e taxa de eficiência proteica (TEP), o que demonstra ineficiência das dietas com altos valores proteicos. Deve-se oferecer aos girinos de rã-touro dietas com 27% de proteína digestível, por apresentarem melhor resultado para taxa de eficiência proteica e para consumo em proteína da dieta.

The taxonomy, zoogeography and ecology of amphibians and reptiles of Hin Nam No National Protected Area (Laos) in comparison with data from Phong Nha – Ke Bang National Park (Vietnam).

In this thesis the mostly unknown herpetofauna in Hin Nam No National Protected Area Laos in the northern Truong Son Range was for the first time intensively investigated, and its diversity was compared to the bordering, and well-investigated Phong Nha - Ke Bang National Park in Vietnam. Twelve new vertebrate species were described comprising 11 geckonids (Cyrtodactylus bansocensis, C. calamei, C. hinnamnoensis, C. jaegeri, C. rufford, C. sommerladi, C. soudthichaki, Gekko boehmei, G. bonkowskii, G. sengchanthavongi, G. thakhekensis, Lycodon banksi and one colubrid snake (Lycodon banksi). Seven species were discovered for the first time in Laos including three frogs (Gracixalus quyeti, G. supercornutus, Rhacophorus maximus), two geckos (Cyrtodactylus cryptus, C. pseudoquadrivirgatus) and two snakes (Lycodon futsingensis, L. ruhstrati abditus). The main hypothesis that the Truong Son Range acted as a biogeographic barrier for the distribution of amphibians and reptiles could be confirmed at least for karst adapted gekkonids. Compared to other herpetofaunal groups the number of gekkonids in karst formations was particularly high (seven bent-toed geckos, four true geckos). By comparing the relative amounts of shared species in Hin Nam No and Phong Nha - Ke Bang, it is interesting to note that fewer reptile species (38%) than amphibian species (66%) were shared between both regions. This might indicate that the Truong Son Range acts as a stronger biogeographical barrier for reptiles than for amphibians. Two pairs of karst-adapted cryptic gecko species (i.e. species with distinct genetic differences, but a similar phenotype) occurred on both sides of the Truong Son Range. Only in one case these were sibling species (Crytodactylus sommerladi in Laos versus C. roesleri in Vietnam), but not in the other (C. hinnamnoensis in Laos versus C. phongnhakebangensis in Vietnam). On the Laotian side, nine gecko species (Cyrtodactylus bansocensis, C. calamei, C. darevskii, C. hinnamnoensis, C. khammouanensis, C. multiporus, C. sommerladi, G. boehmei, G. sengchanthavongi) currently have to be regarded as endemic to the Hin Nam No region. On the Vietnamese side, seven species including two bent-toed geckos (Cyrtodactylus phongnhakebangensis and C. roesleri), three skinks (Lygosoma boehmei, Sphenomorphus tetradactylus and Tropidophorus noggei), and two snakes (Hebius andreae and Boiga bourreti) are currently only known from Phong Nha - Ke Bang and adjacent regions. These high numbers of potential endemic species together with the cryptic species complex in Cyrtodactylus provide strong evidence that the karst formations in the northern Truong Son Range represent a hot spot of reptile diversity and of speciation in Crytodactylus in particular. Correct species identification is a fundamental requirement for conservation measures. The discovery of cryptic species complexes poses a challenge for alpha taxonomy and species conservation, because the true distribution ranges of the species are in fact much smaller than previously assumed. Species conservation in this area of Laos is facing a number of further problems. New and potentially endemic species were discovered in highly populated and disturbed areas. Conversion of the Ho Chi Minh Trail into a highway provided easy access for farmers and still continues to accelerate the destruction of remote forest areas. Southern Hin Nam No with its high diversity of endemic species was identified as the first priority area for conservation. Also Ban Soc, an area isolated from Hin Nam No, should be among the conservation priorities because this region houses a so far overlooked population of the critically endangered Siamese crocodile. Efforts to establish a legal conservation status for this habitat are in progress.