1. Introduction 1.1 Long term monitoring Modern amphibian declines and extinctions are incomparable to that of any other class over the last few millennia (Stuart et al 2004). The global loss of amphibians demonstrates how the world is changing, and how conservation practices must also adapt. Detailed information on amphibian populations in decline is often not available (Gascon et al 2005). Therefore it is crucial that long term monitoring of populations and areas with high species richness, such as the Amazon rainforest, are monitored for the effects of decline. Long term monitoring of populations is an effective technique in understanding the extent and cause of the widespread decline of amphibian populations (Caughley and Gunn1996). The Lago Preto Concession Area is situated on the Peruvian side of the Yavari River in the region of Iquitos. It has a tremendous diversity of frogs and there is opportunity for ongoing research. Therefore, it is an ideal location to monitor the effects of amphibian decline due to climate change, chytridiomycosis and other impacts. This study examines anuran abundance and diversity at the Lago Preto Concession Area and investigates ecological trends across different habitat types. Data collected are compared with that of previous studies to get a more accurate picture of species abundance and this baseline data can be used to help a long-term monitoring project in the area. 1 1.2 Conservation status of Amphibians Amphibians, which comprise frogs, salamanders and caecilians, all descend from an ancient ancestry of organisms and form integral roles within the world’s ecosystems. Amphibians are both predators and prey; they help control populations of insects that transmit diseases and feed on crops, and the tadpoles of many frogs play a major role in aquatic ecosystems (Mendelson, 2006). The most successful, widespread and numerous of amphibians are the anurans, but they are more commonly known as frogs and toads. There are more than 5,500 species of anurans that live in various habitats from mountain slopes to deserts and can be found on every continent excluding the polar regions (Attenborough, 2008). The vast majority of species occurs in the Amazon rainforest and the Montane forests of South America. At a single site in the upper Basin of the Ecuadorian and Peruvian Amazon 81 species of frog have been found, which equals the total number of species in the whole of North America (Duelman 1992). The Amazon Basin is the most diverse and species rich place on the planet for frog fauna. Over the past few decades scientists have observed a significant number of population declines and extinctions, which suggests a general Global Amphibian Decline (GAD) is taking place (Young 2001, Mendelson 2006 ). At present almost one-third of the worlds 5,743 described amphibian species are threatened with extinction, at least 122 species are believed to have already gone extinct since 1980, and 130 species have not been found in recent years and are presumed extinct. The scope of these declines and extinctions is far greater than mammals and 2 birds over the last few millennia. Declines have spread geographically and the numbers of species involved is still increasing. (Mendelson, 2006). 2. Reasons for decline. 2.1 Habitat loss, alteration and fragmentation. At present the greatest danger to amphibians is habitat loss, which is impacting 90% of threatened species (Beebee, 1996, Mendelson, 2006). Frogs need a variety of habitat types throughout different stages in there life process. Many amphibians are habitat specific. Pearman (1997) observed that species richness and diversity can vary in response to precipitation levels, elevation, forest type and structure. As a consequence, if one part of the ecosystem is altered it can seriously upset the behaviour and survival of a population, due to specific habitat requirements needed for reproduction and population growth. Amphibian diversity can be significantly altered in response to habitat alteration, as many species are restricted to different habitat types due to complex life cycle, terrestrial and semi aquatic breeding regimes, the predation of eggs and the necessity of keeping the skin moist. Furthermore, many amphibians have specialised feeding behaviour; therefore they are susceptible to changes in the abundance or composition of food sources, potentially leading to geographical isolation or local extinction (Pearman, 1997). During the 1990s, South America lost 4% of its total forest cover, due to the increasing population and the demand for land for agriculture and housing. However, this figure does not include the destruction of mature forests that are being replaced by secondary growth as loggers 3 selectively remove trees. (Young et al., 2004). Habitat fragmentation caused from habitat destruction and alteration results in population isolation on small pockets of suitable habitat surrounded by larger areas of unsuitable habitat. These small pockets are also known as ‘islands,’ and are often too small to sustain effective breeding populations (Kricher, 1997). Over time, populations begin to lose genetic diversity, lacking the capability of responding to environmental changes, such as climate change, and increasing the likelihood of extinction (Frankham, 2005). Unfortunately, habitat destruction is continuing at a fast pace. Estimates predict that South America will reach 768 million inhabitants by 2050, resulting in a higher demand for resources and intensifying the threat to amphibians in Latin America (Young et al, 2004). 2.2 Climate change Habitat destruction, alteration and fragmentation are thought to be the main reasons for the current high rates of extinction within amphibians (Beebee, 1996, Mendelson, 2006). However, a large proportion of species that are extinct or critically endangered are species that have declined in seemingly undisturbed environments (Pound, 2006). The causes for decline have remained unclear, partly because of the complexity of amphibian life cycles and biology (Alford and Richards, 1999). Although disease is thought to be a major cause, its relationship to environmental change is not well documented (Berger, et al. 1998; Blaustein and Kiesecker, 2002). At present, evidence from several scientist ( Kiesecker, Blaustein, 2002; Pound, 2007) suggests that climate change may be one of the causes. Pound et al (2007) found evidence that suggests 4 increased levels of UV-B exposure caused by climate change are increasing the probability of frog embryos becoming infected by the fungus, Saprolegnia ferax. Reductions in water depth due to altered precipitation patterns caused by climate changes expose embryos to damaging UV-B radiation, increasing the chance of a lethal infection (Pound et al, 2007). Over the past three decades, the averaged air and sea-surface temperatures of earth have increased dramatically, due to anthropogenic factors (Parmesan and Yohe, 2003). The tropical Pacific and Atlantic Ocean are vital in controlling climate and levels of precipitation over Central and South American Tropics, where the vast majority of amphibians exist. Therefore natural populations could be vulnerable where local climate is heavily influenced by the tropical Pacific and Atlantic Ocean. In addition, intense warm episodes caused by El Niño have created extreme climatic effects that have detrimentally affected biological communities, such as those which live in the Amazon rainforest (Root et al, 2005). Rising temperatures have also begun to affect changes in amphibian behaviour. Increases in temperature are thought to be causing frogs to call and breed earlier in the year (Pound et al, 1999; Gibbs, 2001). Though no detrimental effects have been recorded due to changes in breeding season, it is important that long term research is implemented and more knowledge is gained. 2.3 Chytridiomycosis During the 1990s, significant worldwide declines in amphibian populations became noticeable with significant losses seen in Costa Rica, Australia’s east coast and western North America (Mazzoni et al; 2003; Welldon et al; 2004). In 1999 the cause of this decline was identified as a 5 new species Batrachochytrium dendrobatidis (Bd), which infects amphibians and causes the potentially fatal disease chytridiomycosis. The fungus excludes a lethal toxin or suffocates the frogs by clogging skin pores (Halliday 2008). Since the findings, Bd has been linked with amphibian population declines on every continent inhabited by amphibians, and is now seen as one of the biggest threats to amphibian extinction worldwide (Weldon et al; 2004). Museum specimen from the 1930s identify the disease as originating from South Africa. It is thought the spread of the disease began with the commercial trade of the clawed frogs (Xenopus laevis) (Berger et al, 1999). When Bd is introduced to a new site, it begins to spread through water courses and amphibian-to-amphibian contact at a rapid rate. A widespread study across Central America on the spread of Bd calculated the rate of progression at 28-100km/yr (Amphibian Ark, 2008). In moist, cool habitats were Bd thrives it is expected that 50% of amphibian species and 80% of individuals can be expected to become extinct within a year (Lips, 2006). At present there is no cure for wild species, although a small number of species seem capable of surviving with a Bd infection. These infected species are expected to serve as reservoirs and carriers for future outbreaks. Furthermore, many of the species resistant to Bd are recognised as invasive pest species for example the marine toads, American bullfrogs and African clawed frogs (Amphibian Ark, 2008). Amphibians in captivity are also under threat from Bd and deaths in both private and zoo collections have been reported during 2006 and 2007 in Australia, Europe, USA and Japan (Berger 1998). At present there are several different treatments that have been used with varying 6 degrees of success, including anti-fungal drugs such as itraconazole. However, New Zealand scientists have found what they believe to be a cure for the disease. A commonly used eye ointment, Chloramphenicol, is thought to cure frogs when they are bathed in the solution, although because of known side-effects in humans it is unlikely that it can be used in the wild (Griggs, 2007). Therefore, if such findings are correct, zoos and private collectors will have more options when trying to control an outbreak or rescue frogs from the wild. The bigger challenge for researchers must be to find a cure that will work in the wild. At present, chytridiomycosis has only been observed in a relatively low number of cases in Peru. However, our understanding of the extent of the declines in Latin America, due to chytridiomycosis is limited to a few well-documented studies, so further research is needed (Young 2001). 2.4 Trade in amphibians The harvesting of wild amphibians represents a significant threat to the survival of many species. The global trade in amphibians for food, biomedicine, and the pet trade is thought to be affecting around 36 threatened species, representing 3% of all amphibians identified (IUCN, 2006; Young et al; 2004). A large majority of amphibians in trade are distributed for frog meat, a practice that is common in Europe, Canada and the United States. Initial reports in the 1990s identified that Europe imported approximately 6,000 tonnes of frog legs, and America imported 10,000 tonnes of frog 7 meat (IUCN, 2006; Jensen et al; 2003). The vast majority of frog meat is harvested from the wild, with the rest coming from farmed populations. The use of amphibians for medicinal purpose is still being developing, so only represents a small proportion of amphibians in trade. Asia and Chinese are the main consumers of amphibians for medicinal use, where it is common for medicinal shops to sell organs, such as dried ovaries and hallucinogenic skin (Schlaepfer, 2005). Research for medicinal use amongst frog species is on the increase; as new breakthroughs in science have discovered that some species secrete peptides that can provide vital biomedicines, including treatments for cancer and HIV. The collection of peptides can cause substantial stress to individuals and could have harmful effects to wild populations (IUCN, 2006). Most amphibians in the pet trade are captive bred; however some species are not and are still collected from the wild. At present, Peru exports 26 different frog species, all of which are on Appendix 2 of CITES and are classified as Least Concern by IUCN (IUCN, 2006). All of the species exported from Peru are within the genus Dendrobatidae and if not managed correctly could pose a threat to members of this group in the near future (IUCN, 2006; Jensen et al, 2003). 2.5 Pollution and amphibians as indictors Amphibians are very susceptible to pollutants within their environment (Mandelson 2006). Amphibians have porous skin, allowing both air and water to pass through it, along with other toxins. Contaminants within air and water are thought to have a strong impact on the many amphibian populations by damaging immune systems and thus causing infertility, and 8 malformations. Contaminants can lead to unhealthy adults entering breeding population, resulting in a more vulnerable population. It can then proceed to these environmental contaminants causing local population declines and extinction. Amphibians are more vulnerable than other species to environmental change because they live out their life in two different habitats, on land and water. In addition, amphibians prey on invertebrates that are both terrestrial and aquatic, therefore are vulnerable to a bioaccumulation of toxins, which can cause death and reproductive abnormalities. In view of this amphibians are often referred to as 'environmental indicators', which is another way of saying they are like 'canaries in the coal mine' (Halliday 2000). Amphibians might be warning us of unsafe environmental conditions that could eventually seriously impact our health. There is still little research on the effects of pesticide in amphibians, but early studies indicate that use of chemicals in farming can negatively affect nearby amphibians which could explain population declines in areas that have not experienced alteration in habitat (Young et al, 2004). 3. Amphibians Contribution to humanity The skin of amphibians is more permeable than ours and allows liquids to pass through it fairly easily; as a consequence they are susceptible to subtle changes in their environment (Mandelson 2006). Although this permeability allows them to live away from water bodies and exploit different environmental niches, it also means they are at risk of absorbing toxins and chemicals directly into their body (Attenborough, 2008). To combat against viruses and microbes that may enter through their permeable membrane, they have developed some unique biological characteristics to protect themselves. Frogs and other 9 amphibians secrete chemicals onto their skin. This complex mixture of bioactive peptides provides a first line of defence against bacteria, fungi and viruses (Bradbury 2005). These peptides have profoundly enhanced the lives of humans in numerous ways. They provide vital biomedicines, such as compounds that are being refined for antibiotics, stimulants for heart attack victims, and treatments for diseases including depression, stroke, seizures, Alzheimer’s, and cancer. Furthermore, new research indicates that the Australian red-eyed treefrog (Litoria chloris) gives us a compound capable of preventing HIV infection (Amphibian ark, 2008). Although much of this research is in the early steps, it signifies the necessity of protecting species not only for their importance to ecosystems and their intrinsic value, but for the benefit of human life. 4. BIOLOGY 4.1 Biological features Frogs and toads are classed as amphibians in the order of Anurans. They differ physically from other amphibians, having a much shorter backbones and at most only nine vertebrae. No adults have a true tail, revealed by the genus Anura meaning ‘without tail’ (Cogger et al, 1998). The leg and arm bones of anurans are also different to that of other amphibians as both radius and ulna, and the tibia and fibula, are fused together. Also, the anklebones of the hind legs are significantly elongated compared to the front feet allowing them to jump, which is their primary form of locomotion (Cogger et al, 1998). Toes are usually linked by a membrane, helping them to drive forward when swimming in water. Teeth are absent in many species, reflecting the adaptation to insectivorous feeding of most anurans. Feeding is also aided by a proportionally large and unconnected tongue, which has great movement outside the mouth. Like all 10 amphibians, the skin of frogs is permeable to water and must be kept wet, thus most species live in moist habitats were they can replenish their water supply (Rodriguez, 1994). Most species only vary slightly between sexes. The identification is usually based upon the presence of a vocal sack amongst males. Unlike other terrestrial species, anurans do not use scent marks to confirm territories and signal for mates. The majority use sound produced when air is pushed from the lungs over vocal cords, through the mouth into a vocal sack that swells and vibrates (Rodriguez, 1994). Each species of frog has a different call that can only be recognised by the other members of its species. Many species also have ear membranes that can only resonate within their own vocal range therefore can not hear sounds that are equally loud to humans (Attenborough, 2008). The evolution of frogs from water to land has developed a unique way of detecting sound on land. Frogs detect sound using a circular patch of skin called the tympanum, which is held taut over a surrounding ring of cartilage just above the jaw, below the eye. Each is connected to a capsule in the skull by a bone allowing the frogs to hear in air (Attenborough, 2008). 4.2 Reproduction Most North American and European anuran reproduction follows a simple, annual three-step process; first clumps or long strips of eggs are laid in water and then fertilised by the male which grasps the female; secondly the eggs hatch into tadpoles which feed on algae. They then grow legs and metamorphose into froglets; and finally the froglets move onto land where they eat insects and grow into adults which return to water the following year to repeat the process. However, there are many other species, mostly in the tropics, that follow different reproduction processes. In Peru and other South and Central American countries many species breed 11 throughout the year and the females can produce several clutches per year (Rodriguez, 1994). Many Hylidae species mate and live in the trees and place their eggs on plants over ponds and slow moving streams. Once the eggs have hatched they drop into the water. Some eleuthodactylid species lay their eggs on land without having an aquatic stage, whilst some dendrobatydae species develop youngsters within a pouch on their body until they have become froglets (Attenborough, 2008). Regardless of our increasing knowledge about frog species and behaviour, there is still little information that exists on many species, especially those of South America (Rodriguez, 1994). 5. Methods and Material 5.1 Study site In this study I examine the diversity and abundance of anurans between three different habitat types, varzea, terra-firme, and Agujal in a remote area of the Peruvian Amazon. The study was conducted between the 25th May and the 11th June at the Lago Preto Concession Area (4°28’S, 71°46’W, ~90 m elev.). The Lago Preto Concession Area is part of the larger proposed Yavari Reserved Zone, an area of over 1 million hectares that stretches from the Peru-Brazil border in the east, to the Reserva Comunal Tamshiyacu-Tahuaya in the west. The Lago Preto Concession Area is situated near to the mouth of the Yavari Miri River, on the Peruvian side of the Yavari River (Pitman et al, 2003). The climate is seasonal and typical of a humid lowland tropical forest. There is a high annual precipitation averaging between 2000mm to 3000mm. December to March are often the wettest 12 months of the year with June, July and August being the driest. The annual mean temperature is constant throughout the year at 24-26° (Bowler, 2006). However, during the first week of study we experienced a climatic phenomenon, when winds coming north from the Antarctic produced temperatures as low as 10° (Bodmer per communication, 2007). The area between Ucayali and the Yavari River holds the most diverse tree communities in the World (Vasquez-Martinez and Philips, 2000). The area is unique as it consists of three main habitat types - varzea, terra firme and agujal, which are in close proximity to each other (Bowler, 2006). Varzea forests are positioned on the fringes of the main rivers of the Amazon basin and total around 300,000 km² (Ayres et al, 1999, Kvist and Nebal, 2001). Varzea forests are seasonally inundated with white water and large quantities of sediment rich deposits, originating from the Andes (Pires and Prince 1985; in Bowler 2006). The sediment rich deposits result in fertile soils and are of considerable biological significance (Ayres et al, 1999). The undergrowth is modest due to the frequent flooding and the vegetation that exists is adapted to survive partially submerged (Whitmore, 1999). The annual flooding has also resulted in a high degree of endemism amongst fauna and flora (Ayres et al, 1999). Terra firme forests are those that are elevated and not seasonally flooded. They are positioned off the floodplain and make up about 96% of the total Amazon forest (Richer, 1994), and are the most extensive forests in the Yavari Valley (Kvist and Nebal, 2001). As terra firme forests are not flooded, the soil quality is low and lacks the nutrition of varzea and aguajal forests. The 13 foliage ground cover is fairly dense and the average canopy height is between 25-30 m (Kricher, 1994). Aguajal, or palm swamps, are permanently water logged due to the clay soils which retain water. The forest assemblage is very dense with a canopy height of about 20 m and consists of thick undergrowth. The Yavari Agujal is dominated by the palm fruit Mauritia flexuosa which makes up about 25-60% of the floodplain (Pitman et al 2003). Aguajal in the Yavari is common on the flood plains but also occurs in high areas of varzea forest that do not flood profoundly, but do not dry out during periods of low precipitation (Bowler, 2006). 5.2 Data collection Six transects of 500m were surveyed in three habitat types - varzea, terra firme and aguajal. Transects were conducted night and day in varzea and terra firme, though this could not be achieved in agujal due to difficult conditions. To compensate for this, each transect in aguajal was conducted twice during the day. Transects were conducted during the period when amphibians are most active (Rodríguez and Duellman 1994). Day transects were carried out between approximately 7:00am-2pm and night transects took place between approximately 7:15pm-1am. Two - three people conducted surveys during the day and night including a guide to direct through the forest and assist in amphibian detection. To maximise anuran detection the length of time spent on each 500m transect was relatively long and took between 1h 30min-4h. Visual 14 encounter surveys (VES) during the day were carried out using a probe to disturb leaf litter and vegetation (Donnelly et al 2004). During the VES all possible microhabitats were searched, including leaf litter, tree trunks, decayed logs, fallen palm leaves and bromeliads. Due to the cryptic nature of anurans the disturbance of this vegetation using a probe was the most systematic method of detection (Donnelly et al 2004). This was achieved by methodically probing through the area directly in front of the observers, including up to approximately 3m on either side of the trail. On occasion, some amphibians were observed beyond 3m, however as the study aims to assess diversity, these specimens were included. To identify anurans during night transects instead of probing through leaf litter, torches were used to catch the reflection of light from the eyes of anurans. Before each survey the following information was recorded: date, name of observer, place, area searched, weather conditions, start time and finish, and habitat description. When individuals were encountered information was recorded about species type and the time of capture. Upon detection and capture of an individual each specimen was handled carefully and morphological characteristics, such as webbing between fingers, iris and pupil colour, presence of tympanium, were examined and photographed. This was followed by the recording of morphometric measurements; snout to vent length (SVL) measured with a calliper to the nearest 0.1mm, sex (if possible), stage (juvenile, adult), distance along and from the transect, vegetation type and microhabitat use, time recorded and species when possible. When identification of an individual was not possible in the field, photographs and recorded observation could be used and compared to the identification guide produced by Rodriguez et al. 15 (1994). To distinguish between species the following characteristics were observed: body size and shape; iris colour and shape; number and length of toes and phalanges; presence and patterning of dorsolateral, cranial, belly, ventricular, leg and feet markings and presence of webbing. 5.3 Statistical analysis I analysed data using Microsoft Excel and performed statsistical tests using SPSS 15.0 for W indows. Analysis was based upon the use of the arithmetic mean, standard deviation of samples, species richness, abundance across habitat type. Tests used were Shannon’s diversity index, and analysis of variance (ANOVA). 16 6. Results 6.1 Inventory of anurans Class Order Family Scientific Name Amphibia Anura Bufonidae Bufo marinus Bufo typhonius Dendrophryniscus minutes Colostethus marchesianus Colostethus melanolaemus Colostethus trilineatus Dendrobates sp Dendrobates uakari Epipedobates femoralis Epipedobates trivittatus Allophyryne sp Hyla boans Hyla fasciata Hyla geographica Hyla lanciformis Hlya parviceps Hyla rhodopepla Osteocephalus leprieurii Osteocephalus planiceps Osteocephalus taurinus Scarthyla ostinodactyla Scinax garbei Scinax rubra Scinax cruentomma Andenomera sp. 1 Andenomera sp. 2 Andenomera hylaedactla Eleutherodactylus altamazonicus Eleutherodactylus buccinator Eleutherodactylus croceoinguinis Eleutherodactylus delius Eleutherodactylus diadematus Eleutherodactyus malkini Eleutherodactylus martiae Eleutherodactyus ockendeini Eleutherodactylus peruvianus Eleutherodatylus sp Eleutherodactylus sulcatus Eleutherodactylus variabilis Eeutherodactylus Visarsi Hemiphractus scutatus Ischnocnema quixensis Leptodactylus diodrs Leptodactylus pentadactylus Leptodactylus rhodanutus Leptodactylus rhodomystax Leptodactylus wagneri Vanzolinius discodatactylus Syncope antenori Dendrobatidae Hylidae Leptodactylidae Microhylidae Table 1. Inventory of anurans 2003, 2006 and 2007 Number of Individuals recorded in each year 2003 2006 2007 1 22 38 34 4 1 3 6 5 1 2 1 10 1 2 1 1 2 1 2 3 1 5 3 2 1 2 1 1 8 3 18 8 1 1 5 1 1 1 2 4 1 13 3 2 10 3 1 8 1 1 2 1 2 1 1 1 4 1 2 5 14 2 1 1 3 1 2 3 1 1 1 4 1 1 2 4 1 34 49 31 1 1 17 With reference to Table 1, a total 49 species have been collectively reordered in the years of 2003, 2006 and 2007 at the Largo Preto Concession Area. In 2003 a total of 142 individuals were recorded representing 26 species and 4 anuran families. In 2006 the largest number of individuals was recorded, with 158 individuals representing 24 species and 5 anuran families. The fewest number of individuals were recorded in 2007 with a total of 132 individual captured. However, in 2007 the number of species captures increased representing 33 species and 5 anuran families. All individuals recorded were used in the survey results. The anuran survey of 2007 added fourteen new species to the inventory list of the Lago Preto Concession area. Of the species, one species was recorded in the Bufonidae family (Bufo marinus), three species in the Dendrobatidae family, (Dendrodbates sp, Dendrobates uakari, Epipedobates femoralis) two species in the Hylidea family, (Osteocephalus taurinus, Sinax rubra) and eight species in the leptodactylidae family (Eleutherodactylus buccinators, Eleutherodactylus diadematus, Eleutherodactylus malkini Eleutherodactylus sulcatus, Eleutherodactylus visari Hemiphractus scutatus. Leptodactylus pentidactylus, Leptodactylus rhodantutus). Although fourteen species were added to the inventory, several species were not recorded that had been in the previous years. The most noticeable of these species was Eleutherodactylus peruvianus. In 2003 E. peruvianus was recorded fourteen times; this declined dramatically in 2006 to only two recordings, and in 2007 no E. peruvianus were recorded. Andenomera sp 1 also declined in a similar pattern. In 2003 thirteen individuals were recorded followed by only three in 2004 and two in 2007. There was only one species that significantly increased in the number of recordings (Osteocephalius leprieurii). In 2003 Osteocephalius leprieuri had eight recordings 18 followed by a decrease in 2006 with three recording, and in 2007 it increased to eighteen recordings. Finally two species were consistently the most abundant in all of the three surveys Bufo typhonius and Vanzolinius discodactylus. 6.2 Family Numbers Most individuals captured in each year were in the leptodactylids family (Fig….). In 2003 a total of 76 leptodactylids were recorded, followed by 87 leptodactylids in 2006 and 56 leptodactylids in 2007. In the year of 2003 the second largest family was the hylids family with 38 individuals followed by the bufonids family with 26 individuals. Though, in years of 2006 and 2007 the Bufonids family was the second largest family to be recorded. In 2006 the Bufonids family represented 39 individuals and in 2007 a total of 38 individuals were recorded. In 2003 the Leptodactylids family and the Hylids family collectively represented 80.28% of total number of individuals recorded. Whereas in 2006 it was the leptodactylids family and Bufonids family that collectively represented 79.75 % of total number of individuals recorded. In 2007 this percentage slightly decreased with the leptodactylids family and the bufonids family collectively representing 71.21% of the total number of anurans recorded. In 2003 the bufonids family represented 18.3% with 26 individuals recorded leaving 2 individuals in the family dendrobatids representing 1.4%. No microhylids species were recorded in 2003. The dendrobatids family in 2006 represented 12.02% with 19 individuals recorded. This was followed by the hylids family which had dropped significantly since 2003. In 2006 it represented only 7.59 % compared to 26.76% in 2003. Only one individual form the microhylids family was found in 2006 representing 0.63% of individuals recorded. Finally, in 2007 the percentage of hylids recorded had increased with 26 individuals recorded representing 20% of the population. The 19 dendrobatids family represented 8% with 11 individuals recorded, leaving the family microhylids with only 1 individual representing 0.8% of the individuals captured. 100 90 80 No. of Individuals 70 60 50 40 30 20 10 0 1 Bufonidae 2 Dendrobatydae 2003 3 Hylidae 2006 4 Leptodactylidae 5 Microhylidae 2007 Figure 1. A comparison of family numbers across three studies at Lago Preto (all species pooled). 20 80 70 60 no.of Individuals 50 40 30 20 10 0 Varzea Tirre firme Agual Habitat 2003 2006 2007 Figure 2. Origin of individuals collected all species pooled. 6.3 Origin of individuals Anurans were collected in three different habitats – varzea, tirre firme and aguajal. There were clear differences in the number of individuals recorded in each habitat. Figure 2 shows that over the three years most individuals were found in the varzea habitat. This correlates with the data found in 2006 and 2007, though in 2003 most individuals were recorded in terra firme. Over all, 21 aguajal produced the least number of individuals recorded. This is clearly seen in the years of 2007 and 2006; however in 2006 fewer individuals were recorded in terra firme than in aguajal. 6.4 Species Richness In total 49 species have been collectively reordered in the years of 2003, 2006 and 2007. Terra firme was the most species rich habitat over the three studies and in study year retrospectively. All years combined there were 37 species in total recorded in terra firme. Varzea was the second most species rich over the three studies and in each study retrospectively with 25 species in total. Aguajal was the least species rich with only 12 species recorded over the three studies. 25 No. of Species 20 15 10 5 0 Vazea terre firme aguajal Habitat 2003 2006 2007 Figure 3. Comparison of Species richness between habitats 22 7.2 Abundance of Species 2007 Results for the varzea habitat show that it was dominated by two species, Bufo typhonius and Osteocephalusleprieurii. Bufo typhonius was the most abundant species within varzea followed closely by Osteocephalus leprieurii. The third most abundant species was Vanzolinius discodatactylus. The remainder of species have relatively low abundance within the varzea. 0.0035 0.003 Abundance (Individuals/km) 0.0025 0.002 0.0015 0.001 0.0005 ae m El e ut h op hr y ni sc an ol el en dr D ol os te t hu s. M m hu s. C us er us od m ac in ty ut lu es s. El d ia eu de th m er at od us ac El ty lu eu s. th Vi er sa od rs ac i ty us .m H al yl ki a. ni G eo gr ap hi Le ca pt H od yl ac a. ty bo lu an s. s pe nt O ad st eo ac ce ty lu ph s al O us st .le eo pr ce ie ph ur Va al ii us nz .p ol la in ni iu ce s. ps di sc od at ac ty lu s us an es i ar ch ty p C ol os te t Bu fo . p G er a. no m An de iu ho n an dm a s 0 Species Figure 4. Abundance of individuals within varzea 2007 23 no m er a. G p B a uf D en o. ndm d t El a y ro eu ba pho th ni te D er od end s. s us p ro ac ba eci ty El es te eu lus s. th a El u eu ero ltam ak a El eu the dac azo ri ro t th n yl d ic er us us od act ac ylu sul El c s at .b eu tylu us u s th er . cr cci na od oc to eo Ep act r in y ip gu ed us. i o n o c is H bate ken em de s. i i Is phr Fem ni Le chn actu or a o pt s od cne sc lis u ac m ta a. tu Le tylu s. quix s pt pe od Le nt ens ac a i pt od tylu da s ct s. ac y rh ty od lus l an Le us. r h ut pt u od od O ac om s st ty ys eo ta ce lus x .w O ph st a gn eo alu er s. c le Sc ep pr i ha ar i e lu th u s. yl ta rii a. u o rin Sc st u i in ax nod s ac .C ru t en yla to m S Va Sy cina ma nz nc x. ol r in iu ope ubr s. an a di sc t od eno r at ac i ty lu s An de Abundance (Individuals/km) (Figure 5). More species were identified within terra firme than in varzea and aguajal, though like varzea, terra firme is dominated by Bufo typhonius. However, when both habitats are compared, the abundance of Bufo typhonius is higher in Varzea than in terra firme. The second most abundant species in terra firme was Eleutherodactyus ockendeini, but it was considerably less abundant that Bufo typhonius. The remainder of species have relatively low species abundance within terra firme. 0.0025 0.002 0.0015 0.001 0.0005 0 Species Figure 5. Abundance of individuals within terra firme 2007 24 Only two species were recorded in Aguajal, Bufo marinus and Vanzolinius discodatactylus. The most abundant species by far was Vanzoliniu discodatactylus. Bufo marinus had a low abundance with only one individual identified (Figure 6). 0.0035 0.003 Abundance (Individuals/km) 0.0025 0.002 0.0015 0.001 0.0005 0 Bufo. marinus Vanzolinius.discodatactylus Species Figure 6. Abundance of individuals within aguajal 2007. 25 6.6 Habitat diversity indices The species diversity in each habitat is illustrated in figure 7 below. The Shannon Weiner results show that the terra firme has the highest level of diversity. Following close behind is varzea, then Aguajal, which has a comparatively very low level of diversity. 2.5 2 Diversity index 1.5 1 0.5 0 Varzea Terrefirme Aguajal Habitats Figure 7.Diversity of species within three different habitats 2007. (Shannon Weiner Index). 26 Habitats Mean N Std. Deviation varzea 1.9697 33 4.59949 Tirra firme 1.4848 33 2.50151 .5758 33 3.13279 1.3434 99 3.53456 Agaujal Total Table 2. The mean number of individuals within Varzea, Terra firme, and Agujal for 2007. Table 2 confirms that varzea and terra firme have a significantly higher average number of individuals than aguajal. Varzea has the highest average number of individuals followed by terra firme and then aguajal. Sum of Squares Mean df Square F Sig. Between 6.092 1 6.092 Within Groups 59.908 97 .618 Total 66.000 98 9.864 .002 Groups Table 3. ANOVA Results 27 The ANOVA table above shows that there is a significant difference in the species abundance of anurans between the three major habitats. This can seen as the significance is above 0.05. 7. Discussion 7.1Family numbers Most individuals were recorded in the leptodactylid family, which was somewhat expected due to the survey techniques used, which favoured terrestrial species. Also, the leptodactylids are the largest family of anurans within the tropics of South America. The second most abundant family in the tropics of South America is hylids (Huyse 2005). However, very few species of this family have been recorded at Lago Preto in comparison to what has been found in other areas of the Iquitos region. Rodríguez and Duellman (1994) identified over fifty species of the hylid family within the Iquitos region in comparison to only fourteen species that has been found at Lago Preto. Although it needs to be taken into consideration that Rodríguez and Duellmans’ (1994) findings were over a much larger area, their findings still highlight the possibility of finding more species within the hylid family at Lago Preto. The major reason for not recording more species within this family is likely due to the bias of the survey methods. All three studies concentrated on finding species that were found within the leaf litter, which reduced the chance of recording arboreal Hylidae species. The second most abundant family recorded at Lago Preto over the three studies was the Bufonidae family. However, the Bufonidae family had relatively few species recorded with in its family at Lego Preto. This family was significantly dominated by one species alone, Bufo typhonius, with only two other species within this family being recorded, Bufo Marinus and Dendrophyryniscos minutes. The domination of Bufo typhonius within this family at Lago Preto 28 could be explained by the breeding characteristics of this species. Bufo typhonius is an explosive breeder (Wells, 1979) so, as all three studies have been conducted at approximately the same time of year and most Bufo typhonius individuals recorded have been juveniles, it is likely that the surveys have coincided with the end of the breeding season. Such findings of amphibian distribution and activity have long been recorded to be influenced by abiotic and biotic factors (Alonso et al, 2001). Low numbers of microhylids and dendrobatids have been found at Lago Preto. This was expected as both families are relatively small in number and in physical size making them difficult to record. 7.2 Abundance of Anurans in Varzea, Terra firme and Aguajal. In the studies from 2003, 2006 and 2007 on anuran abundance at the Lago Preto anurans were found in all three major habitats. However, there were differences in the assemblages of the species found in each habitat. The abundance research from 2003 and 2006 showed that the abundance of species was greater in varzea and in terra firme than in aguajal. This was confirmed again in the results of 2007 where the abundance of individuals were found to be greatest in varzea. The cause for this in 2007 was the high numbers of Bufo typhonius and Osteocephalus leprieurii within the varzea habitat. Nearly all recordings of the two species were juveniles and as both of these species need water for breeding and the initial stages of their lifecycle, varzea habitat is ideal (Rodríguez and Duellman 1994). Furthermore, as survey methods were biased towards terrestrial species and Bufo typhonius is a terrestrial species it is no coincidence that it was the most abundant species found. 29 Terra firme was the second most abundant habitat in 2007 and like varzea it was dominated by Bufo typhonius. All other species within this habitat were evenly spread, all having relatively low levels of abundance. The most abundant family within terra firme was the leptodactylids, accounting for at least three quarters of the population of anurans. When comparing the three studies form 2003, 2006 and 2007 all studies suggest that the aguajal habitat produces the least amount of individuals. In 2007 this was clearly observed as very few individuals were recorded. Another interesting note regarding species abundance is that aguajal is the only habitat where Bufo typhonius was not the most abundant. Vanzolinius discodactylus was the most abundant species and besides one recording of Bufo marinus it was the only species recorded. Again like Bufo typhonius in the varzea habitat all recorded individuals of Vanzolinius discodactylus were in the early stages of their life cycle. The underlying factor for the high level of Vanzolinius discodactylus juveniles in aguajal are likely to be linked to high production of fruits. The high production of fruit results in abundant numbers of insects, on which many of the Vanzolinius discodactylus individuals were seen feeding on. Though the majority of the insects are relatively small their high densities provide ample food for juvenile anurans. Furthermore, the fact that the majority of insects feeding on the fruit are small is advantageous to juveniles, which require large amounts of small prey (Thomson 2006). An explanation for why so few individuals and species were recorded in aguajal is the difficulty to manoeuvre through the habitat whilst conducting a survey. This is because of difficult conditions caused by swampy ground and poor visibility due to thick foliage (Bowler 2006). Contributing to the low levels of abundance and diversity of species in aguajal is the lack of data 30 for night transects. Only one night transect was possible during the survey of 2007 as conditions were too difficult. This could have seriously affected the results as most anaran species are nocturnal in habit (Rodriguez, 1994). However, it is possible that low levels of abundance and diversity exist due to agujal being an unstable habitat, because of constant flooding, resulting in less ecological niches, food and suitable microhabitats for species to live in (Pitman et al 2003). 7.3 Species richness and diversity The surveys conducted in 2003, 2006 and 2007 all indicate that species richness is greatest in terra firme followed by varzea and then aguajal. These findings correlate with previous studies indicating that terra firme habitats should contain the highest proportion of species. This is due to the increased diversification of vegetation, arthropods and floral species. The increase in diversification of fauna and flora, combined with the stable environment that terra firme provides, creates many more ecological niches allowing more species to live within one habitat (Pitman et al, 2003). Although the species inventory was increased by fourteen species in 2007, resulting in 47 species in total recorded at Lago Preto, the level of species richness recorded is much less than in other studies conducted in the Amazon Basin. At a single site in the upper basin of the Ecuadorian and Peruvian Amazon 81 species of frog have been found (Duelman 1992). Evan closer to the study site, in protected areas along the Rio Yavari Miri and the Rio Yavari, levels of anuran diversity encountered by Perez et al (2007) were over 71 species in the same habitat types. 31 The lower level of species diversity recorded at Lago Preto could be due to several reasons. One reason could be that the study began at the beginning of the dry season when amphibian activity is not at its highest. Therefore amphibian diversity can be expected to be less than what would be expected during the wet season (Young et al, 2004). Amphibian activity in the Neotropics is seasonal with an increased productivity and reproduction during the early months of the rainy season. This is believed to ensure maximum reproductive productivity, avoiding the possible obliteration of tadpoles during the drier months (Alonso et al, 2001). As the studies of 2003, 2006 and 2007 were conducted during the start of the dry season, levels of anuran diversity can not be expected to be at their highest. Unusual weather conditions during the first week of the 2007 survey could also have affected levels of anuran diversity. Temperatures as low as 10° coming north from the Antarctic were produced by a climatic phenomenon that only occurs approximately every ten years. (Bodmer per communication, 2007). Climatic changes during the beginning of the 2007 survey could have been unfavourable for amphibian activity resulting in a decrease in observation (Pechmann 1991). 7.4 Trends in species abundance 2003, 2006 and 2007 When comparing the abundance of individual species in each study there have been some noticeable trends. Throughout the studies Vanzolinius discdactylus have been the most abundant species, though it seems that each species has a preference in habitat. Vanzolinius discdactylus appears to prefer aguajal where it has been recorded in all three studies to be the most dominant. 32 While Bufo typhonius appears to be less restricted in the studies in 2006 and 2007 it was clearly more abundant in varzea. However, there is also evidence of declines in species abundance. In 2003 Eleutherodactylus peruvianus was the most abundant species within terra firme. This declined dramatically in 2006 and then in 2007, no individuals were recorded in either habitat. A similar decline has occurred in another leptodactylid, Adenomera sp.1. It too was recorded as one of the most abundant species in 2003 and has since declined. There are a number of possible reasons for the declines, including climate change, chytridiomycosis, or any of the other threats that amphibians face, but none can be ascertained from this data alone. More research would be needed. On the other hand, with each year of comparative data, there have been species added to the inventory of Lago Preto, and in one case a species’ abundance appears to have increased (Osteocephalus leprieurii). 8. Further research Studies lasting more than two years are uncommon (Skelly et al 2003), but as amphibians are globally declining, it is imperative that long term monitoring of anuran diversity and abundance at Lago Preto continues, as it is a strong hold for many species. Long term monitoring has been identified as the ideal way to collect and document changes in abundance and distribution of species (Caughley and Gunn 1996). Historical data is a necessary step in understanding past actions and detecting the effects of climate change, chytridiomycosis and other relevant impacts and in formulating management strategies (Skelly 2003). 33 Long term monitoring for amphibians is even more important as presence and absence and distribution can change from year to year. There are two possible explanations for fluctuations. Year to year fluctuation in sampling could be a result of inadequate surveying and the cryptic behaviour of anurans. Secondly, changes in distribution could reflect anuran behaviour. Anurans are known to stop breeding during periods in which climate is unfavourable reducing their abundance and then rebound when climate is favourable (Pechmann et al 1991; Skelly 2003). Consequently, consistent long term monitoring of anuran populations such as that of Eleutherodactylus peruvianus and Andenomera sp.1 is necessary to understand the true fluctuation in abundance and distribution. In addition to the continuation of the long term monitoring using the study methods of the three previous studies, it is important that new study techniques are used to favour arboreal species, primarily those within the Hylidae family, most of which are nocturnal and only descend to breed around lakes, ponds and streams for breeding. A suggested solution to monitor arboreal species would be to identify species through audio surveillance, as most species have their own individual call (Donelley 2004). This could be combined with surveys of breeding grounds such as lakes, ponds and slow flowing stream. A variety of survey method techniques has a better chance of increasing the reliability of data collected for long term monitoring and future management strategies. 34 9. Limitations A few limitations existed during this study which may have influenced the results and amount of data I was able to collect. The short time period for study (from 25th May to 11 June) affected the amount of data that could be collected. The time of year in which the study and previous studies have been conducted is likely to have affected the abundance and diversity of anurans recorded, as all studies were conducted at the beginning of the dry season, when anuran activity is not at its highest. In addition different structures of vegetation and ground conditions between habitats created different levels of visibility, skewing the chance of recording an individual and the use of semi-permanent trails is likely to have some impact on diversity. And finally, the bias of survey methods towards terrestrial species is likely to have affected results. 10. Conclusions Amphibian populations are affected by a number of different factors, from natural fluctuation due to disease and climatic/breeding preferences, to anthropogenic factors such as climate change, and habitat to destruction. Data collected in 2007 correlates with that of the previous years and other research conducted on anuran abundance and diversity within the South American Tropics. Anurans were found to be the most abundant in varzea whilst terra firme was the most diverse, with aguajal appearing to be the least abundant and diverse. However, there 35 may be influential variables which have not been accounted for, such as the different levels of visibility between habitats and the time of year at which all three studies have been conducted. It is important to minimise the impact that amphibians face, not only for their intrinsic value and for their importance within the ecosystem, but because they can be used as environmental indicators of ecosystem health. The conclusions drawn from this study and those of previous years can be useful in providing base line data for the long term monitoring of anurans at Lago Preto. Although anurans at Lago Preto face relatively little amount of physical disturbance from human activities due to habitat destruction, it is still important to monitor changes in abundance and diversity, as it is still possible that numbers may be declining due to chytridiomycosis and climate change. Therefore it is essential that consistent and long term monitoring continues at Lago Preto. 36 11. List of References Alford, R. A., Richards, S. J., (1999) Global amphibian declines: a problem in applied ecology. Annul. Rev. Ecology Systems. 30, 133–165 Attenborough, (2008). Life in Cold Blood. BBC Books. Amphibian Ark, (2008). Frightening statistics. Available at: http://www.amphibianark.org/statistics.htm Alonso A., Dallmeier F. and Campbell P. (2001). Urubamba: The Biodiversity of a Peruvian Rainforest, SI/MAB Series #7. 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