The Impacts of Historical and Ongoing Human Activities on Amphibian Diversity in Bilsa Biological Station, Western Ecuador

by Gregor F.M. Jongsma

Thesis submitted in partial fulfillment of the requirements for the Degree of Bachelor of Science with Honours in Biology Acadia University 2012

Copyright by Gregor F.M. Jongsma 2012

This thesis by Gregor F.M. Jongsma is accepted in its present form by the Department of Biology as satisfying the thesis requirements for the degree of Bachelor of Science with Honours. Approved by the Thesis Supervisor __________________________ ____________________ (Tom Herman) Date Approved by the Head of the Department __________________________ ____________________ (Soren Bondrup-Nielsen) Date Approved by the Honours Committee __________________________ ____________________ (Pritham Ranjan) Date

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I, Gregor F.M. Jongsma, grant permission to the University Librarian at Acadia University to reproduce, loan or distribute copies of my thesis in microform, paper or electronic formats on a non-profit basis. I, however, retain the copyright in my thesis. _________________________________ Signature of Author _________________________________ Date

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Acknowledgements
I am grateful for the assistance provided by staff of the Jatn Sacha Foundation and Bilsa Biological Station, particularly C. Aulestia and J. Bermingham, and by field researchers D. Caberera, L. Carrasco, F. Castillo, and J. Olivo. For assistance with species identification many thanks go to G. Vigle and M. Ortego-Andrade. At Acadia, T. Herman and S. Bondrup-Nielsen provided valuable input throughout the writing process of my thesis. Research was supported by the National Science Foundation (OISE-0402137), National Geographic Society, Conservation, Food and Health Foundation and Disney Wildlife Conservation Fund.

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Table of Contents
List of Tables............................................................................................................vi List of Figures ......................................................................................................... vii Abstract .................................................................................................................. viii Introduction ............................................................................................................... 1 Materials and Methods ............................................................................................... 5 Results ..................................................................................................................... 11 Discussion ............................................................................................................... 15 Literature Cited ........................................................................................................ 21 Appendix 1............................................................................................................................................ 24 Appendix 2..............................................................................................................25 Appendix 3......................................................................................................26 Appendix 4......................................................................................................30

List of Tables

Table 1: Number of individual amphibians recorded across seven habitat categories in Bilsa Biological Station, Ecuador.

Table 2: Total individuals, diversity (Shannon-Wiener and Simpsons respectively), observed and expected species richness for the seven habitat categories.

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List of Figures
Figure 1: Three maps showing where Ecuador is located in South America, where the Mache Chindul Reserve is located in Ecuador and where Bilsa (where the study was conducted) is located within the Mache Chindul Reserve. Figure 2: Expected species accumulation curves for amphibians captured in different habitat categories in Bilsa Biological Station, according to the Mao Tau index. The vertical hatched line indicates the minimum abundance level recorded for comparison across habitat types. Curves were re-scaled to show accumulation of species in relation to number of captured individuals.

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Abstract
Human activity in western Ecuador is extensive in the form of logging and slash-andburn agriculture, however the impacts of these activities on amphibian diversity is almost completely unknown. During a nine-month study in the Bilsa Biological Station situated in the province of Esmeraldas, Ecuador I attempted to evaluate the impacts of these activities. Amphibian diversity was estimated using visual encounter surveys along transects in pristine sites (forest and river habitat that has no history of human activities) and historically disturbed sites (forest and river habitat that was logged between 1989 and 1995) as well as along roads with varying levels of ongoing human activity. The results of this study indicate that the influence of disturbance on the diversity and composition of amphibian assemblages in the Bilsa Biological Station varies with habitat. Amphibian assemblages along rivers were the richest as well as the least impacted by human activity. Diversity and species richness were lower in secondary than in primary forest suggesting that amphibian assemblages in interior forest habitat are more vulnerable to disturbances caused by logging. Road habitat around Bilsa harboured a very different amphibian assemblage from that of forest and river habitats. The results of this study show that amphibian assemblages vary markedly in vulnerability to disturbances by human activity; recognising these differences is critical for developing land management plans that promote amphibian diversity in north western Ecuador.

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Introduction
Evidence is mounting that humanity is driving a sixth mass extinction event that is notable for its unprecedented rate of species loss (Wilson 1992). Amphibiansfrogs, salamanders and caecilians are the oldest group of terrestrial vertebrates, and are currently facing risk of extinction world-wide (Stuart 2004; Wake and Vredenburg 2008). Of the 6,800 known amphibian species approximately one third are listed as threatened and at least 43% are in decline (Collins et al. 2007). In the past three decades, the rate of amphibian extinction has exceeded the mean extinction rate of the last 350 million years 200-fold (Roelants et al. 2007). Amphibians are well suited as indicator species for assessing the health of ecosystems and because they inhabit most of the worlds fresh-water and terrestrial biomes, they may serve as ideal indicators of global biological health and predictors for world-wide species extinctions (Collins and Halliday 2005).

There are many factors causing amphibians to decline world-wide and the complex interactions among these factors are poorly understood (Collins et al. 2007). The factors causing the decline include: environmental contamination, invasive species, disease, climate change, and deforestation (Stuart 2004). All of these factors result in the alteration of natural habitats and all are linked to the activities of our ever-growing human population (Wake and Vredenburg 2008). One of the most prominent forms of habitat alteration, the conversion of forests to agricultural lands, plantations, and cattle ranches, is occurring at an alarming rate in the tropics. It would be difficult to find a region in the tropics where this is more evident than in the Choc biogeographic region in north-western Ecuador. Today, conservationists refer to the Choc region as the Tumbes-Choc-Magdalena hotspotan area covering 274,597 km, extending along the coast of Peru north through coastal
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Ecuador and Columbia to southern Panama. The region has been coined the hottest of biodiversity hotspots, one demanding immediate international attention as a conservation priority (Parker and Carr 1992; Mittermeier et al. 1998). Rampant deforestation set this region apart from other hotspots around the world as less than two percent of the original forest cover remains (Dodson and Gentry 1991). Even these remnant stands are under siege by a combination of corporate operations (logging, cattle ranching, African palm and banana plantations) and the activities of impoverished Ecuadorian farmers (slash and burn agriculture and poaching).

Threats Logging has played the most pervasive role in transforming the Mache-Chindul an area of the Tumbes-Choc-Magdalena in north coastal Ecuador both directly and indirectly (Hamilton et al. Unpublished). Clear-cutting and selective logging transform the landscape by removing the trees; roads created in the process allow access to land that becomes occupied by farmers whose activities further transform the area. Cattle pastures and banana and African palm plantations, as well as small-scale subsistence farms, are prevalent in the region. These activities inhibit forest regeneration and further deplete the already nutrient-poor soil. As a result, the Mache-Chindul landscape is now a matrix of pasture-land dotted with patches of primary and secondary forest mostly on steep hillsides unfit for grazing cattle or growing crops. The severe fragmentation of this forest landscape has many implications for amphibians and other flora and fauna local to the Choc.

Endemism An endemic species is one that is restricted to a single locality (Hickman et al. 2008).

Coastal Ecuador harbours some of the highest levels of endemism on earth and amphibians in the region are no exception. Of the 213 recognized amphibian species in the Tumbes-ChocMagdalena, 40 are endemic (Conservation International). Endemic species are inherently at greater threat of extinction due to their naturally restricted distributions (David 2005). Most of the worlds amphibians live in the tropics, where they occupy small geographic ranges and have specific micro-habitat requirements (Wake and Vredenburg, 2008). These characteristics leave amphibians vulnerable to rapid extinction. Some species, like the glass frog Cochranella mache, have only been recorded along a few mountain streams in Western Ecuador (IUCN). Human encroachment into this small area could very well eliminate the entire species. Given the intensity of historical and current habitat alteration in the region, many species have almost certainly perished undocumented and will likely continue to be lost. Habitat destruction in regions with such high levels of endemism significantly reduces global biodiversity, and conservation in these regions should be made an international priority.

Conservation Successful species management and recovery require a number of steps. Amphibian conservation in the Mache-Chindul is under-developed and a number of initiatives are still required: (1) current amphibian diversity and population trends need to be accurately assessed, (2) areas of high conservation priority (i.e. areas with high amphibian diversity or immediately threatened by human activity) and corridors connecting them need to be identified and protected, (3) suitable amphibian habitat needs to be allowed to regenerate (Young et al. 2001). Unfortunately, reserves in Ecuador, as in most of the developing world, are poorly protected. Illegal logging and slash and burn agriculture in the Mache Chindul are widespread

even within protected areas. If the three initiatives above are to be successful Ecuadors government must provide adequate funding for reserves and hold corporations or individuals who break the law accountable. Equally important, government and NGOs must provide local residents with sustainable economic alternatives that promote amphibian diversity, rather than degrade it. Any successful amphibian conservation initiative will require the collaboration of scientists, government, NGOs, and, above all, local people.

Objectives The objectives of this study were two-fold. The first objective was to document the species diversity of amphibians at Bilsa Biological Station. Over a nine month period amphibian diversity was estimated using visual encounter surveys along transects in pristine and historically disturbed forest sites as well as along roads with varying levels of human activity. Because amphibians in this area are poorly known, these basic surveys contributed to the first complete inventory of species composition and richness that is critical to any future conservation efforts. The second objective was to gain insights into the influence of habitat on amphibian diversity and abundance. To address this question, amphibians were sampled from a range of habitats including forests, roadsides, and waterways. In this component of the study, I also addressed how anthropogenic activity may be affecting amphibians by comparing each habitat type (e.g., forest, road and river) in pristine conditions and in disturbed conditions.

Materials and Methods

Study Area The Mache-Chindul mountain range of north-western Ecuador harbours some of the greatest biodiversity on earth with a high degree of floral and faunal endemism. Two major forest categories are found on this mountain range: (1) Humid Pre-Montane forest at elevations between 500 and 1800 m, and (2) Humid Tropical rainforest below 500 meters. As part of the Tumbes-Choco-Magdalena (TCM) biodiversity hotspot, international pressure to preserve the regions unique ecosystems resulted in the creation of the Mache-Chindul Ecological Reserve (MCER) in 1996 (http://www.ambiente.gov.ec/paginas_espanol/4ecuador/docs/areas/mache.htm). Based on the authors personal observations little is being done to curb illegal logging, agriculture or poaching and much of this 70,000 hectare area remains under threat. As a result, the park today is largely deforested and consists mainly of small scale farms, cattle ranches, and African-palm plantations, interspersed with small forest fragments. A smaller reserve inside the MCER, Bilsa Biological Station, is privately owned and managed by the Jatun Sacha Foundation and is sufficiently protected. This study of amphibian diversity was carried out over nine months in 2007 in the Bilsa Biological Station (79 45 W, 0 22 N, 330-730 meters above sea level) in the province of Esmeraldas, Ecuador (Figure 1). The Tumbes-Choco-Magdalena region is one of the wettest places on earth, and Bilsa receives on average more than 300 cm of rain per year. The temperature is very stable. During the rainy season from January to June the average temperature ranges between 24C and 25C. During the dry season from July to December the average temperature is between 21C and 22C. With elevations ranging from 300m to 730m

Bilsa offers protection for both Humid-Pre-Montane forest and Humid Tropical forest. Being one of the largest patches of remnant forest in North-Western Ecuador, Bilsa protects a mosaic of primary and secondary forest covering an area of 3300 hectares, 80% of which is primary forest; the remaining 20% of forest has been allowed to regenerate over the past 12-18 years. This makes Bilsa one of the most representative areas in terms of existing or former diversity in the surrounding valleys and rivers of the Mache-Chindul.

Selecting Sites Rivers: River transect sites were selected based on the type of forest habitat surrounding them (i.e. primary or secondary). Primary forest (never logged) and secondary forest (logged 1218 years previously) were identified with the help of experienced park staff. In addition to forest habitat type, selection of sites required year-round water-flow and sufficient safety for investigators to access and sample. Wherever possible, river sites with the most similar bankwidth and water velocity were selected; however, given the high heterogeneity of the area this was not always possible. In all cases eight common environmental variables, described in detail below, were recorded for each river transect. In total, 36 sampling sessions were conducted across 18 river transects in primary forest habitat and 34 sampling sessions were conducted across 17 transects in secondary forest habitat. Interior Forest: Forest transect sites were selected in areas that had been characterized by a team of ornithologists as either primary or secondary; this ensured sampling was conducted in both forest habitat types equally (Appendix 1). Sixteen transects were set up within 6 primary forest sites and sixteen transects were set up within 6 secondary forest sites. Transects ran perpendicular and parallel to walking trails, at random distances within the sites identified by the

team of ornithologists. There were a total of 32 sampling sessions in primary forest and 32 sampling sessions in secondary forest. Road: To investigate the influence of ongoing human disturbances three different road categories were identified and sampled: (1) roads bordered by secondary forest on both sides (16 sample sessions along 8 transects), (2) roads with secondary forest on one side and pasture land on the other (14 sample sessions along 7 transects), and (3) roads with pasture land on both sides (17 sample sessions along 8 transects). The roads were unpaved and inaccessible by vehicles approximately ten months of the year due to the muddy conditions.

Figure 1: Three maps showing where Ecuador is located in South America, where the Mache Chindul Reserve is located in Ecuador and where Bilsa (where the study was conducted) is located within the Mache Chindul Reserve. Sampling Scheme Amphibian sampling occurred from March to November 2007. For all habitats (rivers, interior forest, roads), sampling was conducted using visual encounter surveys (VES) along transects (Heyer et al. 1994). This involved walking slowly along 50 meter transects, intensively searching with a stick through all leaf-litter and on all leaves and stems within transects for amphibians. Transects in interior forest were 2m wide, while river transects were as wide as the

river-bank (1-3m) and road transects were as wide as the road-side (1-4m). Each transect was laid-out at least two weeks prior to sampling to avoid any effects of disturbance. Each transect was sampled twice: once in the morning and again at night. All amphibians encountered during morning sampling sessions were captured and only released after the night sampling session was complete. In addition to capturing individuals encountered during the morning sample session, the location of each transect was changed each month. Employing this approach is a less invasive way to avoid re-sampling individuals than toe-clipping, which many studies have shown to negatively impact frogs (Davis and Ovaska 2001; McCarthy and Parris 2004; Waddle, et al. 2008). Changing the location of transects each month was also important to avoid modifying the habitat along transects caused by repeated sampling in the same area. This is particularly an issue in the tropics where the forest floor very readily turns to mud when disturbed. Four river transects were sampled twice each month (two in secondary forest habitat; two in primary forest habitat) for a total of 36 sampling sessions along rivers in primary forest and 34 along rivers in secondary forest (sampling along rivers in secondary forest was missed one month due to health complications). River banks, defined as the area between the water and the forest edge, were intensively searched for amphibians along both sides of the river for 50 m. All leaves and branches overhanging the river banks (as high as 3.5 m) were also searched at each river transect. Similarly, four interior forest transects were sampled twice each month (two in primary forest; two in secondary) for a total of 18 transects in each habitat. For the forest transects, two 50 m lengths of string were laid down in parallel rows two meters apart and all leaf litter, logs, branches and leaves inside the two strings were searched for amphibians. Each of the three road habitat categories was sampled twice each month.

Environmental Variables For each transect, the weather conditions, number of people searching, and time spent sampling were recorded. For each frog encountered, the species, age classification (adult, adolescent, juvenile), and substrate were recorded during the sampling session. Depending on the habitat, the appropriate environmental variables were recorded. Environmental variables recorded along river transects included: bank-width, waterwidth, water velocity, depth, understory density (1) over the bank and (2) over the water. All of these variables were measured at 10 m intervals along each transect and then averaged. Water velocity was measured by dropping a ping-pong ball in the river and timing how long it took to travel one meter. Relative understory density was obtained by averaging the number of contacts of vegetation (leaves and branches) with a 2 m tall stick placed vertically every 10 m along a transect. This was measured at the edge of the forest and the edge of the water. The number of waterfalls and pools was also recorded for each river transect. Leaf-litter depth and understory density were measured at each forest transect at 10 m intervals and averaged. The road side width, road wetness, type of vegetation and average vegetation height were recorded for each road transect. Vegetation type was categorized as either grass or shrubs. Road wetness was ranked on a scale of 0-5, with 0 being completely dry and 5 being more water than road.

Species Identification During the first two months of amphibian sampling, all individuals encountered along transects were captured and photographed for subsequent identification. Photographs were shared with herpetologists in the US and Ecuador who had previous experience identifying amphibians in the tropics. Dr. Greg Vigle has studied herpetofauna in Western Ecuador for the

past 25 years and carried out the first herpetological inventory of Bilsa in 2000. Dr. Vigle provided help identifying amphibians encountered during this study via email. Marcelo Escobar from the Natural History Museum in Quito has made numerous trips to Bilsa and is working to create a complete herpetological inventory for the Mache-Chindul. Mr. Escobar offered invaluable support in identifying the amphibians encountered over the course of this study. So, although little work had been done on the assemblages of amphibians in altered versus pristine forests in Bilsa, sufficient inventory work had been done in the park to identify the species encountered during the course of this study with confidence.

Statistical Analysis
To examine how complete sampling was in different types of habitat and to compare species richness across habitat types while controlling for differences in sample size, expected species accumulation curves (also called sample-based rarefaction curves, in which a sample is one sampling occasion for a given station), were computed using the analytical formulas of Colwell et al. (2004) as implemented in EstimateS v. 8 (Colwell 2006). Virtually any sampling methodology is imperfect in completely detecting all the species present in a site, and several indices have been developed to estimate true species richness based on sampling data (Colwell and Coddington 1994). As an estimation of the total number of species present in each habitat category, the mean and range of four commonly employed abundance-based estimators (ACE, Chao1, Jack1 and Boostrap), were calculated using EstimateS v. 8 and based on 1,000 randomizations of samples without replacement. Two diversity indices were calculated using EstimateS v. 8: Shannon-Wiener and Simpson`s. The Shannon-Wiener index places more weight on rare species; conversely, Simpson`s index places less weight on rare species (Krebs 1999).

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Results
Species Abundance and Diversity In total 933 individuals were recorded: 613 along river and forest transects, and 320 along road transects (Table 1). Pristimantis achatinus dominated six of the seven habitat categories and comprised more than half of the total of the individuals encountered during the study. Hyloxalus awa was the most abundant species encountered along primary rivers and was the second most abundant along secondary rivers.

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Table 1: Number of individual amphibians recorded across seven habitat categories in Bilsa Biological Station, Ecuador.
Species Bolitoglossa Spp. Chaunus marinus Total 1 1 1 1 4 2 1 37 8 27 114 45 1 48 2 1 20 535 3 2 4 37 31 7 933 Forest 1 2 0 0 0 0 0 0 0 0 1 1 0 3 0 0 0 1 18 50 1 0 0 0 0 0 75 0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 97 2 0 3 0 0 0 104 River 1 2 0 0 1 0 2 2 1 23 2 16 79 21 0 18 0 0 1 45 0 0 0 9 19 0 239 1 0 0 0 2 0 0 14 2 10 35 21 0 30 0 0 1 39 0 0 0 28 12 0 195 Pasture 0 0 0 1 0 0 0 0 1 0 0 0 1 0 1 0 0 91 0 1 0 0 0 1 97 Road 2 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 114 0 0 0 0 0 3 119 Mix 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 99 0 1 1 0 0 3 104

Caecilia leucocephala
Caecilia nigricans Cochranella cf. albomaculata C. Mache C. Spinosa Centrolene prosoblepon Craugastor longirostris Epipedobates boulengeri Hyloxalus awa H. sp. nov. Hypsiboas pellucens H. picturata Leptodactylus labrosus L. pentadactylus Oophaga sylvatica Pristimantis achatinus P. parvillus P.subsigillatus P. walkeri Rhinella margaritifer Rhaebo haematiticus Smilisca phaeota Total

Primary habitat is represented by 1, secondary habitat by 2.

A sample-based rarefaction curve shows the accumulation curve of each habitat category (Figure 2). In Bilsa Biological Station river habitat harboured the highest species richness according to the Mau Tau index. Primary forest habitat harboured the third highest richness levels, followed by the road habitat category, Pasture-Pasture. The latter two habitat categories also had the steepest accumulation curves, which suggests that there are still more species to be
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sampled in these habitats, particularly along roads surrounded by pasture. The remaining three habitats had lower and similar richness levels.

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No. species

12 10 8 6 4 2 0 0 50 100 150 200 250 300

River, Primary River, Secondary Forest, Primary Forest, Secondary Road, Secondary-Secondary Road, Secondary-Pasture Road, Pasture-Pasture

No. individuals
Figure 2: Expected species accumulation curves for amphibians captured in different habitat categories in Bilsa Biological Station, according to the Mao Tau index. The vertical hatched line indicates the minimum abundance level recorded for comparison across habitat types. Curves were re-scaled to show accumulation of species in relation to number of captured individuals.

Secondary river habitat had the highest diversity (Shannon-Wiener and Simpsons), followed by primary river habitat (Table 2). Primary forest had higher amphibian diversity than secondary forest. Road bordered by pasture had the highest diversity of the three road-categories. The expected species richness (based on the mean of ACE, Chao1, Jack1, Bootstrap indices) was most striking for the pasture-road habitat.

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Table 2: Total individuals, diversity (Shannon-Wiener and Simpsons respectively), observed and expected species richness for the seven habitat categories. Richness Diversity Total Ind. Obs Expected Shan-W Simpsons Primary 7 0.9 2.0 75 13.1 (8.6-19.9) Forest 4 4.4 (4.0-5.0) 0.3 1.2 104 Secondary Primary River Secondary Pasture Secondary Mix 14 12 7 4 4 17.2 (15.5-19.5) 14.2 (13.0-15.5) 16.4 (9.1-22.0) 5.8 (4.8-7.7) 5.8 (4.8-7.7) 2.0 2.1 0.3 0.2 0.2 5.7 7.4 1.1 1.1 1.1 239 195 97 119 104

Road

Number of species observed (Obs), expected species richness (Expected) is the mean of four common species richness indices (ACE, Chao1, Jack1, Bootstrap), Shannon-Wiener diversity index (Shan-W), and Simpsons diversity index (Simpsons).

Environmental Variables The mean understory density (USD) in primary forest (1.6) was much lower than the mean USD of secondary forest (2.8) (Two sample t-test, p = 0.005). There was no significant difference between any environmental variable in primary and secondary forest. Vegetation-height along roads in pasture land was significantly lower than along roads in secondary forest or roads bordered by pasture and secondary forest (One-way ANOVA, p = 0.052). No environmental variable recorded had a significant correlation with diversity. Descriptive statistics (mean, standard error, standard deviation, skewness, sample size) for each habitat category are included in Appendix 3.

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Discussion
Historical Disturbance Throughout the tropics, alteration of forest habitat by logging and fragmentation commonly results in reduced amphibian diversity and species richness (Pearman 1997; Krishnamurthy 2003; Walting and Donnelly 2008). The results from this study show that the influence of disturbance on the diversity and composition of amphibian assemblages in the Mache-Chindul varies with habitat. Amphibian assemblages along rivers were the most diverse as well as the least impacted by human activity in the form of logging 12-18 years prior to this study. Diversity and species richness were lower in secondary than in primary forest suggesting that amphibian assemblages in interior forest habitat are more vulnerable to disturbances caused by logging. Avian diversity sampled at the same forest sites in Bilsa exhibited the reverse response and had higher diversity in secondary forest than in primary forest (Karubian unpublished). This demonstrates how different groups of animals can respond very differently to disturbance and suggests that amphibians in the Mache-Chindul are more negatively impacted by human activities, like logging, than birds, in terms of overall diversity and species richness. All forest sites in this study were part of a mosaic of habitat with direct contact zones between primary and secondary forest. This type of setting makes it possible for amphibian populations in primary forest to serve as a source in repopulating secondary forest following a major disturbance like logging. Despite this, there was still a significant difference in diversity between primary and secondary forest. What is more alarming about the apparent vulnerability of interior-forest amphibian assemblages is that one could expect to find even lower amphibian diversity in isolated secondary forests lacking available source populations.
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The different responses between forest and river habitats may be explained by regular disturbances along river-banks caused by rising water-levels during the wet season. This natural disturbance results in a similar forest structure growing along both primary and secondary rivers. This was demonstrated by the identical understory densities (USD) in both secondary and primary river habitat. Conversely, mean understory densities differed greatly between primary and secondary forest. Amphibian diversity richness is influenced by a number of environmental variables (canopy cover, soil moisture, distance to nearest stream, invertebrate density) (UrbinaCardona et al. 2006). Recording more of these variables during this study may have helped identify necessary microhabitat characteristics that support greater amphibian diversity or richness at Bilsa.

Ongoing Disturbance Road habitat around Bilsa harboured a very different amphibian assemblage from that of forest and river habitats and of the nine species encountered along roads six were found in no other habitats. Typically, amphibian assemblages in pasture land have lower diversity and species richness (Tocher 1996; Pearman 1997, Urbina-Cardona et al. 2006) but roads in pasture land surrounding Bilsa actually harbour the greatest species richness of the three road-categories. Road wetness was similar across all road categories; however, vegetation height was significantly shorter in pasture-road habitat. It is possible that species went undetected in secondary-road and mixed-road habitats, despite rigorous sampling efforts. Continued sampling in this habitat is needed to more accurately determine the actual diversity and species richness. Additional sampling should be done in pasture away from roads to determine whether the species encountered during this study are restricted to roads or are widespread throughout pasture lands. This information, in combination with pasture diversity in
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relation to distance from forest edge, will help gauge the overall amphibian diversity in the Mache-Chindul as more and more forests are converted to pasture.

Amphibian Detection Species detectability as defined by Boulinier et al (1998) is the probability of detecting at least one individual of a given species in a particular sampling effort, given that individuals of that species are present in the area of interest during the sampling session. This is a crucial factor to consider for any study involving species richness or diversity estimates. Naturally, not all species are equally detectable. Additionally, not all species are equally detectable across different habitats. Fortunately, some species richness estimators take detectability into account. Abundance-based Coverage Estimator (ACE) for example, separates common and rare species from a sample and uses the rare subset to predict how many species are likely missing due to lowered detectability (Chazdon 1998). The issue surrounding detectability is highlighted by canopy dwelling species in the tropics. Canopy dwelling species deserve more consideration when assessing amphibian diversity in the tropics. Utilizing bromeliads as high-rise nurseries, some species may never have to descend from the canopy and thus would never have been encountered in this study. Consequently the true amphibian diversity of the forest may be under-estimated. Two species in Bilsa exemplify the real possibility that amphibian assemblages, particularly frogs, could occupy the canopy without ever descending. Cochranella albomaculata, has only been encountered four times in Bilsa and each encounter was of a mating-pair on a leaf overhanging a river (1-2 m above the water). C. albomaculata is commonly encountered low on vegetation along rivers in Central America

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(Savage 2002). Guayasamin (2007) proposed that what is currently identified as C. albomaculata may actually be several species, as individuals from Columbia and Ecuador exhibit larger spots on the dorsum than in Central America. These isolated encounters in Bilsa support the possibility that C. albomaculata represents different species in Ecuador and, in Bilsa at least, appear to have taken to the canopy, only returning to lower strata of the forest to procreate. Only during mating was it possible to account for this species and it is likely that many individuals were not recorded because they were too high in the canopy. Oophaga sylvatica, is a ground-dwelling frog that uses the water trapped in bromeliads for rearing its tadpoles. Secondary forest supports far fewer epiphytes than primary forest (Turner 1997) which may explain why O. sylvatica was never encountered in secondary forest during this study. The combination of O. sylvaticas use of bromeliads and C. albomaculatas canopydwelling life-style demonstrates that amphibian assemblages may exist undetected in the canopy of primary forest, or in forest where sufficient epiphyte growth has occurred. As such, efforts to detect canopy-dwelling amphibians should be undertaken more often when assessing diversity in the tropical forests. This theory is supported by the discovery of Ecnomiohyla phantasmagoria in Bilsa in 2009 (Patricio M. Olmedo, unpublished. See photograph in Appendix 4). E. phantasmagoria is a canopy-dwelling species and it is likely that is was never detected during this study or any previous ones, because of this. New developments in bioacoustics recording devices may be the best solution to surveying amphibians in the canopy.

Amphibians as Indicator Species At Jatun Sacha Biological Station in Amazonian Ecuador, Pearman (1997) found that

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species richness of the genus Pristimantis declined with proximity to pasture and suggested that Pristimantis may be a useful indicator assemblage for assessing the quality of Tropical Wet Forest. The Pristimantis spp. at Bilsa Biological Station, however, exhibit the greatest richness in pasture and secondary forest habitat. All Pristimantis species encountered in and around Bilsa but one (P. achatinus) were very rare. These findings suggest that Prismantis has little value as an indicator assemblage for assessing the quality of Pre-Montane or Humid Tropical Forest in the Mache-Chindul. Four species (Cochranella mache, C. spinosa, Caecilia leucocephala, and Leptodactylus pentadactylus) encountered during this study were found exclusively in pristine habitat (primary forest or river). All of these species were only encountered once or twice over the course of nine months, thus making them poor indicator species. Oophaga sylvatica, however was encountered 18 times in pristine habitat and never in secondary forest habitat and may be a good candidate for an indicator of pristine forest habitat in the Mache-Chindul since it (1) was the most abundant amphibian found solely in pristine forest during this study, (2) is easily identified, (3) vocal during the day and (4) is wide-spread in the region. All of these traits make its detection easy and feasible. Surveys for the presence of O. sylvatica, in other pristine (and disturbed) forest habitat across its range in the Tumbes-Choc-Magdalena are needed before adopting this species as a suitable indicator of quality pristine forest habitat.

Conservation Implications The results of this study in Bilsa Biological Station show that amphibian assemblages vary markedly in their vulnerability to disturbances by human activity. Recognising these differences is critical for developing land management plans that promote amphibian diversity in

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north western Ecuador. The resilience of riparian amphibian assemblages to historical human activity has strong implications for the conservation and restoration of amphibian diversity along rivers in MacheChindul and possibly throughout the tropics. Conserving existing forest along rivers and promoting forest growth along rivers in deforested areas has potential, not only to encourage and sustain amphibian diversity, but also to provide corridors connecting fragmented interior forest amphibian populations. The extent of forest surrounding a river necessary to support diversity levels similar to those in Bilsa as well as to serve as an effective corridor is still unknown and will require further investigation. The results of this study show that interior forest amphibian assemblages in Bilsa are more vulnerable to disturbance than are riparian amphibian assemblages. Pristine, unlogged forest harboured greater species richness and diversity than secondary forest. In order to conserve intact interior-forest amphibian assemblages any remaining remnant forest must be protected. For successful amphibian conservation to be achieved more research is needed into amphibian responses to habitat size in order to create land-management plans that will sustain amphibians well into the future.

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Literature Cited
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McCarthy, M. A., K. M. Parris. 2004. Clarifying the effect of toe clipping on frogs with Bayesian statistics. Journal of Applied Ecology 41:780-786. Mittermeier, R. A., N. Myers, J. B. Thomsen, G. A. B. da Fonseca, S. Olivieri. 1998. Biodiversity hotspots and major tropical wilderness areas: approaches to setting conservation priorities. Conservation Biology 12:516-520. Parker III, T.A., and J. L. Carr. 1992. Status of forest remnants in the Cordillera de la Costa and adjacent areas of southwestern Ecuador. Conservation International RAP Working Papers 2:172. Pearman, P.B. 1997. Correlates of amphibian diversity in an altered landscape of Amazonian Ecuador. Conservation Biology 11:1211-1225. Roelants, K., S. J. Gower, M. Wilkinson, S. P. Loader, S. D. Biju, K. Guillaume, L. Moriau, F. Bossuyt. 2007. Global patterns of diversification in the history of modern amphibians. Proceedings of the National Academy of Science 104:887-892. Savage, M. J. 2002. The amphibians and reptiles of Costa Rica: a herpetofauna between two continents, between two seas. University of Chicago Press. Tocher, D. M. 1996. The effects of deforestation and forest fragmentation on a central Amazonian frog community. University of Canterbury Press. Turner, I.A., Y. K. Wong, P. T. Chew, A. Bin Ibrahim. 1997. Tree species richness in primary and old secondary tropical forest in Singapore. Biodiversity and conservation 6:537-543. Urbina-Cardona, J. N., M. Olivares-Perez, V. H. Reynoso. 2005. Herpetofauna diversity and microenvironment correlates across a pastureedgeinterior ecotone in tropical rainforest fragments in the Los Tuxtlas Biosphere Reserve of Veracruz, Mexico. Biological Conservation 132:61-75. Waddle, J. H., K. G. Rice, F. J. Mazzotti, H. F. Percival. 2008. Modeling the effect of toe clipping on treefrog survival: beyond the return rate. Journal of Herpetology 42:467-473. Wake, D. B., and V. T. Vredenburg. 2008. Are we in the midst of the sixth mass extinction? A view from the world of amphibians. Proceedings of the National Academy of Science 105:11466-11473. Watling, J. I., M. A. Donnelly. 2008. Species richness and composition of amphibians and reptiles in a fragmented forest landscape in northeastern Bolivia. Basic and Applied Ecology 9:523532. Wilson, O. E., 1992. The Diversity of Life. W. W. Norton & Company. New York. Young, B. E., K. R. Lips, J. K. Reaser, R. Ibanez, A. W. Salas, J. R. Cedeno, L. A. Coloma, S. Ron, E. L. Marca, J.R. Meyer, A. Munoz, F. Bolanos, G. Chaves, D. Romo. 2001. Population
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declines and priorities for amphibian conservation in Latin America. Conservation Biology 15:1213-1223.

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Appendix 1
Quoted from Jordan Karubians methods (unpublished): Forest type characterization Along >15 km of trails that cover about a third of the Bilsa Station area, we establish sampling points every 200 m (total: 84 points) where we visually classified the forest type around it as primary (48 points), altered (16 points), or secondary (20 points, Fig. 1). To confirm if our visual classification reflected actual differences in habitat structure, in each of these points we recorded: the number of trees with DBH >10 but < 50 cm (within a 10 m-radius circle), number of trees with DBH 50 cm (within a 20 m-radius circle), canopy height (in meters), amount of light reaching the understory (measured with a densiometer, average of four measures), and elevation (in meters). We then used a Discriminant Analysis (DA) to assess if sites could be confidently assigned to forest type categories based on these structural variables.

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Appendix 2
Table 1: Species richness results, calculated using Estimate S program: ACE, Chao 1, Jack 1 and Bootstrap.
Habitat Category Samples
Individuals (computed) Sobs (Mao Tau) Sobs 95% CI Lower Bound Sobs 95% CI Upper Bound Sobs SD (Mao Tau) Sobs Mean (runs) ACE Mean ACE SD (runs) Chao 1 Mean Chao 1 95% CI Lower Bound Chao 1 95% CI Upper Bound Chao 1 SD (analytical) Jack 1 Mean Jack 1 SD (analytical) Bootstrap Mean Bootstrap SD (runs)

River Forest Road Primary Secondary Primary Secondary Pasture Forest Mix 36 34 32 32 17 16 14 239 14 10.58 17.42 1.74 14 19.51 0 15.5 14.17 26.89 2.29 17.89 1.86 15.74 0 195 12 8.49 15.51 1.79 12 15.54 0 13 12.09 23.06 1.87 14.91 1.63 13.24 13 7.96 44.4 7.08 10.88 1.84 8.58 0 75 7 3.24 10.76 1.92 7 19.89 104 4 3.06 4.94 0.48 4 4 0 4 4 4 0 4.97 0.97 4.62 97 7 2.76 11.24 2.16 7 22 0 22 10.32 74.78 13.48 12.65 3.34 9.14 5 4.08 17.27 2.17 5.88 1.28 4.83 5 4.08 17.27 2.17 5.86 1.26 4.82 0 119 4 1.79 6.21 1.13 4 7.67 104 4 1.79 6.21 1.13 4 7.67

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Appendix 3
Descriptive statistics of environmental variables: Table 1: Descriptive statistics for primary and secondary forest habitats. Number of Surveyors
Mean Standard Error Median Mode Standard Deviation Sample Variance Kurtosis Primary Forest Skewness Range Minimum Maximum Sum Count Confidence Level (95.0%) Mean Standard Error Median Mode Standard Deviation Sample Variance Secondary Kurtosis Forest Skewness Range Minimum Maximum Sum Count Confidence Level(95.0%) 2.00 0.13 2.00 2.00 0.72 0.52 -0.96 0.00 2.00 1.00 3.00 64.00 32.00 0.26 2.09 0.16 2.00 2.00 0.89 0.80 -0.56 0.39 3.00 1.00 4.00 67.00 32.00 0.32

Slope
2.18 0.11 2.00 2.00 0.60 0.36 -0.46 0.08 2.00 1.00 3.00 61.00 28.00 0.23 1.75 0.11 2.00 2.00 0.60 0.35 -0.59 0.12 2.00 1.00 3.00 56.00 32.00 0.21

Understory Density
1.55 0.10 1.50 1.00 0.58 0.34 -0.39 -0.26 2.21 0.29 2.50 49.62 32.00 0.21 2.77 0.23 2.67 2.83 1.08 1.16 0.50 0.93 3.70 1.50 5.20 60.92 22.00 0.48

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Table 2: Descriptive statistics for primary and secondary river habitats.

Surveyors (#) Mean Standard Error Standard Deviation Sample Variance Kurtosis Skewness 1 River Range Minimum Maximum Sum Count Confidence Level(95.0%) 2.47 0.22 1.34 1.80 3.60 1.68 6.00 1.00 7.00 89.00 36.00 0.45 Slope 2.72 0.10 0.59 0.35 0.32 0.80 2.00 2.00 4.00 98.00 36.00 0.20 USD Forest 2.91 0.13 0.75 0.56 -0.78 0.27 2.62 1.58 4.20 104.8 8 36.00 0.25 USD water 1.28 0.13 0.78 0.61 0.48 0.87 2.75 0.25 3.00 46.22 36.00 0.26 Width Bank 4.63 0.23 1.07 1.15 -0.31 -0.90 3.40 2.40 5.80 101.9 4 22.00 0.47 Width Water 2.51 0.11 0.68 0.47 0.24 0.57 2.62 1.54 4.16 90.26 36.00 0.23 Pool 2.39 0.35 2.09 4.36 1.17 0.45 6.00 0.00 6.00 86.0 0 36.0 0 0.71 Water falls 1.39 0.17 1.02 1.04 -1.21 -0.19 3.00 0.00 3.00 50.00 36.00 0.35 Depth Avg (cm) 24.95 2.65 15.87 251.99 -0.93 0.73 46.20 6.80 53.00 898.20 36.00 5.37 Veloci ty 0.19 0.02 0.12 0.01 5.04 2.24 0.50 0.09 0.59 6.92 36.00 0.04

Mean Standard Error Standard Deviation Sample Variance Kurtosis 2 River Skewness Range Minimum Maximum Sum Count Confidence Level(95.0%)

2.06 0.20 1.15 1.33 3.85 1.77 5.00 1.00 6.00 70.00 34.00 0.40

2.50 0.07 0.39 0.15 1.06 0.82 1.50 2.00 3.50 85.00 34.00 0.14

2.92 0.15 0.88 0.77 -0.77 0.11 3.00 1.42 4.42 99.26 34.00 0.31

1.35 0.13 0.77 0.60 -1.16 0.35 2.34 0.33 2.67 45.82 34.00 0.27

4.28 0.32 1.51 2.27 1.28 1.13 5.58 2.30 7.88 94.08 22.00 0.67

1.65 0.11 0.66 0.44 -0.16 0.38 2.53 0.61 3.14 56.22 34.00 0.23

1.65 0.33 1.91 3.63 1.20 0.65 5.00 0.00 5.00 56.0 0 34.0 0 0.66

0.18 0.07 0.39 0.15 1.23 1.78 1.00 0.00 1.00 6.00 34.00 0.14

15.72 1.95 11.35 128.77 -0.66 0.87 34.80 3.50 38.30 534.34 34.00 3.96

0.18 0.01 0.06 0.00 -0.01 0.88 0.22 0.09 0.31 6.04 34.00 0.02

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Table 3: Descriptive statistics for pasture, forest and mixed road habitats. Surveyors (#) Slope Mud
Mean Standard Error Median Mode Standard Deviation Sample Variance Kurtosis Skewness Range Minimum Maximum Sum Count Confidence Level(95.0%) Mean Standard Error Median Mode Standard Deviation Sample Variance Kurtosis Skewness Range Minimum Maximum Sum Count Confidence Level(95.0%) Mean Standard Error Median Mode Standard Deviation Sample Variance Kurtosis Skewness 2.59 0.32 2.00 2.00 1.33 1.76 1.86 1.43 5.00 1.00 6.00 44.00 17.00 0.68 2.14 0.25 2.00 2.00 0.95 0.90 -0.69 0.31 3.00 1.00 4.00 30.00 14.00 0.55 2.21 0.24 2.00 2.00 0.89 0.80 1.00 1.04 1.17 0.17 1.00 1.00 0.41 0.17 6.00 2.45 1.00 1.00 2.00 7.00 6.00 0.43 1.42 0.14 1.25 1.00 0.47 0.22 -1.93 0.38 1.00 1.00 2.00 17.00 12.00 0.30 1.22 0.15 1.00 1.00 0.44 0.19 0.73 1.62 28

Veg. Height

Road Side Width

353.33 30.26 450.00 450.00 117.21 13738.10 -1.03 -0.71 300.00 150.00 450.00 5300.00 15.00 64.91 314.29 12.21 350.00 350.00 45.69 2087.91 -1.56 -0.66 100.00 250.00 350.00 4400.00 14.00 26.38 170.77 29.04 125.00 125.00 104.70 10961.86 2.51 1.95

Pasture

Pasture, Forest

Forest

2.00 41.62 0.35 14.10 2.00 20.00 1.50 20.00 1.26 50.83 1.58 2583.26 -0.28 2.97 0.07 2.09 4.00 142.00 0.00 13.00 4.00 155.00 26.00 541.00 13.00 13.00 0.76 30.71 2.00 49.40 0.41 12.86 1.75 35.00 2.00 30.00 1.31 40.65 1.72 1652.71 -0.58 1.09 0.41 1.60 4.00 108.00 0.00 17.00 4.00 125.00 20.00 494.00 10.00 10.00 0.94 29.08 1.50 55.89 0.22 13.67 2.00 40.00 2.00 30.00 0.71 41.00 0.50 1680.86 0.57 -0.49 -1.18 0.76

Range Minimum Maximum Sum Count Confidence Level(95.0%)

3.00 1.00 4.00 31.00 14.00 0.52

1.00 2.00 1.00 0.00 2.00 2.00 11.00 15.00 9.00 10.00 0.34 0.51

117.00 3.00 120.00 503.00 9.00 31.51

300.00 100.00 400.00 2220.00 13.00 63.27

29

Appendix 4

30