Dra. Ana Cristina Mendes Oliveira (UFPA) • E-mail: firstname.lastname@example.org
East Pará - Dra. Ana Cristina Mendes Oliveira (UFPA)
West Pará - Dr. José Reinaldo Pacheco Peleja (UFOPA)
Amapá - MSc. Claudia Silva (IEPA)
Maranhão - Dr. Tadeu Oliveira (UEMA)
Mato Grosso - Dr. Rogério Rossi (UFMT)
Groups of interest and diversity of species evaluated per grid: Non-flying mammals: 48 species (Didelphimorphia 8, Xenarthra 4, Primates 7, Carnivora 2, Perissodactyla 1, Artiodactyla 2, Rodentia 23, Lagomorpha 1).
Biological role of the group: Mammals are one of the most studied groups in terms of biological diversity, the results of such studies being used to support programs for subsidizing activities related to conservation strategies. A number of authors have proposed various methods for monitoring biodiversity, the results, based on knowledge obtained on different types of mammal representatives, being included in environmental impact analyses (EAGLES, 1988; CERQUEIRA, 1991). Mammals have become of particular interest due to their high level of interaction into various sites throughout known distribution areas and because of variations in feeding habits, forming relevant niches within the biological dynamics of the forest systems. These animals contribute to the equilibrium of ecosystems through seed dispersal, biological control and by acting as storage reservoirs for infectious agents (DEANE; MARTINS, 1952; DEANE; DAMASCENO, 1961; ALMEIDA; DEANE, 1970; VOSS; EMMONS, 1996; EMMONS; FEER, 1997; NOWAK, 1999). Furthermore, mammals represent one of the main sources of protein for a number of native populations (AYRES; AYRES, 1979; REDFORD; ROBINSON, 1987, 1991; REDFORD, 1997; BODMER et al., 1997; ROBINSON; BODMER, 1999; NOVARO et al., 1997; Bodmer; Pezo, 1999; VALSECCHI, 2005). However, environmental change and degradation in tropical regions has made the presence and biological variability of mammals uncertain in the future. Among western countries, Brazil is the leader in terms of mammal diversity, the Amazon region having the largest proportion, including a large number of endemic species. Such numbers have been on the increase due to the intensification of field work in this particular region, through descriptions of new species, corrections to geographic distributions, and the reinstatement of taxa. These characteristics enable mammals to be successfully used as environmental bioindicators, including areas changed through human activity.
Technique 1. Qualitative and quantitative surveys
The group will be inventoried and have its relative abundances evaluated through the line transect method (BURHAM, 1980), according to the standard procedure established for studies of tropical forest diurnal mammal populations (e.g. EISENBERG et al. 1979; EMMONS, 1984; BROCKELMAN et al., 1986; PERES, 1996, 1997B; BODMER et al., 1997). This is the most widely used method for the census of Neotropical mammals, being employed during surveys taking place in the Brazilian State of Rondônia (FERRARI et al., 1995, 1996; LEMOS DE SÁ, 1996; RONDÔNIA, 2000; MESSIAS, 2001, 2002a,b, 2003, 2004a,b, 2005).
This method consists of slowly walking along a trail at an average and constant speed of approximately 1.5 km/h, recording all sighted mammals. The maximum amount of time for recording data for each sighting will be 10 minutes in an attempt to avoid large census time variations between lines and researchers. The following information will be recorded for each sighted mammal, or for the first mammal sighted (in the event of social groups): time; location of the observer within the trail system (plot number); location of the animal on the trail (left or right of the trail), placement in relation to the side of the trail (left, right, front or back); type of vegetation in which the mammal was observed (primary solid ground forest, primary Igapó forest or secondary forest); method of detection (visual or audible); observed species; activity of the animal at the moment of sighting (fleeing, moving, foraging, resting, socially interacting or emitting sounds); number of individuals observed (in relation to social species); count type (partial or total); age-sex composition (where possible); estimated height in meters of the first animal seen in relation to the ground; distance in meters of the first animal seen in relation to the observer (r) and in relation to the trail (x), gauged with measuring tape; angle of the animal sighting in relation to the trail (θ°), by using a compass; in addition to other relevant observations, such as food item consumed, description of any type of strange behaviour, etc. Traces found along the trails such as footprints, feces and gouge marks will be photographed in scale and collected, where possible, using reverse plaster moulds, or in the case of feces, using the methodology described for carnivores.
Transects must be swept a number of days in advance and before the start of each campaign (dry and rainy), as well as being regularly marked out (preferably every 50 meters) with numbered marker posts. Removal of forest floor debris is necessary in order to reduce any noises made during the walk, facilitating the detection of mammals by means of sound.
Surveys will go ahead on a daily basis, both in the morning and in the afternoon, at a pre-defined time. The start time of activities in the morning will depend on factors such as visibility, weather and trail access conditions, ideally commencing between 6:30 and 7:00 a.m. During the afternoon, the survey can start any time from 1:00 p.m. onwards. The survey finish time for both the morning and afternoon periods must be before 12 p.m. (midday) or 12 a.m. (midnight). An interval between the end of the morning census and the start of the afternoon survey must be employed so that the observer (and the trail) can rest for snacks or meals, as well as for checking data and organizing any necessary recordings (CULLEN; RUDRAN, 2004). We suggest a distance between researchers/technicians at the end of the trail of at least 100 meters.
The censuses should be temporarily halted in case of low light or heavy winds and rains, remembering to note down the time and the position on the trail where the research was stopped. The noise caused by wind and rain often make detection of animal vocalizations or other audible signals difficult, leading to potential errors in direct and indirect observations. It is normally only necessary to wait a maximum of 30 minutes before continuing with the census in the area in which it was interrupted. If the rain persists, it is advisable to terminate the census at a specific point and continue it again on another day at the same place of final interruption and at the same time period during the day. If this occurs during the first 1000 meters, it is suggested that the interrupted census be cancelled, the entire route (5 km) being walked on another day.
The identification of the taxa in the field should be based on field guides (e.g. Auricchio, 1995; Eisenberg & Redford, 1999; and Emmons e Feer, 1997) or on standard literature that addresses group systematics and taxonomy (CERQUEIRA, 1980, 1985; COSTELLO et al., 1993; FORD, 1994; FOODEN, 1963; FRÖELICH et al., 1991; GREGORIN, 1995; HERSHKOVITZ, 1949, 1950, 1977, 1979, 1983, 1984, 1985, 1987, 1988, 1990; HILL, 1960; HIRSCH et al., 1991; KELLOGG; GOLDMANN, 1944; KONSTANT et al., 1985; NOWAK, 1991; ROOSMALEN et. al., 2002; RYLANDS et al., 2000; SILVA JÚNIOR, 1992, 2001; THORINGTON, 1985; TORRES DE ASSUMPÇÃO, 1983; VIVO, 1985, 1988, 1991; WEKSLER, 1996; WILSON; REEDER, 1993; XIMENES, 1999). Confirmation of animal identifications will be made, as necessary, through direct comparison with zoological collection specimens. The expected geographic distribution will also be considered in addition to the morphological characteristics of the sighted specimens during the identification process.
Mammals are very often identified during the census through clear and direct sightings. However, there are a number of situations in which mammals are indirectly detected by means of vocalizations, broken branches, the rustling of shrubs, paths and feces found in the forest floor debris, etc. Sightings will be considered as indirect on occasions in which the researcher and technician identify such traces and therefore conclude the presence of an identifiable species, providing that they know exactly where the first animal presence indication was observed, so that distances, the perpendicular position and the sighting can be measured. It is still necessary to record the gender when it’s unclear as to what sort of species has been sighted, making it possible to calculate density for that specific gender.
Sampling unit: A distance of 10 km, or total km traveled.
Sampling design: The sampling effort to be employed will be a total of 300 km of covered trail, a total of 150 km for each climatic season (dry and rainy). Three trails will be used, each one parallel to the grid. These trails will be of 5 km in length and will be separated by a distance of 1 km, one from the other. Each pair of census takers (researcher and field technician) will cover one trail per day, a total transect of 10 km being covered per day, corresponding to an outward (morning) and return (afternoon) trip during the same day. A sampling force of 50 census kilometers for each one of the six transects will be used per climatic season, totalling a distance of 25 km during the morning and 25 km in the afternoon.
Sampling will be carried out by a pair of census takers who will visit the trails during a fixed sequence. After all of the lines have been visited for the first time, the same sequence must then be repeated until all of the lines have been visited five times each. In this way, the gap between two visits along the same line will remain fixed and will total two days at the minimum. Such criteria are needed in order to increase the randomness of the recorded information, in addition to promoting the return of mammals that might have been scared away from the trails during previous censuses.
In the event that there is more than one pair of census takers, it is suggested that the pairs change lines during each sequence of visits, i.e. if censuses are to be taken during the visit on lines A, C and E - during the second visit, lines B, D and F must be censored. This increases the time interval that each census taker requires for repeating the census within a specific line, helping to avoid memorization of locations along the trails where animals may have been previously sighted.
Data analysis: The relative abundance of all the recorded species will be estimated and then expressed as - rate of sighting / 10 km traveled. For species that have been sighted more than 20 times, absolute density will be estimated based on two different methods, based on the number of sightings recorded for each species within the grid.
For species that have been sighted more than 40 times, the calculation of population density will be based on the Fourier series expansion, or other types of functions (Buckland et al., 1993). For species with a total of 20 to 39 sightings, the population density calculation will be based on the definition of the reliable visibility distance per species, following the Kelker method (reliable perpendicular distance), requiring a minimum of 20 (twenty) sightings. For this particular method, the width of the reliable range (2w) is defined by analyzing the general sighting frequency of each species in accordance with the animal-trail (x) distances. The (w) value is estimated by plotting the frequencies and identifying the distance at which there is a well-defined decrease in the frequency of sightings, i.e. the distance at which the probability of sightings reduces significantly.
The formula is as follows: D = n / 2Lw
Where D is the absolute density, i.e. the number of individuals/km2;
‘n’ is the number of sightings within the trail’s distance range, established as animal/reliable range (but discounting all sightings that are not within the reliable range);
‘L’ is the length of the transect, and ‘w’ is the trail’s distance range, defined as the animal/reliable range.
Technique 2. Footprint traps
A total of 104 footprint traps, comprising of fine sand will be set out in accordance with the methodology proposed by Pardini (2003), as well as a few modifications. Three types of bait will be used in the following sequence: control (without bait), scented banana bait, and sardines for felines. The 2m2 traps (1 x 2 m) will be set out at 200 m intervals along four of the six transects, in an east-west direction. A total of 26 footprint traps will be primed per transect, i.e. a total 104 traps per expedition, totalling 208 m² of sampled area per collection day, representing around 3120 m² of sampled area per expedition in total, taking place over a period of 15 days. Each trap will have a minimum of 15, 24-hour exposure repetitions. The area to be used for the laying out the trap must be cleared, removing about 2 to 3 cm of the substrate, carefully filling the area with finely sieved sand (not compacted) in order to improve the quality of the footprint impressions left by small animals. The traps will be visited on a daily basis, the footprints then being photographed to scale and then measured, with some of the prints being recorded using reverse plaster moulds. The footprints will be carefully smoothed over after the procedure has finished. The olfactory bait will be replaced every three days, sometimes less often during rainy periods, with the food bait needing to be replaced every day, as necessary. These trails will be exclusively used for this methodology during each campaign.
Sampling unit: a trap, exposed for a period of 24 hours.
Technique 3. Photographic devices
Collection method: Photographic devices can be used for the detection of mammals. The cameras (devices) will be visited on a periodic basis, the collected images then being transferred and batteries changed.
Sampling design: Nine cameras will be set out in a semi-regular design, closely positioned at the corners of the trails of each grid. The cameras can be set out in any position, within a radius of 300 m, from the corner serving as the reference point for the sampling area. The position of each of the cameras may be changed during each inspection and visit. The placement area for each camera will be chosen based on the best available quality wildlife corridors within each sampling radius. The time taken between each camera inspection has no bearing on the sampling design, but is relevant to the total sampling time. The overall intention is to carry out a minimum of three months sampling in each of the corners. Another possibility is to reduce this time in half by doubling the number of cameras. Assuming more cameras are chosen, the devices need to be set out in accordance with the already used procedure, maintaining a regular sampling design by distributing inner circles in which the cameras can then be placed.
Technique 4. Pitfall traps with drift fence
Standardized sampling, appropriate for the sampling of anura, reptiles, mammals, crustaceans, spiders, beetles, Orthoptera (grasshoppers and crickets), in addition to other types of forest floor animals. This technique provides quantitative data for comparison between different areas, in which the number and the size of the collection buckets, the distance between them and the standard trap assembly processes are explained. Four 60 liter buckets will be used at each sampling point, laid out in the form of a “Y”, in which the central bucket will be 10 m away from the three peripheral buckets. The peripheral buckets will be connected to the central bucket by means of plastic sheet fencing of 50 cm in height, crossing the centerline of each bucket.
The buckets must be visited on a daily basis during the sampling period so that the trapped animals can be collected. The buckets may be left out in the field after the sampling period has expired, with branches being placed into the openings so that any fauna falling into the buckets can get out. Due to the efficiency of this method not being restricted to a specific group of fauna, contact with other protocol teams should ideally be sought in order to make full use of all captured animals.
Sampling design: The samples will be linked to 15 of the 30 plots within the PPBio grid, each of these incorporating two “Ys”, each consisting of a set of four buckets. Fifteen plots will be used, incorporating the use of 30 “Ys” and 120 buckets. The buckets will then be sampled for 15 consecutive days, totalling 1800 samples. The plots to be sampled will be distributed across three parallel trails, two kilometers apart. Two “Ys” will be placed in each of the plots, with 10 meters separating each of the buckets.
Two points will be marked out along the grid trail, 50 meters away from the point marking out the beginning of the plot, with both points on opposite sides.
A 10 meter line will then be extended out from both of these points, perpendicular with the grid's trail, then placing the first bucket belonging to the “Y” design. The other buckets will be laid out in the form of a “Y”, avoiding contact with the trail itself. The “Ys" should preferably be laid out on the same side of the trail as the plot itself, but still being possible to use the other side of the trail, if necessary. The same rule applies to the “Y” itself, being placed and rotated in such a way so that it avoids any obstacles found within the environment. One of the factors that must be considered during the positioning of the "Y" is whether the design will enter the limited access regions of the plot, this sometimes happening if the plot is very close to the trail running along the grid. The plot layout map can be used to avoid this. This type of design is shared with the herpetofauna protocol (protocol 10), signifying that the utilized field effort may be shared between both protocol teams.
Sampling unit: A bucket, although the complete “Y” must be used as the sampling unit for the majority of analyses,
Technique 5. Capture using mist nets
Sampling design: A line of 10 mist nets, measuring 12 x 2 m, will be set out over a single hectare within each of the 30 plots, demarcated by a period of 2 days. The nets will be opened at dusk, around 6 p.m., and closed at 1 a.m., the nets then being inspected for the removal of bats at intervals of around 20 minutes. The captured bats will be placed in individual cloth bags so that they can be inspected and screened, afterwards being released at the end of the collection session, except for the specimens selected as part of the reference collection or those where identification is particularly difficult, accumulating the total of necessary hours/net within each sub-plot. Between three and four observers, working every two days within the five, one hectare sub-plots, set out along the same trail, will facilitate the monitoring of a total of 50 mist nets on a daily basis. One day will remain free during the sampling of the two sets of five, single hectare plots, so that the 50 mist nets can be taken down and repositioned. The logistics for this type of methodology will therefore require 3 to 4 exclusive observers for the sampling process itself, as well as a total of 50 mist nets; this being the operational scheme that best utilizes human resources and mist nets in terms of the number of days for sampling bats (n = 18). The points sampled during the rainy season must be repeated during the dry season of the same year.
Technique 6. Capture by active search for shelters
Even though the use of mist nets is very popular for the study of bats, various authors have demonstrated the need to use other, supplementary techniques, due to the difficulty in capturing bats using nets at ground level. In order to supplement the data obtained using mist nets, the shelter capture technique may also be used.
This procedure is adapted from the work of Simmons and Voss (1998). A route of 800 m in length is set out within a 100 x 100 m (1 hectare) sub-plot, containing 5 longitudinal trails spaced at every 20 m (Figure 1a). Five sub-plots of 100 x 100 m will be established along the 5 km trail previously set out for capture using mist nets, each one of these being a square of 1 x 1 km. Two technicians will walk along the 8 lines of 100m each, identifying and marking out any shelters found at a distance of up to 10 meters from the guideline. Afterwards, the shelters will be revisited by 3 collectors who will then capture the bats using appropriate equipment, e.g. through the use of ladders and nets, including mist nets for surrounding the shelter.
Figure 1. a) Sub-plot of 100 x 100 m, outlining the 800 m route, shown by arrows; b) PPBio 5 x 5 km grid, showing the 5km perpendicular trails.
Supplementary data: The list of species present in each area can be supplemented with information supplied by local communities, obtained by means of semi-structured interviews. Traces found along the trails such as footprints, feces and gouge marks will be photographed in scale and collected where possible using reverse plaster moulds, or in the particular case of feces, using the methodology described for carnivores.
Collection of zoological material: The collection of medium and large size mammals using firearms, both during the day and at night, will only take place after the 300 km census sampling effort of the third campaign has come to an end. The collection of certain species (mainly primates) will be aided through the use of decoy calls (“playback”), helping to reduce the random nature of the searches. The collection of small mammals will take place throughout the standard capture method sampling effort. In accordance with IBAMA’s rules of collection, a maximum of six (06) specimens from each species will be collected within each grid. The collected specimens will be deposited in trustworthy Amazon depository collections.
Collection of genetic material: The collected mammals will also be used for basic genetic studies at the molecular biology laboratories of MPEG or UFPA, in situations in which the Institution responsible for the grid does not have its own Genetic research group. In the event of multiple specimens of the same species being collected, the additional material may be sent to the research centers of interested partner Institutions. The liver, as well as a number of collected muscle samples, will be fixed in absolute ethanol and then stored in dark plastic tubes, afterwards being preserved at an average temperature of 4°C.
Utilization of secondary data: A systematic analysis of already available records within literature and scientific collections will be carried out in order to compile a valid knowledgebase on mammal diversity within the areas being studied.
Environmental variables: Variables such as air humidity, air temperature (minimum, average and maximum), rainfall and soil classification, may be used, as appropriate and depending on availability, for each one hectare plot.
Restrictions on activities that could harm protocol development: Samples must be inventoried with the concomitant presence of personnel who work with other types of taxonomic groups, in order to ensure the successful collection of bat samples within the grid.
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