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ZOOLOGY

Dogs working in the forest

Dogs help identify deer habitat areas by tracking the scent of droppings for DNA extraction

Brown brocket deer: monitored through tagging and a radio transmitter

Eduardo CesarBrown brocket deer: monitored through tagging and a radio transmitterEduardo Cesar

Granada the dog has already found over 1,800 deer dropping specimens, but if she were to see one of these animals in nature, she probably wouldn’t know what it is. Although she has contributed to research on species including the Brazilian dwarf brocket (Mazama nana), the red brocket (M. americana), the small red brocket (M. bororo) and the brown brocket (M. gouazoubira)—four of the eight deer species catalogued in Brazil—Granada has but one goal: to locate the scent for which she was trained, thereby winning praise in the form of affection and a tennis ball from the members of the Deer Research and Conservation Center (NUPECCE) at São Paulo State University (Unesp) in Jaboticabal. Theirs is the only Brazilian research group focused on the study of these animals.

During the dog’s most recent outing with researchers led by Professor José Maurício Barbanti Duarte of the School of Agricultural and Veterinary Sciences at Unesp, they explored 46 protected areas of southern and southeastern Brazil. Owing in part to the droppings found by Granada and her predecessor Apolo, the researchers were able to define the area of occurrence of the four species in the Atlantic Forest by identifying deer DNA in 555 fecal specimens collected at 30 of those locations. They discovered that the small red brocket, a species described in 1996 by Duarte (see Pesquisa FAPESP Issue nº 65) and still little studied, is closely associated with the 37,517 square kilometers of dense forest in the rainy region along the Brazilian coast, which shows its dependence on a limited vegetation type and climate for species survival, according to an as-yet-unpublished paper. “It is Brazil’s largest endemic animal, and possibly the one with the smallest geographic distribution among all neotropical deer described to date,” Duarte says.

The use of sniffer dogs is one of the NUPECCE researcher and group leader’s greatest triumphs. He has been using dogs in field collections since 2002. Before Granada, a mixed breed, the searches were done by Apolo, a Springer Spaniel, who died in 2007 at the age of 12. Sniffer dogs are used in research to locate droppings of maned wolves, jaguars, cougars and anteaters in Brazil, as well as foxes and even whales, in other countries—in the latter case, aboard a ship whose pilot follows the signals of a canine partner to steer towards floating feces.

Without the dogs, we would probably know even less about Brazilian deer, whose sharp hearing and keen sense of smell cause them to jump and run at the slightest sign of danger. “It does no good to stake them out; they pick up our scent,” says Duarte, who has been in the habit of going into the forest in search of these mammals—usually without success—since he finished his undergraduate studies. “In a research project on the small red brocket deer, we captured just three individuals in four years of searching,” he comments.

060-063_Cervos_235Added to the difficulty in finding them is the fact that species of the genus Mazama are cryptic, that is, they look so similar that one can only distinguish the difference between them with any certainty through DNA testing. Samples of blood and other tissue are ideal for extracting the genetic material of any animal, but since deer are rarely captured in nature, the researchers’ only other option has been to use droppings. In 2013 they showed this to be a feasible method for research on deer, in a paper published in Genetics and Molecular Research.

And that involves Granada, who was trained by the police corps. The training is similar to that used for drug-sniffing dogs at airports, but with a different target scent. “When looking for drugs, the dogs are on a leash and have only a small area to search, such as a room or a few suitcases. Here, we have forests or entire fields,” says Márcio Leite de Oliveira, a Ph.D. student at the Luiz de Queiroz College of Agriculture at the University of São Paulo (ESALQ-USP) who is doing research at NUPECCE.

Part two of the dogs’ training, therefore, is done with the researchers themselves, walking through the forest off leash. After about a week of going into the field every day, the animals begin to learn what they are supposed to do. In a month, they are already helpful to the researchers. “When we’re tracking, the dog searches for droppings in a radius around us, and this greatly expands the search area,” explains Pedro Peres, a researcher at NUPECCE. “She can find a single coprolite underneath vegetation without even seeing it,” he says, using the term that refers to pellet-shaped droppings.

Even after droppings are collected, however, problems remain. While DNA extraction from blood and tissue is a well-established technique, the same cannot be said about fecal matter. One of the difficulties lies in obtaining enough DNA from the samples, because amounts are limited, and it degrades rapidly in that type of material. “Enzymes and microorganisms degrade the DNA as soon as the droppings hit the ground,” Duarte explains. To add to the problem, each sample contains not only the genetic material of the animal that produced it, but also that of the plants it ate and the bacteria that were present in its digestive system and in the soil. “Any hair from another animal that was there, or even from the person who collected it, can contaminate the sample,” he points out.

Therefore, in addition to searching for fresh samples in which the DNA is less degraded, the researchers need to separate out what is actually the genetic material of the deer. To do just that, in 2002 Duarte’s team created the first genetic markers specific to the genus Mazama, which enabled them to differentiate the genus and its five species in any sample.

Once they find the droppings, mark the coordinates on the GPS and identify the species found there, the NUPECCE researchers have a valuable set of data on the deer’s area of occurrence. In addition, they can link the animals to the environment where they are found (vegetation type, humidity, temperature). The compiled datasets yield an estimate of where the animals are found and what their habitat is—essential information on which to base conservation policies such as the designation of protected areas.

Granada: rewarded with a ball and affection when she finds deer droppings

Eduardo CesarGranada: rewarded with a ball and affection when she finds deer droppingsEduardo Cesar

On the trail of deer
Some of the data from the most recent paper, on Atlantic Forest species, is the product of the doctoral research being carried out by Oliveira, who is defining high-priority areas for conservation of the red brocket and the Brazilian dwarf brocket and is expected to defend his dissertation in the next few months. “By learning where they are located and which vegetation type they live on, we can provide data that will enable these areas to be protected,” he says. Brazilian deer are endangered mainly through habitat loss, but livestock-transmitted diseases, hunting and predation by dogs are also factors.

Encounters between wildlife and dogs are common at the boundaries between conservation units and human-occupied areas. Even when alone, dogs can detect deer and pursue them for hours. Deer are known for their agility and quick reaction, which are ideal for fleeing jaguars (Panthera onca) and cougars (Puma concolor), but not dogs, which can run for long distances. Even when they are not caught, deer can die from capture myopathy, a syndrome caused by intense physical activity during flight. Since Granada has been trained to locate only droppings, she doesn’t pose a threat to deer. Another consequence of contact with human-occupied areas is transmission of bovine diseases such as bluetongue and epizootic hemorrhagic disease, which do not kill cattle but cause fatal hemorrhaging in deer.

The research on fecal DNA is producing data that could change the very definition of species that live on Brazilian soil. “We already have evidence to propose a division of the genus Mazama, which is present from Mexico to Argentina,” Duarte notes. He says that at least two genera will probably be created to accommodate the current Mazama nemorivaga, known as the Amazonian brown brocket, and M. gouazoubira, the brown brocket. “We may get descriptions for another four or five new cryptic species of animals that are already known but classified as other species,” he says. In order to get nationwide coverage and the number of individuals necessary for the reorganization, droppings may be the only option.

The researchers are also doing an extensive historical review of the literature and are gathering DNA samples from material in natural history museums to genetically confirm what was described through morphology. Some of the work is being done by Aline Mantellatto, a doctoral candidate who obtained bone fragments from about 400 deer skeletons of the genus Mazama from the Atlantic Forest that are held in the collections of 10 Brazilian museums and at the Natural History Museum in London.

Detective work
Half of the fragments have now been classified, and 20 of them have been shown to belong to Mazama bororo but were identified in the museums as other species. Mantellatto also discovered that the species described by Duarte in 1996 had already been recorded in 1901 as a subspecies, M. americana jacunda. If this finding is confirmed, M. bororo will likely get its name changed to Mazama jacunda.

The field and laboratory work done by the researchers is receiving additional support from an arboretum at NUPECCE, where 58 individuals from seven species that occur in Brazil serve as a genetic repository and enable research studies without the need to capture wild animals. This living collection can help researchers review the classification of these animals, though with some limitations. The very species that was described by Duarte, for example, is not represented there due to the difficulty of finding them in nature.

Also hard to find, at least in the state of São Paulo, is the Pampas deer (Ozotoceros bezoarticus). If the species is not sighted in the next few years, it may become classified as regionally extinct. At Santa Barbara Ecological Station, where the state’s last known population of the species lived, Peres recently collected 73 fecal samples with Granada’s help—all of them from brown brocket deer. The Pampas deer may no longer be present in that area. During a break-in in early August 2015, the only female of the species in captivity in Brazil disappeared at NUPECCE, where researchers had hoped to begin a breeding program.

In an effort to keep the marsh deer (Blastocerus dichotomus) from suffering the same fate, the group at Jaboticabal has successfully produced embryos from a captive population in order to implant them in a wild female. The animal was captured in the wild and brought to NUPECCE, where she will receive an embryo following hormonal treatment. After impregnation, the female will be returned to the original location to give birth and raise the surrogate’s fawn in the wild. The phases of the pregnancy will also be monitored through droppings, since the researchers have developed a method that now enables them to recognize the different hormones excreted during pregnancy, as shown in a 2012 paper in Animal Reproduction Science. The idea is to increase genetic diversity and the breeding chances for this species, which is vulnerable to extinction, largely due to flooding of its habitat by hydroelectric dams. The operation even involved a helicopter to locate and capture the future mother in the wild. This time, Granada stayed home.

Project
The gray brockets in Brazil (Mammalia; Cervidae; Mazama): detection of the genetic, morphologic and ecologic variants to explain the complex evolution in this group (nº 2010/50748-3); Grant Mechanism Thematic project; Principal Investigator José Maurício Barbanti Duarte (FCAV-Unesp); Investment R$808,564.00.

Scientific articles
OLIVEIRA, M. L. & DUARTE, J. M. B. Amplifiability of mitochondrial, microsatellite and amelogenin DNA loci from fecal samples of red brocket deer Mazama americana (Cetartiodactyla, Cervidae). Genetics and Molecular Research. V. 12, No. 1, p. 44-52, January 16, 2013.
KREPSCHI, V. G. et al. Fecal progestins during pregnancy and postpartum periods of captive red brocket deer (Mazama americana). Animal Reproduction Science. V. 137, No. 1-2, p. 62. February 2013.

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