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On the tracks of a new species

Method of chromossomal differentiation makes it possible to identify, in the Atlantic Rain Forest of the south of São Paulo, a kind of Brazilian deer as yet unknown - but already threatened with extinction

The people from the zoo in Sorocaba are not used to having problems in identifying animals, but, at the beginning of the 90s, there arrived a member of the Cervidae family that took them all by surprise: some thought it was, without a doubt, a red brocket (Mazama americana); for others, it had all the looks of a lesser or dwarf brocket (Mazama nana). To resolve the impasse, they called a veterinary surgeon, José Maurício Barbanti Duarte, who teaches at the São Paulo State University (Unesp) in Jaboticabal, who at that time was already going all over Brazil researching deers. He noticed that the animal had the same patches as the red brocket under the tail and the neck and between its legs. But its weight was halfway between this species and the lesser brocket. Duarte did not know what to say.

It was only some years later that the researcher from Unesp settled on a conclusion: the animal from Sorocaba was no less than a new species of deer, which lives in a limited area of the Atlantic Rain Forest in the south of the state and the northeast of Paraná, and was baptized in Portuguese as veado-bororó de São Paulo, which is something like the lesser brocket of São Paulo (Mazana Bororo). This work by Duarte gives weight to the importance of studying the set of chromosomes (the karyotype) to differentiate species hitherto regarded as one and the same. At the same time, he proposes a more accurate study of the mechanisms for the evolution of animals. In the case of these brockets, he thinks that there has been a rearrangement or packaging of chromosomes, leading to compression of the karyotype and favoring the conservation of the species.

The differences
Duarte was only able to nurture the suspicion that he had found in Sorocaba a species as yet unrecorded, when he compared its karyotype with those of the red brocket and the lesser brocket. The number andthe arrangement of the chromosomes – the structure of cells that contains the DNA (deoxyribonucleic acid, which carries the genetic code) – were quite distinct, much more so than he could imagine. The red brocket has 54 chromosomes, most of them acrocentric – the centromere, the litle knot that links the twoparts of the chromosome, is at one of the extremities. The lesser brocket has 38 chromosomes, of which 20 are metacentric – with the centromere right in the middle.

The one from the zoo that had raised the doubt originally has 36 chromosomes, close to the lesser brocket indeed, but with a very different structure: only two are metacentric and eight are submetacentric, with the centromere closer to one of the extremities, a characteristic that reflects an evolutionary history of its own. “The differences are glaring, they couldn’t be the result of crossing different species”, was the researcher’s finding.

Nor did he think it could be a chromosomal aberration or a variation within the same species, such as happens with the red brocket – in which the number of chromosomes varies from 44 to 54, but the general pattern of the karyotype does not vary. That is why he asserts: should they manage to cross, these animals would produce sterile offspring – hybrids, like the mule, the product of crossing a he-ass and a mare, which has only one different pair of chromosomes.But how to explain that the three animals being studied are so similar outwardly? “This similarity is a chance outcome of the evolutionary process”, says the researcher.

New proofs
But one animal on its own does not make a species. Duarte worked in silence, and ended up finding another three specimens – a couple with its kid – free in the back yard of a house of a breeder in Barra do Turvo, on the border with Paraná, and they all had the same chromosomal pattern as the animal from the zoo in Sorocaba. Afterwards, he examined another three specimens – two in Maringá, Paraná, and another in Passo Fundo, Rio Grande do Sul -, but he came to the conclusion that they were hybrids, the sterile offspring of the mating of a red brocket with the lesser brocket. From the size, the color of the fur and the patches, they were at first confused with the supposed new species. The difference is that the hybrids had more chromosomes: 46, the average of the number of chromosomes of its parents.

Still not enough. On June 15th last year, Duarte found stronger evidence: he and his team of three biologists and veterinarians, after years of searching, captured a specimen of the new species that was living freely in the State Park of Intervales, a reservation in the Atlantic Rain Forest in the southwest of the state. It is remarkable that the discovery is now being made of a new species of a mammal of some size – especially in the most populous Brazilian state, where the natural landscape has been much modified by cities, farming and pastures. An expedition recently found new species of rodents, but in the east of Tocantins, an area little studied. Even so, Duarte’s team knew well what they were getting into: they would still have to collect more evidence.

Linnaeus questioned
The first complication: the deer of the Mazama genus, to which the red brocket and the lesser brocket belong, live in thick forest and are extremely jumpy. For this reason, there is still no estimate of the populations of either of them, as there is with better known deer. Brazil, for example, has about 130,000 specimens ofthe pampas deer or campeiro deer (Ozotocerus bezoarticus) and 40,000 of the marsh deer (Blastocerus dichotomus). Both live in open spaces – in the cerrado (type of wooded savanna) or in meadows – and they can be counted and studied from low-flying aircraft. Furthermore, the comparison of karyotypes is not a universally accepted method. A kind of acid test, it is used a lot to differentiate animals that are very similar morphologically, above all reptiles, rodents and, more recently, primates like the cebids.

Put forward by the Swedish naturalist Linneaus – Carl von Linné (1707-1778) -, the method of classification (or taxonomy) most accepted takes into account the external characteristics (morphology). Duarte has some restrictions to make on this method, which differentiates the species by physical features like the shape and the size of the paws, the kind of fur, among others. “In the Mazama genus, the morphological differences are subtle, compared with the karyotypical ones”, he says. An adult red brocket weighs about 30 kilograms (kg), and a small red brocket some 25 kg, although both have the same patches and the same color of fur, whilst the lesser brocket weighs between 13 and 15 kilos and has no patches.

Merger of chromosomes
Duarte’s hypothesis for the differences in karyotype in the Mazama genus is that chromosomes have merged. The brown brocket (Mazama gouazoubira) has therefore kept its ancestral 70 chromosome karyotype, all of them small and with the X- knot at the end. Then the small red brocket has the more compact version, with a larger reduction in the number of chromosomes. Duarte thinks that karyotypes with larger and less numerous chromosomes are more stable and lessen the possibility of combinations that lead to diseases and the loss of individuals.

The most surprising case of the merger of chromosomes is to be found in a genus of Cervidae in Asia, the muntjac, which lives in regions of creeping vegetation between Malaysia and the Himalayas. One species with 46 chromosomes, the Muntjacus reevesi, is, external looks, absolutely the same as another, with just six chromosomes (in the female), the Muntjacus muntiac. The mechanisms by which the chromosomes of the first species merged to arrive at the second, without losing genetic information, are already known. It is estimated that each merger may take about 300 years to fix itself permanently in a population.

A species may also arise through mutations – genetic differentiation -, while the karyotype remains stable. Look at the case of the marsh deer and the pampas deer, totally distinct, but with karyotypes that differ by only one pair of chromosomes: the marsh deer has 66 chromosomes and weighs between 100 and 150 kg, while the pampas deer, of another genus, has the same chromosomes plus two, and rarely goes beyond 40 kg.

In July, Duarte intends to go back to Intervales, for further study of the new found deer. His dream is to have specimens donated by people who rear them in captivity, since it is forbidden to take animals out of the reservations, and to start a conservation program in captivity. He may then be able to enrich the evidence of reproductive potential and to arrive at a definitive finding: to get fertile offspring from the species and infertile ones from crossings with the others.

Frozen material
With an eye to the future, Duarte keeps 150 samples of the semen of two males in liquid nitrogen, which may make assisted reproduction possible: “The characteristics of this material are excellent, from the point of view of the morphology and motility of the spermatozoids”, he says. He is also keeping in the laboratory samples of blood and skin of 850 deer – material that may be valuable for research into cloning, as they are diploid cells (with the genetic material of the father and of the mother).

After such a struggle to locate and capture animals, the Unesp team is also working with material that is still little appreciated, feces, but which has proved to be a rich source of biological information. “With their feces”, Duarte points out, “we can assess the animals from the genetic, endocrinological, health and nutritional points of view, without touching them”.

Studying the droppings of deer, the Unesp group discovered how the levels of testosterone (the male hormone) determine sexual development. “When the testosterone level is low, the antlers of the pampas deer fall off “, says the researcher. “When it is high, the antler is unsheathed and the male is ready to fight over the possession of the females.” Cells from plants excreted in the feces can indicate what the diet consisted of, in the same way that microorganisms makes it possible to assess the state of health.

But there is cause for lament: all the specimens of the new species that he has studied have already died. “As far as I know, no zoo in São Paulo has received any specimen of this species since 1992”, he comments. The difficulty of finding specimens is increased by the finding that the new small red brocket should be treated as something very rare and is threatened with extinction – not least because it lives in a limited area of the Atlantic Rain Forest.

Seven red brockets?
His bet is that the study of the karyotypes may result in new discoveries, ruling out the possibilities of variations within the same species. Duarte maintains that there may be seven species of the red brocket. “We have already established some differences in karyotypes between populations from the Paraná Basin, Rondônia, Acre, central Amazonia, northern Amazonia and Pará”, he says. That is where the danger may increase for species like this one, where the populations seemed sufficient to ensure their conservation. According to him, a probable new species of the red brocket lives exclusively in the Iguazú National Park, on the border between Brazil, Argentina and Paraguay.

Setting a trap for the animal

To find one or more animals to characterize the existence of a new species of deer, the researchers from Unesp prepared lures (bait) with fruit and peanut paste, and scattered them over the State Park of Intervales, which showed most signs of the occurrence of the species. This attracted the tapir (Tapirus terrestris), the paca (Agouti paca) and the tayra (Eyra barbara) – an animal with a long body that is reminiscent of the otter. Not a single deer. Another attempt: attracting them with urine and feces of animals held in captivity. That did not work either. They even tried bits of wood with the secretion from the glands at the base of the antler or vaginal secretions. To no avail.

With time, the researchers found deer tracks or trails. Then, using hides with cameras, they discovered they feed on the fruit of the guava tree (Eugenia sp.). Back they went, months later, and perched themselves in the treetops for five nights in a row, with a weapon called a netgun, which fires a net. “It is not easy to spend the night at the top of a tree, without talking, drinking or eating anything, and in a temperature of 2°C”, Duarte admits. Even so, without success. Afterwards, they imagined that the smell of human beings could be frightening away animals that are already jumpy. Then came the last resort: impregnating their clothes with feces and urine from the animals held in captivity. It was useless.

Some time later, they tried enclosing with bamboo fencing the trail that the animal followed, forming a corral where it could be trapped during its walks in the forest. Then, alleluia! They managed to capture a 24 kg male, now characterized as belonging to the new species.

They put a radio collar on him and monitored him for weeks. They discovered how jumpy he was: he would only leave his hiding places at night, and the walks would always include stretches of river – a way of leaving no trail of scent to attract predators. “He is a specialist in escaping”, Duarte recognizes. The end was desolating: two months later, they found the animal they had captured dead. It had been partly devoured by a puma (Puma concolor), identified by the footprints it left.

The Projects
Biological Study of the Mazama bororo species of the state of São Paulo (nº 96/10246-0); Modality Regular line of benefits for research;
Coordinator José Maurício Barbanti Duarte – Unesp in Jaboticabal; Investment R$ 54.635,2
2. Monitoring the Annual Reproductive Activity of of Red Brocket (Ozotoceros bezoarticus) Males Living Freely (nº 00/04493-1); Modality Regular line of benefits for research; Coordinator José Maurício Barbanti Duarte – Unesp in Jaboticabal; Investment R$ 32.083,00