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The origin of the species

Study of fish that live in caves reveals the mechanisms of progressive differentiation that register their evolution

MARIA ELINA BICHUETTE / USPLapa do Angélica, one of the São Domingos caves in Goiás: environment preserves genetic mutationsMARIA ELINA BICHUETTE / USP

Permanent darkness and scarcity of food lie at the base of the transformations that differentiate cave fish from their relatives outside. Discovering how these transformations occur and from what point they constitute a new species was the challenge for a team from the Institute of Biosciences of the University of São Paulo (IB-USP), coordinated by Eleonora Trajano, who researched caves in Goiás. This work, which helps to understand the role of the environment in the formation of new species, has already borne fruit in other places: thanks to it, the blind catfish of the Ribeira valley were declared as being threatened with extinction, and the Poço Encantado (Enchanted Well) in the Chapada Diamantina, Bahia, has been closed for diving.

The scenario for the research is the State Park of Terra Ronca, located in the municipality of São Domingos, northeast of Goiás, in the region of the upper Tocantins river. There, the waters have sculptured in the limestone about 30 caves, at least five of them over 5 kilometers in length. The area boasts the richest fauna in troglobite (cave living) fish in Brazil: of the 14 species recorded in the country, five are to be found in this region. There are dry caves in the park, some with rock paintings, but those that interest the team from USP are those that are crossed by rivers, such as Angélica, Bezerra, Passa Três and São Vicente.

“Our struggle is for the preservation of this sanctuary in Goiás”, Eleonora emphasizes. The State Park of Terra Ronca was listed by Unesco as World Heritage and the area is being expropriated and demarcated, but due to the lack of funds for the purchase of the land, the sources of the rivers remained outside the delimited space. “If the sources are damaged by forest clearing, forest fires and pollution, all the life in the place will be threatened”, the biologist warns.

The study includes a survey of the biodiversity of the caves and their surroundings, and the characterization of the species – not only of the fish. The group is investigating molecular biology, morphology, ecology and animal behavior: “Very often”, the researcherobserves, “molecular biology tells us that two animals belong to the same species, while the morphological and behavioral analyses clearly indicate that we are dealing with different species”.

On being characterized, a fish is compared with close relatives that live outside the caves and to unrelated cave species that inhabit other regions. The method makes it possible to distinguish characteristics common to the unrelated species, which show converging responses to the same environment – in this case, to the subterranean surroundings.

Function and regression
According to the researchers, when the exchange of genes is interrupted between the populations of cave-dwelling fish and those from the outside world (called epigaeous), the successive generations in the caves undergo a progressive reduction in their visual apparatus and in their dark (melanotic) pigmentation. A reduction in bodily size may also occur and behavioral modifications, such as the loss of photophobia , of the circadian (daytime) rhythms of activity, and of the habit of hiding.

Other noteworthy modifications in the fish studied are the narrowing of the snout and a reduction in the relative size of the head.All of this is the result of genetic modifications which, under the specific conditions of the caves, are not eliminated by natural selection as they would normally be. In an illuminated environment, a fish that was born blind, for example, would have little chance of survival.

These mutations affect such features as the eyes, pigmentation and circadian rhythmicity, which lose their function in the permanently dark environment of the caves and may recede after many years of isolation in this habitat. According to Eleonora, other features, such as those involved in obtaining food, generally scarce in caves due to the absence of plants, “are selected positively, resulting in greater efficiency in locating, capturing and making good use of these foods”. In one stage of the changes, the accumulation of divergent characteristics defines a new species, exclusively subterranean or troglobite.

Borderline cases
But there are borderline forms, which conceptually represent a challenge. Maria Elina Bichuette, a member of the team, says: “There are troglobite populations that are completely blind and without pigmentation. Others show these traits in just a partial manner. They are cases where regression has not yet been completed”.

There are borderline cases in which regression seems to be at such an incipient stage that it is difficult to distinguish completely the fish from the cave from those from outside. This is what happens with the electric fish Eigenmannia vicentespelaea, of the same order as the electric eel, but which does not have such a strong discharge. Its classification as a species arouses doubts: there are no pronounced morphological differences to be seen between the subterranean species and its relatives from the epigaeous environment. The researchers observed in the cave-dwelling species not only specimens with very small eyes and no pigment, but also individuals with skin pigmentation and well developed eyes. These doubtful species are the biggest riddle they have come across so far.

The fish are seen in various situations. Some live in caves that have become illuminated as a result of landfalls, but are totally blind and without pigment – a proof that their differentiation took place beforethe opening of these wide-open contacts between the caves and the outside. Others inhabit dark caves, but are just starting to differentiate themselves. It may be a sign that they colonized the place a short time ago, but not always. “It can happen that the animal has been inhabiting the cave for a long time, though its genetic isolation is a recent fact”, says Eleonora.

“Only when they stop exchanging genes with their epigaeous relatives is it that the troglobite start to differentiate themselves”. The loss of vision, for example, progresses through four stages: reduction in the size of the eyes, with the structures preserved; disorganization of the crystalline lens and the associated structures; disorganization of the retina; and atrophy of the optic nerve.

Mysterious case
“We found troglobite catfish, from the Trichomycteridae family, with relatively well developed eyes”, reports Maria Elina. “From the macroscopic examination, we believe that they are between the first and second stage.” The reduction of the eyes, however, only tends to progress, because, according to her, there is no external population capable of contributing with genes associated with the development of sight.

If in this case it is easy to understand the dynamics of differentiation, the same cannot be said of the Ancistrus cryptophthalmus catfish, which has the possibility of contact with external populations, and which is nevertheless diverging. It is a mystery. “Perhaps we are faced by a rare case of parapatric speciation (without isolation)”, says Maria Elina. “Speciation by isolation, called allopatric, explains the characteristics of the majority of Brazilian populations, but not all of them”.

Allopatric speciation follows a known course. Initially, there would be a single species, which occurs both inside and outside cave. Next, a change in climate could eliminate the external population. The population in the cave, protected, is preserved, but, without the external population, the exchange of genes ceases. The mutations will then start differentiating these fish, and they end up forming a new species.

“On the basis of this model”, says Eleonora, “it would be to be expected that there would be more troglobite species in regions that have undergone many paleoclimatic modifications than in those that have had few of them.” The São Domingos caves confirmed this expectation for terrestrial invertebrate fauna, but belie it for the fish. The region went through some , but not very intense paleoclimatic modifications, and nevertheless, five species of troglobite fish are found there, more than in any other karstic area – formed by soluble rocks where caves develop – in Brazil.

The problem is that the allopatric model is not enough to explain this situation. Be that as it may, says the biologist, “the exceptions are of extreme importance for the development of the models, because they provide parameters. The more a model is delimited, the greater its capacity for prediction”.

Body shape
To distinguish species, the team used geometric morphometry: it recorded digitalized images of the animals by a video system hooked up to a computer, and then it applies over them a Cartesian grid, the deformation of which indicates how and by how much the figure under study differs from the standard shape. The method was applied tothe study of four populations of the Ancistrus cryptophthalmus catfish from the caves, comparing them with the epigaeous population of Ancistrus in the region.

A graph of this survey shows the gradual transformation from the epigaeous population to the one in the caves. “Starting with the principle that the epigaeous species currently found in the region is a little modified form of the ancestor of the troglobite populations, one may conclude that, beginning with an ancestor with a large head and short body, a broad snout and big eyes, populations originated that gradually underwent a reduction in the size of the eyes and in pigmentation, a narrowing of the head and a relative increase in the body”, sums up Eleonora.

Another part of the research project implies catching, marking and returning each animal to its habitat – tasks that are not easy with dark surroundings, small fish, and rivers that disappear into cracks. This work is fundamental for an assessment of the environmental impacts, identifying the species that are potentially threatened, and for drawing up strategies for conservation. “The troglobites in general”, says Eleonora, “show a long life cycle, coupled with low fecundity and delayed maturity”.

This results in a slow renewal of the population. Consequently, population losses caused by perturbations in the environment are difficult to replace, which makes troglobite organisms particularly susceptible to modifications in the environment.

The Project
Study of Brazilian Subterranean Fish: Ecology and Behavior of Ancistrus cryptophthalmus, Cave-dwelling catfish from the Region of São Domingos, Goiás (nº 99/00376-1); Modality Regular Line of Research Benefits; Coordinator Eleonora Trajano – Institute of Biology of USP; Investment R$ 41,120.98 and US$ 1,082.87