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Biology

Music in the marshes

Frogs, toads and tree frogs exhibit 70 calls and 29 of sexual reproduction

CÉLIO HADDAD/UNESPHypsiboas bischoffi, a tree toad the Atlantic RainforestCÉLIO HADDAD/UNESP

At nightfall the “frog army” awaits at the edge of the lagoon. One sound breaks the silence, “croak, croak, croak”. Leptodactylus notoaktites, says Célio Haddad. This is the Iporanga’s white-lipped frog. Little by little, more and more croaks: deep, sharp, in chorus, solo, trills, metallic beats, from various points of the lagoon or from the surrounding vegetation. Haddad accompanies: Hypsiboas faber, Dendropsophus minutus… “We have to find the Phyllomedusa“, he beacons. They are green tree toads with red or orange hind legs stripped with purple, and they remain in the vegetation close to the water. The croak sounds like a stomach growling.

Haddad, from the São Paulo State University (Unesp) of Rio Claro, is coordinating a project on the mapping of various anuran amphibians (frogs, toads and tree toads) in the State of São Paulo. The Atlantic Rainforest houses a huge abundance of anuran amphibians, of diverse sizes, colors and sounds. They are green, nut brown, golden, the pajama hyla has stripes and spots, which vary from one individual to the next, and the horned leaf-frog appears like a dry leaf. As well as this, the diversity involves dozens of reproductive strategies, life cycles, chemical compounds, states of conservation, etc. In order to study this immense multitude, professor Haddad is working with students and collaborators who together are attempting to unmask the natural riches of this Brazilian forest, one of the regions with the highest biodiversity in the world, and which is seriously threatened by the advance of human occupation.

To explore the forest
Part of the work involves walking through the forest and then waiting at the edge of marshes. Listening with attention reveals the species in reproductive activity, since the males croak in order to attract the females. Experienced researchers listen to the cacophony of a lagoon and quickly recognize the members of the choir. In order to guard and transmit this knowledge, one needs to record the croaks of the representatives of the studied species. These recordings are stored in a sonorous collection and give origin to graphic representations, the sonograms, which allow for differentiating the sounds with more detail than human hearing can capture. The description of a species, nevertheless, includes data about its appearance, its genetic make up and its croak as well.

As part of the project, professor Haddad, along with three of his students, has produced a CD with samples of the croaking of 70 species of frogs, toads and tree toads from the Atlantic Rainforest. This sonorous guide brings a croak in each range, which in the accompanying notebook corresponds to its scientific and common name, as well as a photo and information about the animal’s habitat. In the opinion of professor Haddad, “the guide has a lot of worth for researchers, but also for laymen and could be used for teaching”. Now anyone can go out into the wild night or listen to the tree toads that lives in the shower room and attempt to discover its identity.

CÉLIO HADDAD/UNESPPajama hyla (Hypsiboas latistriatus), from the town of Campos do JordãoCÉLIO HADDAD/UNESP

On encountering frogs in the vegetation there is innumerable information to collect. Observing them, to see where the male croaks, how the female reacts, how they mate, what they do with their eggs and much more. Record their calling. Capture them. And it is important to photograph them, preferentially in the wild; in this manner preserving information about their colors and shape as well as the environment in which they live.

One of the consequences of so much observation was a revision of the reproductive modes of the anuran. When one thinks about the reproduction of frogs, tadpoles in ponds spring to mind, which little by little create legs and lose their tail, and finally come out of the water. The better observers will have seen eggs being laid, like long collars of black beads or in masses of foam stuck to the vegetation. But these are not the only cases: until recently 29 modes of reproduction were known in the world, of which 21 occur in tropical America, the champion in ecological strategies due to its diversity of environments. Reproductive modes involve the description of where the eggs are laid, the development of the embryos and if there is any type of parental care. Some species carry the eggs on their back. “From inside them tadpoles come out, deposited by the mother in bromeliad puddle water”, related professor Haddad, who showed off the tree toad recently found on his field trip. Some mothers swallow their eggs and the tadpoles develop in their stomach. The revision done by professor Haddad and Dr. Cynthia Prado, during her post-doctoral work in his laboratory, has added a further 10 reproductive modes, all from the Atlantic Rainforest. There are nests in sub-aquatic traps or dugouts away from the lagoons, even masses of eggs deposited directly in the humid soil; tadpoles that come out of eggs stuck to rocks near to water and that develop adhering to the wet rock.

Unfortunately it is necessary to sacrifice some individuals in the name of science. For this reason, Haddad underlines that “it’s necessary to collect the maximum possible information so as to value that life”. The animals are measured; a liver sample is removed and preserved in alcohol for genetic analysis. From that point onwards, they are then collection specimens: they receive a label that will identify them in accordance with where they were collected, by whom and all of the information available about the species. They will be preserved in formal and alcohol and transferred to a museum. Unesp’s amphibian collection at Rio Claro is one of the most important in Brazil, in part thanks to ongoing projects.  Haddad tells that three years ago the collection had around 6,000 specimens. Today that number has reached nearly 15,000. As well as this, exchanges with the Zoology Museum of the University of Sao Paulo (USP), the National Museum of Rio de Janeiro and the State University of Campinas (Unicamp) have contributed to the enriching of Unesp’s collection.

Many people are shocked to see zoological collections, but they are essential for the advance of knowledge about nature. In Haddad’s laboratory it is common to find researchers in need of help to identify some animal. The herpetologist (a specialist in reptiles and amphibians) arrives with the frogs in a conserving liquid, perhaps with photos and a recording of the call. Haddad will have a look, listen to the call and ask where the animal was found. All of these bits of information are parts that fit together and lead to identification. Or not. The researchers compare their specimens with the museum’s specimens and the calls from the sonorous collection. Perhaps they might not reach a decisive conclusion. Who knows, could it be a new species? “The Brazilian biological diversity is so great and so little is known that new species appear every day”, advised professor Haddad. And there are discoveries even in inhabited areas, such as the hyla of the genre Phyllomedusa which lives in the outskirts of Sao Paulo city and has still not been officially described.

CÉLIO HADDAD/UNESPThe hidden leaf-frog or Phyllomedusa tetraploidea, from Ribeirão Branco, in the state of São PauloCÉLIO HADDAD/UNESP

Invisible diversity
Often not even experience and comparisons with museum species are enough. Certain species have a very similar appearance and can only be distinguished by what we do not see. What commands in nature is evolution, not our eyes. For this reason, two groups of animals could well appear similar on the outside, but genetically they are so different that they would not manage to mate and reproduce. Perhaps a more careful examination might reveal differences, for example, in behavior or physiology. This diversity must be known and preserved.

For this reason one needs to investigate genetic material, extracted from those liver samples and preserved in alcohol. Starting from there, the genetic sequence that will be compared to other animals is unlocked. The result leads to the so called phylogenetic trees that are genealogies of species. In collaboration with researchers from other countries, last year professor Haddad published monographs that reorganized the classification of amphibians and changed many of the scientific names. “This is going to produce a shake up in this research area, which is somewhat stagnant. To answer to the changes, more work will be needed, which will bring advances”, he forecasts.

Another source of variety is the chromosomes, little packages in which the genes are organized. The number and arrangement of these structures is important because during fertilization the chromosomes of the spermatozoid have to align themselves with their counterparts in the egg. Starting from that, an embryo is formed, and each cell division depends upon these chromosome pairings. There are mechanisms with which organisms can get around this problem. But, in the majority of cases that alterations in the number of chromosomes occur, a distinctly new species is originated. At Unesp of Rio Claro, Sanae Kasahara is coordinating a research line that represents a form of understanding evolutionary processes: the study of the appearance of new chromosomes and of how they behave during fertilization and cellular division.

Time and space
In order to unravel evolution, there is more to it than distinguishing species, which are like a static image of a dynamic process – like pressing the pause bottom when watching a film. But how to go beyond?
One way is to understand spatial processes. Kelly Zamudio, from Cornell University in the United States, is working in collaboration with professor Celio Haddad. She has a project financed by the North American National Science Foundation (NSF), which is comparing three species with different levels of ecological specialization: one that lives only in bromelias, another that circulates throughout the Atlantic Rainforest and a third that depends on more humid areas in order for it to reproduce. According to professor Kelly, “this ecological specialization is correlated with movement and has detectable effects in the genetic diversity of each one of the species”. Or that is to say, if we capture more mobile examples of tree toads in various points of their distribution, there will not be a large difference. But in those that do not move away from their bromeliads, we shall detect genetic differences that are more marked between populations. With this work, the researchers is showing that “aspects inherent to the ecology or biology of the animals can help us to understand differentiation and in the end specialization”.

Philogeography goes even further. This is the geographical study of genetic divergence, which allows for interfering in the history of populations in time and space. A central presupposition is that, if the genetic constitution of a population is more diverse, it is because it perhaps has existed in that location for a longer time, in relation to more homogeneous populations. This is the thinking used by João Alexandrino, a researcher associated to Unesp of Rio Claro. His project involves comparing the philogeographic standards of six species of anurans that occur widely throughout the Atlantic Rainforest. The researcher is looking to understand more than just their evolution. “I”m pretending to identify the areas that over thousands of years remained sufficiently stable to sustain viable populations of frogs and their relatives”, explained researcher Alexandrino. These areas have a greater chance of continuing to flourish throughout environmental fluctuations, and consequently it is essential that they be preserved.

“Frogs, oh my!”, this is what lots of people say. With lots of good intention, some concede that at least they eat flies and mosquitoes. Prejudice apart, they are animals full of surprises and marvelous. And as they are more fragile than birds and mammals, knowing them help to detect which areas of our forests are more under threat.

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