Appearances are deceiving

Integrated genetic and ecological studies reveal evolutionary processes

C. brevipedunculata

Barbara Leal / Unesp C. brevipedunculataBarbara Leal / Unesp

Visitors to Ibitipoca State Park in southeastern Minas Gerais State might be rewarded with glimpses of bright red flowers hanging from trees. They are orchids of a genus quite commonly seen, even in supermarkets. The curious thing is that observant experts tend to see in them something akin to mannequins with interchangeable parts: foliage characteristic of one species having flowers that look more like another. Despite their striking appearance, which makes them susceptible to collection for commercial purposes, they bear witness to the fact that much remains to be discovered and understood about these plants.

It is nothing new that Nature mixes together parts of different organisms, generally through hybridization. That was the expectation in the mind of biologist Bárbara Leal when she decided to investigate the enigma during her master’s research under botanist Eduardo Borba of the Federal University of Minas Gerais (UFMG). Based on prior identifications, she expected to find the orchid species Cattleya coccinea and C. brevipedunculata, as well as hybrids of the two. But that is not what she found in the process of genetic analysis—and for one simple reason. Only the latter of the two species exists in Ibitipoca, and its appearance varies according to the environment, as she and colleagues describe in a paper published in the Botanical Journal of the Linnean Society in August 2016.

Ibitipoca: plant parts typical of C. coccinea

Barbara Leal / Unesp Ibitipoca: plant parts typical of C. coccineaBarbara Leal / Unesp

The hypothesis that the existence of plants having a mixture of traits from two species could be explained by hybridization came from research coordinated by botanist Samantha Koehler of the Institute of Biology at the University of Campinas (IB-Unicamp). It was part of the doctoral dissertation written by ecologist Jucelene Rodrigues of the Luiz de Queiroz College of Agriculture at the University of São Paulo (ESALQ-USP), under the advisorship of agronomist Elizabeth Veasey. When the group investigated the species delimitation of C. coccinea and C. mantiqueirae using genetic data, they found that the population in Ibitipoca, located in the municipality of Lima Duarte, appeared to be more closely related to C. brevipedunculata, as described in a paper published in 2015 in the journal Plant Systematics and Evolution. “Several floristic studies show that the vegetation of that area has similarities to that of the Serra do Espinhaço Mountains,” Koehler says. She points out that the soil composition denotes a rupestrian grassland environment favorable to the vegetation of the inland areas of the state. “The distribution of C. brevipedunculata expanded and moved into Mantiqueira, where it found a favorable environment.” It is a peculiar circumstance, because C. coccinea occurs in a much closer region, in the Serra do Mar Mountains in the state of Rio de Janeiro. “It’s just an hour away from Lima Duarte,” Koehler notes. This gave rise to the suggestion of a possible hybrid zone—a process that can lead to the emergence of a new species (see Pesquisa FAPESP Issue No. 212)—a scenario that Leal immediately investigated and refuted.

The red orchids in Ibitipoca Park actually belong to the species C. brevipedunculata, which inhabits the blistering-hot rupestrian grasslands of the Serra do Espinhaço Mountains. “The species was known only in open environments, and there was no indication of development of features similar to those of another species when placed in the shade,” Borba says. Shade accompanied by moisture is the natural environment of C. coccinea, a species of the Atlantic Forest. Besides color—pink for orchids exposed to sunlight and red for forest specimens—the most striking feature that differentiates the two species is their pseudobulb, a water storage structure at the base of the leaves. In C. coccinea it is elongated, and in C. brevipedunculata it is spherical—a more efficient shape when water is scarce. The plants that live in more sunny areas also have thicker, firmer leaves, often with a reddish coloration due to pigments that protect them from sunlight, as opposed to long, flexible leaves. It is one more defense strategy against adverse conditions.

Orchids_248Finding one species disguised as another was a surprise that leaves it quite clear that relying solely on appearance can lead to mistaken identity—a point that may be obvious but is frequently ignored. “Hundreds of hybrids are inferred based solely on morphology,” Borba notes. “The lesson to be drawn from this is that differences can be the product of phenotypic plasticity.” The term is a reference to variations in features in response to environmental conditions, independently of genetics. Ibitipoca, located in the Serra de Mantiqueira Mountains, is fertile terrain in this case, due to its mosaic of rocky areas and typical rupestrian grasslands vegetation interspersed with patches of dense, moist Atlantic Forest.

In Borba’s opinion, the findings call attention to the need for expanding plant classification procedures beyond just one technique. It is not enough to study the visible features, or phenotype. Nor does it suffice to consider the geographic distribution. In the case of plants, it is clear that several sources of information, including genetics, physiology and chemistry, add up to improved species delimitation, and the same holds true for most organisms. What can be called integrative taxonomy is far from a new idea, but quite often that procedure is not used, as has happened so far with the orchids in Ibitipoca. Borba calls attention to conservation of orchids, which regularly show up on lists of endangered species, due to either habitat destruction or over-collection of ornamental plants for commercial purposes—or a combination of the two factors. “A more suitable delimitation of populations and species would help us draw up plans for conserving and preserving orchids, either in germplasm banks or in situ,” he explains.

South to north: yellow spots on P. burmeisteri in the state of São Paulo (above) and the blue thighs of P. bahiana (at left)

Célio Haddad / Unesp South to north: yellow spots on P. burmeisteri in the state of São Paulo (above)…Célio Haddad / Unesp

Meetings and missed encounters
The taxonomic status of C. coccinea is also undergoing a more in-depth analysis, according to Koehler. The 2015 study showed a marked divergence between the populations in the municipalities of Petrópolis and Nova Friburgo in Rio de Janeiro State, and another that is now in São José do Barreiro in São Paulo State. It may signify that they are completely separate populations.

In more or less the same region, a green leaf frog with yellow spots on the sides of its body and the backs of its blue thighs also appears to be hiding a new species, according to biologist Tuliana Brunes, currently a postdoctoral researcher at USP. Genetic analyses conducted during her doctoral research at the University of Porto in Portugal suggest that Phyllomedusa burmeisteri is restricted to an area of the Serra do Mar in Rio de Janeiro State where there is thought to have been a forest refuge about 1.3 million years ago. “The population of this area may have become differentiated from the individuals in the rest of the distribution,” she explains. Brunes says that more in-depth studies are needed, from both a genetic and a reproductive perspective (a specific song is essential for enabling the females to find the right male), in order to confirm that the leaf frogs currently classified as P. burmeisteri in the states of São Paulo, Minas Gerais, Espírito Santo and southern Bahia actually belong to a different species.

... and the blue thighs of P. bahiana

Tuliana Brunes / USP … and the blue thighs of P. bahianaTuliana Brunes / USP

Another curious aspect of these leaf frogs that enables us to draw a further parallel with orchids is a variation in the coloration pattern of the backs of the thighs when comparing P. burmeisteri and P. bahiana—two very similar species that occur from São Paulo State to Sergipe State. This was observed 25 years ago, when zoologists José Pombal Jr. of the National Museum of Brazil and Célio Haddad of São Paulo State University (Unesp) in Rio Claro described a south-to-north trend among these leaf frogs. The ones from São Paulo had many yellow spots on the thighs, while those from Bahia had none. Between the two distribution points, there was a gradient of intermediate forms that suggested a hybrid zone in the middle. Through more extensive and intensive sampling, Brunes and colleagues, including Haddad, have now verified that the distribution of the coloration pattern does not conform to the species limits. Unlike the findings for orchids, leaf frogs do form hybrids, as described in a paper published in the journal Zoologica Scripta in 2014. Brunes found genetic signals of ancient hybridization in Espírito Santo, south of the Doce River, and another current one in southern Bahia.

The surprising thing was that these hybrid zones do not appear to be responsible for what is considered to be intermediate coloration on the animals’ thighs: the study indicates that it is a common pattern in both species. Once more, appearances are not helpful. “We believe that natural selection is likely responsible, with the yellow spots serving as a warning to predators that this frog has toxins on its skin,” she explains. And the toxins are plentiful: a number of chemical substances with pharmacological potential have now been isolated in these animals (see Pesquisa FAPESP Issue nº 133). A snake can swallow the leaf frog and regurgitate it live when it senses the harmful substances in its mouth. According to Brunes, the coloration is on a part of the thighs that is normally hidden, but leaf frogs of the genus Phyllomedusa have a visual communication system in which they extend their hind legs and expose the colors. The advantages for survival would explain a tendency for the yellow pattern to take hold, but that hypothesis will require further study to find corroboration.

Taking these studies as a whole, orchids and leaf frogs are calling attention to an aspect that often receives little heed at a time when scientists are sequencing genetic material to organize plants and animals into compartments: environmental adaptations occur frequently, they are essential to survival, and they do not necessarily give rise to new species.

Molecular systematics, diversification patterns and conservation of Brazilian orchids (nº 2006/55121-3); Grant Mechanism Young Investigators Awards Program; Principal Investigator Samantha Koehler (Unicamp); Investment R$ 266,360.99.
2. Phylogeography, population genetics and species delimitation of the complex Cattleya coccinea (Orquidaceae) (nº 2011/18532-3); Grant Mechanism Regular Research Grant; Principal Investigator Elizabeth Ann Veasey (USP); Investment R$ 112,022.18.
3. Biogeography, phylogeography and diversification patterns of anuran species widespread in the Brazilian Atlantic Forest (nº 2005/52727-5); Grant Mechanism Young Investigators Awards Program; Principal Investigator João Alexandrino (Unifesp); Investment R$ 307,302.27.
4. Speciation of frogs in high-altitude environments (nº 2008/50928-1); Grant Mechanism Thematic Project; Principal Investigator Célio Haddad (Unesp); Investment R$ 1,407,985.13.

Scientific articles
LEAL, B. S. S. et al. When hybrids are not hybrids: A case study of a putative hybrid zone between Cattleya coccinea and C. brevipedunculata (Orchidaceae). Botanical Journal of the Linnean Society. V. 181, No. 4, p. 621-39. August 2016.
RODRIGUES, J. F. et al. Species delimitation of Cattleya coccinea and C. mantiqueirae (Orchidaceae): Insights from phylogenetic and population genetics analyses. Plant Systematics and Evolution. V. 301, No. 5, p. 1345-59. May 2015.
BRUNES, T. O. et al. Species limits, phylogeographic and hybridization patterns in Neotropical leaf frogs (Phyllomedusinae). Zoologica Scripta. V. 43, No. 6, p. 586-604. November 2014.