A theory in motion

Research that looks beyond the gene suggests new directions for understanding how species diversify

Ana Matsusaki

The British naturalist Charles Darwin (1809–1882) didn’t know what a gene was when he described evolution through natural selection in his 1859 book The Origin of Species. But he understood that traits were transmitted between generations and proposed hypotheses that are even today being studied and gradually explained. It wasn’t until 1953 that the structure of the DNA molecule and its capacity for transmitting heredity would be discovered and become the protagonist in the process of change, diversification, and adaptation in evolving species.

In the decades that followed, the mechanisms became more diversified, leaving the branches of the tree of life more intertwined and less hierarchical. Some—polemic—hypotheses argue that DNA isn’t modified by random mutations exclusively, and that the information contained in its sequence isn’t the only data transmitted to the offspring. In recent years, the perspective named extended evolutionary synthesis (EES) has been systematizing a concept that doesn’t refute existing knowledge, but expands on accepted understanding and, in some cases, allows for two-way routes where unidirectional determination was the traditionally accepted view. According to this perspective, an organism’s development during its life has an importance in its construction that transcends what was genetically programmed (see inset). The interaction between organism and environment thus is seen for its mutual effects, not just as adaptation to something preestablished. These have been relevant themes for decades among experts, but EES considers them causes of evolution, whereas in the traditional view they would be considered consequences, according to an article published in 2015 in the journal Proceedings of the Royal Society B. “It’s another way of thinking about evolutionary mechanisms, with a different set of expectations and predictions,” says evolutionary biologist Kevin Laland of the University of Saint Andrews, in Scotland, the article’s lead author.

In an article reprinted in March this year in the newspaper Folha de S.Paulo, Laland compares the evolutionary path to that traveled by a person walking several dogs and being pulled in every direction by leashes of differing lengths. The numerous forces at work in evolution, he says, include natural selection, but are not restricted to it alone. “EES is a tool for thinking,” he told Pesquisa FAPESP. According to the initiative’s website, there are currently 22 projects dedicated to testing the hypotheses that differentiate EES from the traditional view. “It will take us decades to synthesize a new conceptual framework.”

For now they are hypotheses in the testing phase, an essential step for scientific leaps. While some researchers in the field see convincing evidence, others see nothing new. “We shouldn’t rush into a new theory before we know if the traditional view can accommodate the findings,” says evolutionary biologist Douglas Futuyma of Stony Brook University in New York. In 2016, he participated in a symposium organized by Laland and colleagues in London, in the role of opponent, and sees value in this type of exchange. But he’s far from convinced. “Much of what they’re saying has been a part of evolutionary theory for a long time.” He refers to a diversity of studies on the connections between development and evolution, for example, that earned the nickname “evo-devo” (see Pesquisa FAPESP issue no. 152).

The argument, a recent one worldwide, hasn’t yet attracted many supporters in Brazil. Among those interested are biologists Hilton Japyassú, from the Federal University of Bahia (UFBA), and Eduardo Ottoni, from the Institute of Psychology of the University of São Paulo (IP-USP), who are interested in evolutionary aspects of culture and behavior. Others are investigating aspects of evolution that were previously inaccessible due to technical or conceptual limitations, without worrying about remaking theories as examples to follow.

Studies indicate epigenetics acts as an interface between the environment and the biology of bees

More than a sequence
Epigenetics, a phenomenon that has been gaining relevance, alters a gene’s production of RNA or proteins by modifying its activity throughout the life of an organism, without changing the DNA sequence. It is a dynamic regulatory function that depends on a network of molecules refined over the course of evolution, under genetic control.

Under rigorous study over recent decades, epigenetics now seems to be behind something that was a mystery to Darwin to the point that, in his eyes, it put in question his entire theory: the reproductive division in bee colonies. If (according to natural selection) an individual who leaves behind more descendants becomes better represented in a population and has its traits disseminated, how can there be communities in which only one female reproduces, with the help of sterile companions? Darwin justified it by thinking of the survival of the hive as a unit, and later, the very close kinship between the members of the colony became the most accepted explanation. Now epigenetics enters the equation as a mechanism of interaction between the environment and DNA to define, among a group of genetically similar larvae, which will become queen and which becomes a worker bee.

Genes and environment
Groups led by geneticists Klaus Hartfelder and Zilá Simões, from the USP campus in Ribeirão Preto, have studied the differential activity of genes by methylation, one of the principal mechanisms of epigenetics. By analyzing the genome of Melipona scutellaris, a stingless bee native to Brazil, biologist Carlos Cardoso-Júnior, a doctoral student in Hartfelder’s group found that the larvae exhibited interference in many parts of their DNA—thus it’s considered to be hypermethylated. The pattern suddenly changes during the transition from larva to pupa, when the workers have their genetic material modulated by three times more methylation than queens and males. Thus, the portion of the genome affected by epigenetics establishes a functioning pattern in the genes that is determinant in the physiology that differentiates the castes of these insects. In adult life, the expression of these bees’ genes is controlled by changes in their histones, proteins that package the DNA inside the cells—another type of epigenetic alteration, according to an article published in 2017 in the journal Genetics and Molecular Biology. These effects are regulated by food, which defines caste, together with classical genetic inheritance. “Histone modification is a process that allows for rapid adjustments in the regulation of gene activity in response to the environment,” Hartfelder says.

The studies indicate epigenetics acts as an interface between the environment, which includes food and climatic conditions, and the bees’ biology. It took time to recognize the importance of this mechanism in invertebrates, since the genomes of the principal models for genetic studies in this type of animal—the fly Drosophila melanogaster and the nematode worm Caenorhabditis elegans—lack the methyltransferase genes that methylate DNA. This explains why epigenetic studies of invertebrates have been behind for so long, according to Hartfelder.

In vertebrates, rodents help to investigate human functioning. By studying mice, biologist Vânia D’Almeida’s group from the Department of Psychobiology of the Federal University of São Paulo (UNIFESP) investigates how the environment can affect the genetics of pregnant women and their offspring. “Deficiencies in the B-complex vitamins alter the expression of genes linked to Alzheimer’s disease,” says biologist Vanessa Cavalcante-Silva. The finding comes from her doctorate, completed in 2012 under D’Almeida’s guidance, in which she subjected pregnant and lactating females to a nutritional deficiency and observed a propensity to lose memory in their offspring. Alterations in the function of risk genes for Alzheimer’s are a warning sign, according to a 2014 article in PLOS ONE. For the study group, the findings make doctors’ concerns about nutrition in their pregnant patients even more crucial.

For example, a study by biologist Martha Bernardi’s group at Paulista University (UNIP) showed that the diet of rats during gestation can affect the gene activation profile linked to obesity not only in their direct offspring but also in the second generation (see Pesquisa FAPESP issue no. 252). “During pregnancy, three generations are present and subject to the environment,” D’Almeida notes. “The mother, a female fetus, and the ova already forming in its ovaries.”

Between Darwin and Lamarck
Epigenetic mechanisms of regulation, such as those demonstrated in bees and rodents, have already been studied in depth and have begun to be widely accepted as part of DNA functioning. What is still in question is their evolutionary importance. “For the last ten years you didn’t hear much about it,” says Futuyma, who is interested in recent findings on heredity. “Now there are people writing models of population genetics to measure the impact of epigenetics on evolution,” he reports. It isn’t yet possible to evaluate the extent of the phenomenon and its long-term importance separate from the already well-established mechanisms: mutations at random points of DNA as the primary source of variability, with add-ons such as recombination between parts of the genome and jumping genes (genes which change location in the DNA).

Laland refutes the caveat that the transmission of epigenetic markings for more than a few generations has not been documented. “This is the wrong way to think, as if something needed to have gene-like properties to be important.” In his view the epigenetic regulation of the genome is always in action and can renew itself with each generation, in such a way that the puzzle resides in understanding how transient effects can be long lasting. “The extragenetic forms of heredity have received a lot of attention,” he says, using terminology that is still controversial.

Discussing epigenetics as an evolutionary force revives the theory of Frenchman Jean-Baptiste de Lamarck (1744–1829), known for the concept that traits acquired throughout life could be transmitted (see timeline). And it wasn’t exclusively Lamarck’s theory, since Darwin didn’t deny his predecessor’s ideas. “The great and inherited development of the udders in cows and goats in countries where they are habitually milked, in comparison with the state of these organs in other countries, is another instance of the effect of use,” Darwin wrote in The Origin of Species. The first edition of his 1859 work received new translations this year by Brazilian publishers Ubu and Edipro; the latter comes with a preface and notes by biologist Nélio Bizzo, from the USP School of Education. “It should be noted that these changes would not only be caused by the environment, but would become hereditary, in a model very similar to that advocated by Lamarck,” says Bizzo regarding this passage, in one of his notes which highlights Darwin’s acceptance of other mechanisms acting concomitantly with natural selection. (He wouldn’t have been surprised by the example of Laland’s dogs.) As of the middle of next year it will also be possible to read Lamarck’s principal opus, Zoological Philosophy, in Portuguese, which is in the process of being translated by Editora UNESP. “We want to publish classical texts relevant to evolutionary theory,” explains philosopher Jézio Gutierre, the book’s executive editor. “The Lamarck text fills a hole in the collection essential to giving researchers looking into the subject a complete picture.”

The capacity for environment to drive evolutionary processes also appears in more recent studies. When confronted with antibiotics, a reaction called the SOS response increases the rate of mutation in bacteria. “A lot of people confuse it with Lamarckism, but the mutations are random—not driven by the environment,” explains biologist Rodrigo Galhardo of the Institute of Biomedical Sciences at USP (ICB-USP). From the human point of view, it’s problematic because it gives rise to antibiotic-resistant bacteria.

In experiments with ciprofloxacin, an antibiotic widely used for urinary tract infections and an inducer of the SOS response, his group tests for substances that inhibit mutagenesis, as reported in an article published in 2017 in the journal Antimicrobial Agents and Chemotherapy. The pharmaceutical amicacin reduced the mutation rate resulting from the SOS response in Pseudomonas aeruginosa, a bacterium that causes dangerous infections in hospitals. He now wants to dissect its mechanisms of action and understand why one substance interferes with the action of the other. “The dream is to administer the antibiotic together with the SOS inhibitor.”

Biological variation
In the view of American theoretical biologist Sarah Otto of the University of British Columbia in Canada and her doctoral student Linnea Sandell, perspectives that allow for integrating organism and environment should become increasingly prominent in genetics research. In a commentary published in 2016 in the journal Genetics on the occasion of the journal’s centennial, they foresee a trend over the next 100 years toward integrating information in order to understand the diversity of life. The computational capacity and tools available today allow the construction of trees of life in which hypotheses can be tested to predict evolutionary transitions.

Along these lines, biologist Thiago Rangel from the Federal University of Goiás uses computational models to investigate the emergence of biodiversity in South America. Dividing the region into the Andes, Amazon, Atlantic Rainforest, and Patagonia, he recreated the patterns of species diversification over the last 800,000 years using computer simulations. It was done completely virtually and later corroborated by comparison with empirical data of birds and mammals extant in the scientific literature.

The results, published in July in the journal Science, point to the Andes range as the largest cradle of species on the continent. The decisive factor is the diversity of habitats in a relatively small area as a consequence of the abrupt geographic relief with a dramatic climatic diversity. “The Amazon is a climatically homogeneous plain; if there are changes that make the environment less conducive to an organism, it’s 2,000 kilometers from a safe harbor where the climate remains adequate,” Rangel explains. That would make the Amazon a species graveyard, despite the vastness of its forests that allows it to maintain a huge diversity of creatures—which originate in part in the nearby mountains and as a consequence of barriers established by the great rivers. “In the Andes, taking ten steps upwards is enough to escape altered temperatures.” This vertical movement along geological time encourages speciation, since populations can differentiate when isolated on distinct peaks (see Pesquisa FAPESP issue no. 140).

Biologist Fabio Machado, who is also looking at evolution on the broad scale of a phylogeny, studied the diversification of carnivores during his doctorate at USP’s Institute of Biosciences (IB-USP), under the guidance of biologist Gabriel Marroig. His work, which involved making three-dimensional recreations of the skulls of different species to compare how the shapes vary as a whole, is integrated into the line of research at Marroig’s lab, which investigates how the organization of anatomy restricts or facilitates the evolution of certain morphologies (see Pesquisa FAPESP issue no. 230). Darwin already understood the existence of “laws of variation” (correlations of growth, in his words) that linked inherited characteristics. Two dental arches that don’t fit, for example, do not favor survival.

This kind of agreement between Darwin’s thinking and current discoveries is a demonstration of the Englishman’s ability to make unexpected connections and then draw conclusions and explanations that hadn’t occurred to anyone else, Bizzo emphasizes. “Every time I read The Origin of Species, I discover one more thing that he’d already said that I hadn’t noticed,” says Machado, who claims to have a tattoo of Darwin’s portrait. His expectation was to find variability among the carnivore skulls that would be associated with the diversity of eating habits. But he found that the canids—the family of dogs and wolves—show a far greater variation than other carnivores. “Their facial region is more variable than in other mammals,” says the biologist, a postdoctoral intern at the Bernardino Rivadavia Argentine Museum of Natural Sciences in Buenos Aires. “The results indicate that the faces of canines have become modular, becoming more independent from the rest of the cranium.” It’s the type of alteration in the pattern of development that, according to the extended evolutionary synthesis, can drive the adaptation of organisms in a much more dramatic way than localized mutations could achieve.

In parallel to anatomy and biology, behavior is also prominent in the new proposals, and when innovative behaviors transcend generations it has come to be considered culture. These behaviors can spread through learning in a population and differentiate that population from others, as shown in studies done with monkeys by Eduardo Ottoni’s group at USP (see Pesquisa FAPESP issue no. 259). The basis of this thinking lies in the perception that traditions, such as proficiency in breaking hard fruits and the methods used to do it, are part of the inherited package handed down from one generation to another in capuchin monkeys. This context, according to researchers interested in cultural evolution, can influence the physical aspects of animals and alter the substratum of natural selection.

Culture and construction
The modification of the surrounding environment by organisms is a central mechanism in EES, which argues that this capacity—called niche construction—ends up having an evolutionary role. The studies of biologist Hilton Japyassu, from UFBA (see Pesquisa FAPESP issue no. 249), proved to be a perfect case for discussing these issues during a postdoctoral fellowship in Scotland with Kevin Laland, as discussed in an article in 2017 in the journal Animal Cognition. “Spiders, or any organism that builds structures and has an intimate relationship to them, can modify their cognition in accordance with these artifacts,” he says. Such is the case of the spiderweb, which becomes a tool for adaptively processing information about the captured prey, affecting the spider’s behavior. This creates a two-way street—spider and web modify one another. According to Laland, organisms that live in modified environments can leave their constructions, the fruit of lifelong learning, to their descendants, and thus direct their own evolution, which would derive from this constructed context.

The examples presented in this report illustrate approaches that are only possible with recent advances in knowledge and technology. It does not mean that their authors subscribe to one or another theoretical field, because as Darwin demonstrated in the nineteenth century, a breadth of thought and connections is much more productive than erecting barriers. For Japyassu, the changes in perception that the new ideas represent need to be added to evolutionary theory, which may become unrecognizable. The debate will go on for a good while yet, and there are some dates already on the calendar: the “Talking Evolution” meetings, organized by the Max Planck Institute in Plön, Germany, this month (September 26–28), and “Evolution Evolving,” which will be held in April 2019 in Cambridge, England. Laland is one of the organizers.

1. Maternal hyperhomocysteinemia and epigenetic alterations in the fetal programming of genes involved in the etiopathogenesis of Alzheimer’s disease (no. 10/00075-2); Grant Mechanism Regular Research Grant; Principal Investigator Vânia D’Almeida (UNIFESP); Investment R$150,664.69.¬
2. Causal analysis of the development of Apis mellifera—regulatory genes and hierarchical networks of gene expression in the specification of tissues and organs (no. 11/03171-5); Grant Mechanism Thematic Project; Principal Investigator Zilá Luz Paulino Simões (USP); Investment R$2,707,231.97.
3. Modularity and its evolutionary consequences (no. 11/14295-7); Grant Mechanism Thematic Project; Principal Investigator Gabriel Henrique Marroig Zambonato (USP); Investment R$2,026,186.98.
4. SOS response and bacterial resistance in Pseudomonas aeruginosa (no. 17/22430-8); Grant Mechanism Regular Research Grant; Principal Investigator Rodrigo da Silva Galhardo (USP); Investment R$176,049.90.¬

Scientific articles
LALAND, K. N. et al. The extended evolutionary synthesis: Its structure, assumptions and predictions. Proceedings of the Royal Society B. Vol. 282, no. 1813. August 22, 2015.
SILVA, V. C. da et al. Effect of vitamin B deprivation during pregnancy and lactation on homocysteine metabolism and related metabolites in brain and plasma of mice offpring. PLOS ONE. Vol. 9, no. 4, e92683. April 2, 2014.
CARDOSO-JÚNIOR, C. A. M. et al. Epigenetic modifications and their relation to caste and sex determination and adult division of labor in the stingless bee Melipona scutellaris. Genetics and Molecular Biology. Vol. 40, no. 1. March 2, 2017.
SANDELL, L. and OTTO, S. Probing the depths of biological diversity during the second century of Genetics. Genetics. Vol. 204, pp. 395–400. October 2016.
MACHADO, F. A. et al. Evolution of morphological integration in the skull of Carnivora (Mammalia): Changes in Canidae lead to increased evolutionary potential of facial traits. Evolution. Vol. 72, no. 7, pp. 1399–419. July 2018.
RANGEL, T. F. et al. Modeling the ecology and evolution of biodiversity: Biogeographical cradles, museums, and graves. Science. Vol. 361, no. 6399, eaar5452. July 20, 2018.
VALENCIA, Y. V. et al. Ciprofloxacin-mediated mutagenesis is suppressed by subinhibitory concentrations of amikacin in Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy. Vol. 61, no. 3, e02107–16. March 2017.
JAPYASSÚ, H. F. and LALAND, K. Extended spider cognition. Animal Cognition. Vol. 20, no. 3, pp. 375–95. May 2017.