Study discovers new species of green microalga in São Paulo

A group led by researchers affiliated with the Phycology Laboratory at the Federal University of São Carlos (UFSCar) in the state of São Paulo, Brazil, have discovered a new species of green microalga in a reservoir located in the northwest of the state. As a result of the discovery, microalgae of the genus Nephrocytium have been moved to an order belonging to a different taxonomic class and phylogenetically reclassified (placed in a new family).

Green microalgae are found in freshwater and in the sea (where they are known as phytoplankton) and produce more than 80% of the oxygen present in the atmosphere. They are considered primary producers and hence the base of the food chain in aquatic environments. In addition, they have significant potential for commercial applications, such as biodiesel production.

The new species was found in a fishing pond called Muritiba, fed by a spring near the town of Tupã. The species was isolated in 2014 and is now part of UFSCar's Freshwater Microalgae Culture Collection (CCMA). Begun in 1977 by Armando Augusto Henriques Vieira early in his career as a professor in the Department of Botany, CCMA is one of the world's largest and oldest collections of freshwater microalgae, with some 800 strains deposited and conserved for researchers to analyze and potentially commercialize.

The new species was named Nephrocytium vieirae as a tribute to Vieira, who has retired and is now a senior professor at the university. The research that identified it was part of several projects supported by FAPESP (11/50054-4, 12/19520-1, 13/17457-3, 13/18083-0 and 16/07089-5).

The study involved samples collected at more than 300 sites including reservoirs, marches, bogs and lakes in all 22 hydrographic regions of the state. After collection by various scientists under Vieira's supervision, the material was isolated in the laboratory and identified on the basis of morphology. During this process, a particular alga was isolated and identified as a morphotype similar to Nephrocytium. In taxonomy, a morphotype is an individual or set of organisms distinguished within the same species by having a certain physical structure.

Inessa Lacativa Bagatini, now a professor in UFSCar's Department of Botany, was then a postdoctoral fellow under Vieira. After cleansing the specimen of contaminants such as fungi and bacteria, Bagatini found a molecular marker by polymerase chain reaction (PCR) sequencing. The marker placed the strain in the genus Nephrocytium, but pointed to the possibility that a new species had been found.

Thaís Garcia da Silva, first author of an article published on the research, was then studying different algal morphotypes for her doctorate. She sequenced other markers in the specimens and analyzed their morphology under an optical microscope. She then submitted them to transmission electron microscopy, cleaving cells to study the organelles, and scanning electron microscopy, analyzing the cell surface. This part of the research was assisted by Naiara Carolina Pereira Archanjo, also a researcher in UFSCar's Department of Botany and a PhD candidate at the time.

Silva and Bagatini also collaborated with Lenka Štenclová, a researcher at the University of South Bohemia in the Czech Republic, to obtain molecular markers of a Nephrocytium species not available from CCMA at UFSCar. Integration of all this data confirmed that the DNA sequence obtained for the isolated strain was different from those of other species described in the literature. "Microalgae can have very similar morphology in various situations so that molecular markers must be used to decide whether a new species has been discovered, as was the case here," Silva explained.

The next step was the mapping of a phylogenetic tree for Nephrocytium vieirae. Phylogeny traces the evolutionary history of a species, organizing beings into hierarchical taxonomic categories such as kingdom, phylum, class, order, family, genus and species. A phylogenetic tree depicts the lines of evolutionary descent from a common ancestor, rather as a genealogical tree does for human families.

"When we were constructing the phylogenetic tree, we confirmed the suggestion in recent research that the genus belonged to a different class," Silva said. In this stage of the study, the microalga in question was compared with microalgae in all families belonging to the order Sphaeropleales, to which it is allocated, and the researchers conducted a review of the scientific literature in which occurrences of the genus Nephrocytium were mentioned.

Phylogenetic reclassification

In the course of reclassifying the new species, the researchers noted several traits that would not let them keep microalgae of the genus Nephrocytium in the class Trebouxiophyceae, order Chlorellales and family Oocystaceae, as per the scientific literature. Based on the data they obtained in their various analyses, they proposed the creation of a new family, Nephrocytiaceae, belonging to the class Chlorophyceae and order Sphaeropleales.

The article with their findings is published in Taxon, the journal of the International Association for Plant Taxonomy (IAPT), and signed by Silva, Bagatini, Štenclová and Archanjo.

As detailed in the article, microalgae of the family Nephrocytiaceae, including the newly discovered species N. vieirae, can be distinguished from others in the order Sphaeropleales by a combination of morphological traits, smooth cell surfaces, asexual reproduction via autospores, differences in ultrastructure (cell architecture visible at higher magnifications than found on a standard optical microscope), and molecular data. These characteristics corroborated their phylogenetic reclassification and placement in a new family.

Other reports of the new species

Publication of the results enables other researchers in Brazil and elsewhere to use the molecular markers to find out whether specimens can be classified as N. vieirae. "The morphology of N. lunatum closely resembles that of the recent discovery in Tupã. Anyone who has collected a Nephrocytium and not done a molecular phylogenetic analysis is likely to think it's N. lunatum. Publication of our findings showing that we now have this marker could enable others to identify microalgae of the new species in other countries where massive parallel sequencing is used," Bagatini said.

Massive parallel sequencing is widely deployed in research on microalgae. It entails simultaneous sequencing of many samples without observing morphology and comparing the results with sequences stored in a database. "If no matches with other organisms are found, the specimen is placed in the closest genus or taxonomic group," Bagatini explained. The alternative is morphological and molecular analysis of individual organisms.

Correct classification is essential not only for researchers to be able to continue their studies but also for the development of industrial and biotech applications in general. "If you're studying a species to develop a cancer drug, for example, and you want to look at others that maybe could produce more of the compound in question, you'll look for a species that's phylogenetically close," Bagatini said, adding that incorrect taxonomic classification can lead to selection of organisms that lack the desired characteristics.

UFSCar's microalgae collection

The new microalga joins a collection formed from a project that started with the sea. In 1970, Vieira was an undergraduate studying biology at the University of São Paulo (USP) and embarking on an internship at the Oceanographic Institute (IO-USP), where he would organize a marine phytoplankton collection. There he learned how to isolate marine microalgae and managed to mount a collection of some 100 strains. He worked at IO-USP until 1977, when he moved to UFSCar to focus on freshwater microalgae, whose study he decided to prioritize after attending a lecture on these organisms while still an undergraduate. UFSCar's collection began with specimens collected in the vicinity of the university, but microalgae were soon being collected in other areas of the state thanks to funding won by Vieira.

"We improved the quality of the collection as time passed, and some four years ago we implemented a cryogenic system to conserve microalgae in liquid nitrogen," he said.

UFSCar's collection has been used for research on biodiesel and to test swimming pool water treatment systems, as well as in research projects at several institutions around Brazil conducted by master's and PhD candidates, and even by high school students.

Demand was low for a long time but has risen in the last few years because of an increase in potential uses."

Armando Augusto Henriques Vieira

After retiring, he handed over to Bagatini as curator of the collection. "I've continued to help, and I plan to return and resume my support when the pandemic permits. There's still plenty of material in the collection that needs to be studied," he said.

Asked how he felt about having a species named after him, he said, "I'm flattered and grateful." Because of this homage, his name does not appear as an author of the article, even though he supervised the team. "I was lucky to have such good students. They're now fully qualified professionals working in Brazil and abroad," he noted.