As it turns out, I began my first postdoc just to be instantly pushed into unintended homeoffice by a global pandemic. Oh well. Why not use this time to learn more about my new subject and share what is so fascinating about it! Let’s dive together into Proteorhodophytina, one of the newest recognized deep lineages of eukaryotes and a possible key to multiple mysteries of protist evolution.

Red algae (phylum Rhodophyta) are lesser known cousins of green algae and land plants (Viridiplantae) and another small algal group (Glaucophyta), which together form a vast and important eukaryotic supergroup Archaeplastida, characterized by presence of primary plastids – photosynthetic organelles of direct cyanobacterial origin.

You may be familiar with some red algae even without realizing it, as many species have profound ecological, industrial, and culinary importance! Macroscopic red algae represent one third of all seaweeds grown in aquaculture worldwide and create half of the economic value generated by this industry. Among the most commonly used are species of the genus Pyropia (formerly Porphyra; class Bangiophyceae) known as nori and used in Japanese cuisine e.g. to wrap sushi and onigiri. Chondrus crispus (class Florideophyceae) known as Irish moss is harvested for a gelatinous polysaccharide carrageenan used as a stabilizer and thickener in processed food industry, for fining beer and wine, and for traditional production of tasty milk-based desserts in Ireland or the Caribbean. Another polysaccharide of rhodophyte origin, agarose, has similar uses, but is also a ubiquitous material used in biological laboratories. Agarose is isolated from a number of red algal species of the class Florideophyceae commonly referred to as “agarophytes”.

Calcified “coralline” red algae of the class Florideophyceae which deposit calcium carbonate within their cell walls are crucial ecosystem engineers in oceans. Crustose coralline algae mechanically reinforce tropical coral reefs, reducing their erosion. Branched coralline algae provide habitat and shelter for an enormous diversity of marine animals in intertidal environments, protecting them from waves, drying, and predation. Unattached rhodolith coralline algae form extensive benthic communities, known as rhodolith beds or maërl, which are just recently being recognized as unique and important ecosystems on par with seagrass meadows or kelp forests.

On the other end of red algal diversity spectrum are extremophilic single-celled rhodophytes of the class Cyanidiophyceae which thrive in acidic and hot environments around hot springs and fumes. There are currently only three genera with eight species recognized in this class, but environmental sequencing studies hint at much greater undiscovered diversity. Phylogenetic analyses place Cyanidiophyceae as sister to all other red algae, and so their extremophilic nature was proposed to reflect the habitat of the last common ancestor of all rhodophytes. This so called “hot start” hypothesis, if true, could explain some exceptional genomic and functional simplifications observed in living red algae.

Between the large, multicellular seaweeds of Bangiophyceae and Florideophyceae classes on one hand and the extremophilic, simplified, single-celled Cyanidiophyceae on the other lie the long neglected lineages which are the true focus of this story: four classes with delightfully arcane names Rhodellophyceae, Compsopogonophyceae, Stylonematophyceae, and Porphyridiophyceae. Collectively they are known as mesophilic non-seaweed red algae and this unflattering designation easily reveals why they didn’t attract much scientific interest so far: mesophilic means they live in “normal” non-extreme conditions and non-seaweed tells that they don’t form large multicellular bodies, which would be interesting for human use or important as ecosystem creators. Members of these four classes are usually unicellular or form simple filaments, but always are tiny and inconspicuous.

It used to be assumed that mesophilic non-seaweed red algae are paraphyletic, forming several independent lineages branching one by one and representing intermediate steps between Cyanidiophyceae and the two large seaweed classes. However, recent phylogenomic analysis revealed that the four classes actually form one monophyletic lineage (sharing a single exclusive common ancestor) which was classified as a new subphylum Proteorhodophytina.

This result came as a side product of interest in another fascinating aspect of rhodophytes: their plastidal genomes. Plastids, the photosynthetic organelles of algae and plants, are in fact highly integrated bacterial symbionts and as such they usually retain remnants of their own DNA taking care of various essential functions. Plastidal genomes of red algae were generally considered to be primitive, evolutionary stable, small, efficient, and compact. They retain the highest number of genes of all studied plastids, but these genes are organized very frugally without much non-coding sequences taking up space. The structure of plastidal genomes looked very similar across the entire phylum. However, these observations were made mostly on the well-studied seaweed and extremophilic red algae, not on members of Proteorhodophytina. Sergio Muñoz-Gómez and colleagues (2017) started filling in this knowledge gap by sequencing plastidal genomes of previously neglected mesophilic non-seaweed red algae.

What they found was a very different picture. All newly sequenced plastidal genomes are larger than those of previously studied red algae and show a remarkable diversity of sizes between the various Proteorhodophytina species. One of them, Corynoplastis japonica, has the largest plastid genome which also contains the highest number of introns of all studied organisms. The Proteorhodophytina plastidal genomes are generally heavily bloated with non-coding regions and highly varied in their gene arrangement, which contrasts starkly with the conserved, compact genomes observed in seaweed and extremophilic red algae.

So far, we were discussing red algae and also touched on green algae and land plants as well as glaucophytes. All these lineages have a common origin in an ancient cell which engulfed a cyanobacterium and instead of eating it, domesticated it and learned how to use its photosynthetic machinery to make its own food from sunlight and carbon dioxide. This is the origin of the primary plastid – defining trait of the supergroup Archaeplastida. But what about other types of algae? What about kelp, forming vast, cathedral-like underwater forests? What about diatoms, living in tiny houses of glass? What about coccolithophores, whose enormous quantities form basis of chalk sediments like the white cliffs of Dover? What about dinoflagellates, which live in symbiosis with corals and whose sensitivity to climate change is causing devastating coral bleaching? There are numerous groups of algae which do not belong in Archaeplastida, and yet they also have plastids and perform photosynthesis. Most of them are so-called secondary red algae and deep origins of their plastids can be traced back to rhodophytes.

In this case it was not a cyanobacterium who got eaten and tamed, but a fully developed eukaryotic cell already endowed with a well-integrated plastid. The resulting “secondary” plastids have a wide range of forms, each adapted to its new host in a different way. They usually have multiple additional membranes and some even keep a remnant nucleus of their original red algal host, creating an intricate genomic landscape in the cell. Many unanswered questions remain about these complicated plastids and their history. One of the most pressing is: How many times and in what lineages did this happen?

We are not sure whether all these lineages share a common ancestor which already had a secondary plastid (this is called “chromalveolate hypothesis”), or rather only some lineage gained the original secondary plastid, and then other, unrelated organisms have eaten and enslaved them, forming a chain of symbioses, resulting in tertiary, quaternary, or even higher degree plastids (“serial endosymbiosis”). Current understanding of the large-scale eukaryotic evolution favors the latter, more complicated scenario, but the jury is still out.

We also don’t know which lineage of red algae was the original donor of plastid. And was it only one lineage? What if there were multiple origins of secondary plastids from different branches of the rhodophyte tree of life? Phylogenetic analyses seem to endorse the notion of single origin, but the statistical support for this is low and unconvincing and such results can easily be attributed to a low number and limited diversity of red algal genomes included in the analyses. There is a lot of room for surprises. Multiple origins of secondary red plastids might actually elegantly explain certain discrepancies in previously proposed evolutionary scenarios, e.g. concerning numbers of membranes surrounding different types of secondary plastids.

Based on properties of secondary red plastids, we can assume that their ancestors were simple, single-celled, or colonial red algae living in moderate conditions. This description perfectly matches the newly erected subphylum Proteorhodophytina and it is possible that the ancestor (or ancestors?) of secondary red plastids was indeed member of this lineage, or closely related to it. This puts previously neglected mesophilic non-seaweed red algae in center stage of one of the greatest stories in evolutionary protistology!

In my new position in the DEEM team in Orsay, I’m joining a project which aims to gather more genomic data, both plastidal and nuclear, from a broad diversity of Proteorhodophytina and finally bring this lineage into light. We hope our results will help to answer questions about the origins of secondary plastids, as well as red algae themselves, and illuminate the differences in plastid genome architecture among them. And who knows, what other secrets we might discover in genomes of these mysterious, long-neglected creatures?

Lukas, 27/03/2020

• Muñoz-Gómez, Sergio A., Fabián G. Mejía-Franco, Keira Durnin, Morgan Colp, Cameron J. Grisdale, John M. Archibald, and Claudio H. Slamovits. 2017. “The New Red Algal Subphylum Proteorhodophytina Comprises the Largest and Most Divergent Plastid Genomes Known.” Current Biology 27 (11): 1677-1684.e4.
• Yoon, Hwan Su, Wendy Nelson, Sandra C. Lindstrom, Sung Min Boo, Curt Pueschel, Huan Qiu, and Debashish Bhattacharya. 2017. “Rhodophyta.” In Handbook of the Protists, 89–133. Springer International Publishing.

The mineral pyrite, iron sulfide gemstone also known as fool’s gold for its gold-like appearance, used to be a favorite material for alchemists in their futile struggles to create precious metals. Since antiquity it is also combined with silver in the so-called marcasite jewelry, popular especially in the 19^th century, and until today pyrite is used for production of sulfur dioxide for various industrial applications. Whether you are alchemist, Victorian lady, chemical engineer, or anybody else for that matter, you always have a bit of pyrite with you, or rather inside you. The iron-sulfur (FeS) clusters are molecules composed of iron and sulfur atoms arranged in a pattern resembling pyrite crystal structure, which function as cofactors – small molecular “plug-ins” – in a vital group of proteins involved in such important tasks as electron transfer or DNA repair. FeS clusters are found in all living cells and are indispensable for life as we know it. Their ubiquity and importance even led to formulation of a hypothesis saying that pyrite and similar minerals played a crucial role in the very origin of life.

In eukaryotic cells – building blocks of animals, plants, fungi, and microbial protists – FeS clusters are typically produced by two different metabolic pathways. One set of enzymes (ISC) works in mitochondria. The other (CIA), localized in cytoplasm, provides FeS clusters to all the other parts of the cell including the nucleus. The cytoplasmic pathway doesn’t work on its own, but depends on a, yet unidentified, product of the mitochondrial one. Mitochondria are therefore usually essential for the cell and even their most simplified forms tend to have at least this function intact. And so, production of FeS clusters became a central question for our team after we described the first known eukaryote completely devoid of mitochondria, a chinchilla gut-inhabiting protist Monocercomonoides. How can the cytoplasmic pathway build FeS clusters if mitochondria, together with their pathway, were lost? It turns out the answer is lateral gene transfer – sharing of genetic material between unrelated organisms. The ancestors of Monocercomonoides gained another pathway for FeS cluster synthesis, called SUF, from bacteria and recruited it for work instead of the lost mitochondrial one.

We investigated the evolutionary history of this gene transfer. When did it happen? What other organisms share it? And how did the bacterial SUF pathway change in its new home? We sampled a broad diversity of Monocercomonoides’ relatives constituting a group called Preaxostyla. All of them are single-celled microbes which shun oxygen just like Monocercomonoides, but that’s where the similarity ends. For example, Streblomastix resembles a bundle of snuggly packed symbiotic bacteria, only held together by a network of thin lobes – the actual protist cell. Another one, Saccinobaculus, looks like a bag with a snake inside, which constantly wriggles around. The “snake” is in fact a structure of cellular skeleton that helps the cell to move. Preaxostyla are wonderfully weird creatures indeed! We sequenced 10 species, chosen to cover all major lineages, and found the genes for the SUF pathway in each of them, even those species which, unlike Monocercomonoides, still retain mitochondria. Also, none of the organisms harbored the mitochondrial ISC pathway.

All the 5 genes constituting the SUF pathway in Preaxostyla show the same evolutionary history. That means they must have come in one gene transfer event from bacteria, which happened before all the species split – more than 100 million years in the past. This happened before the mitochondrion vanished, possibly representing the final nail in its coffin. When the mitochondrial pathway was replaced with a new substitute, the microbes simply lost any remaining motivation for keeping the costly organelle and got rid of it. We also found out that 3 of the 5 genes are fused together in Preaxostyla, likely producing a large chimeric protein, a situation not observed in bacteria.

Our findings are most interesting from the evolutionary point of view. They show another strong evidence of lateral gene transfer having a dramatic effect on eukaryotes, a notion which is still controversial. However, they might also have a broader impact in the future. The interesting fusion of 3 genes may indicate that the protein products of these particular genes may be closely cooperating, providing a hint on the general functioning of the SUF pathway. Also, remember that still mysterious connection between mitochondrial and cytoplasmic pathways for FeS cluster production in most eukaryotes including humans? Well, now we have a system where both pathways are in cytoplasm and no mitochondrion is involved. Cross-examination of these different arrangements might help us finally identify the elusive link. Maybe one day, microscopic inhabitants of animal guts will help us uncover the secrets of the gems inside us.

Paper: Vacek V, Novák LVF, Treitli SC, Táborský P, Čepička I, Kolísko M, Keeling PJ, Hampl V: Fe-S Cluster Assembly in Oxymonads and Related Protists. Molecular Biology and Evolution 2018, msy168.

This summer we are organizing the 15th International Congress of Protistology in Prague, Czech Republic. For more information please visit the congress web page, join the Facebook event, or follow the International Society of Protistologists and particularly the #ICOP17 hashtag on Twitter. Here I made two simple maps which might help you to plan your stay in Prague. For official information on public transport in Prague as well as for finding a particular connection you can use the public transport company page. Please, feel free to contact me with any (nonofficial =) ) questions about Prague or the congress.