Amanda suggested that alongside archaeal, bacterial and fungal associated hydrolases, we should also look at protozoans. No problem, I’ll just get the taxonomy ID for protozoa and extract another database from UniProtKB as before. Simple! Or so I thought…

The rabbit hole is pretty deep on this one. Feel free to skip my multi-day exploration in to the history of protozoans and their taxonomy and meet me on the other side.

Classification of protozoa appears to be less clear than I had realised. UniProtKB lists only three taxonomy entries for the term:

• uncultured rumen protozoa
• uncultured Canadian Arcott wether rumen protozoa
• uncultured protist

But none of these are really what I’m looking for. UniProtKB uses these psuedo-species as a catch-all for environmental samples that don’t really fit elsewhere in the database. but I want a high level taxonomic rank like a kingdom that encompasses all of the organisms of interest. So what are the organisms of interest? In an early introduction to my project, I described the protozoa as:

[…] single celled micro-organisms that feed from their direct surroundings and have the capacity for controlled movement with a tendency to thrive in moist environments […]

Yet it seems the answer to “What are protozoa?” boils down to who you ask, or rather, whose interpretation of the taxonomic system you ask.

A Very Brief and Biased History of Modern Taxonomy

This section is mostly an attempt to condense J.M. Scamardella’s 1999 paper: Not plants or animals: a brief history of the origin of Kingdoms Protozoa, Protista and Protoctista. I won’t attempt to present a full history and instead summarise the origins surrounding the protozoa. For a full, interesting review I suggest you check it out.

In the 18th century, Carl Linnaeus published works on the biological classification of organisms based on their structural appearances, establishing the three kingdoms of Animale (Animal), Vegetabile (Vegetable) and Lapideum (Mineral) which lend themselves to a guessing game of the same name. Linnaeus’ work, particularly on naming strategies for organisms served as a foundation to modern taxonomy.

However, the class system set out by Linnaeus was designed for identification rather than heritage. Following Charles Darwin’s revolutionary On the Origin of Species a century later in 1859, tree representations of species sharing common descent became increasingly common (although his tree diagrams were by no means the first¹) and as evolutionary theory developed and took hold, scientists attempted to group available fossil records by structual affinity to link ancestry of common species through the ages. It was here we drew the comparison between contemporary bird species and the dinosaurs.

Meanwhile, micro-organisms exhibiting a variety of characteristics with affinity to both known plants and animals were confusing the topic of taxonomy further…

All Hail the Mighty Kingdom of Protista

Before Darwin, in 1820, the first use of Protozoa appeared in literature from Georg Goldfuss as a class for “first, or early animals” inside the established kingdom of Animalia. Other scientists believed that the protozoa were in fact a phylum, or even a kingdom of their own².

Just a few years after Darwin’s publication and heavily influenced by evolutionary theory, in 1866 Ernst Haeckel proposed the addition of a third kingdom³, the kingdom of Protista. Not seeing a reason for organisms to be binarily classified as plant or animal, Haeckel hypothesized that organisms which cannot be classed as plant or animal “without manifest coercion” must “have evolved independent of the lineages of the animal and plant kingdoms”⁴.

Thus, the kingdom of Protista was proposed to contain “doubtful organisms of the lowest rank which display no decided affinities nearer to one side [animals] than to the other [plants]” or organisms possessing “animal and vegetable characters united and mixed”⁵. Haeckel proposed this as a “kingdom of primitive forms” and included the “Monera” (bacteria) as members of this kingdom too.

It’s clear how such kingdoms are later described in 21st century literature as “a grab-bag for all eukaryotes that are not animals, plants or fungi”⁶, these early classifications appeared to focus on removing contradicting taxa that were clouding “pure” definitions of what was truly plant and animal and choosing to conviniently classify difficult organisms on what they are not, as opposed to what they are⁷. The term became a dumping ground for unicellular protozoa that were not quite animals, protophytic algae that were not quite plants and fungal organisms such as slime molds that appeared to be somewhat both (it wouldn’t be until 1969 that Fungi would get a kingdom of their own).

After the invention of the first electron microscope in the early 20th century, confusion was further compounded by the discovery of a distinct cellular nucleus in some unicellular organisms.

The Protozoan Identity Crisis

Although Haeckel later refined his model, adding the now familiar term protozoa as a “sub-kingdom” to his Protista, to represent unicellular animals, another kingdom schema emerged in 1938. Proposed by Herbert Copeland in The Kingdoms of Organisms⁸, Copeland moved the bacteria and algae out of Haeckel’s Protista kingdom to create a new kingdom: Monera.

Copeland would later re-name his kingdom of Protista to Protoctista (“first established beings”) — a term originally coined by John Hogg. As Haeckel model still persisted, with his Protista kingdom still containing bacteria Copeland deemed it “unfit” to continue using the same name in his model. Selecting a new name, Copeland purposefully avoided the term Protozoa due to both its confusing prior use as a kingdom, class and phylum, but also in agreeance with a point made in Hogg’s 1860 manuscript On the Distinctions of a Plant and an Animal and on a Fourth Kingdom of Nature: that the term “can alone include those that are admitted by all to be animals or ‘zoa'”⁹. i.e. The term is inappropriate if it is to be applied to non-animals.

The Rise of Superkingdoms

In the 1960s, microbiologist Roger Stanier propagated an observed “fundamental division of life” between the “prokaryotes” and “eukaryotes”, originally noted decades prior by Edouard Chatton in 1925¹⁰.

Robert Whittaker published his own five kingdom model in 1969 (a revision of his earlier work where he had in fact returned the bacteria to the Protista kingdom, it would take a few more years of research before concluding “this evolutionary divergence in cellular structure [in bacteria] had to be accounted [for]”¹¹), but placing the kingdom Monera beneath a new Superkingdom of Bacteria (placing all the other kingdoms under the new Eukaryotic Superkingdom) and introducing the kingdom of Fungi; a wholly distinct kingdom from Plantae. Whittaker maintained that unicellularity was the most important characteristic for deciding membership of the kingdom of Protista¹².

Around the same time biologist Lynn Margulis began working on her own schema for organising life. After several iterations, Margulis relied more on morphologic and structural observations over Whittaker’s strict unicellular criteria and allowed her Protista (later re-named to Protoctista as per Copeland’s model) to contain eukaryotic organisms that were “either unicellular or multicellular that are not plants, animals or fungi”[Emphasis mine]¹³.

For those still playing along at home, Whittaker’s five-kingdom re-organisation in 1969 left us with this:

In the early 1980s, John Corliss reviewed the work of both Margulis and Whittaker and tried to solve the question of cellular complexity by instead counting the number of “differentiated, functional tissues” an organism exhibits, rather than just the boolean question of whether or not they are unicellular. Corliss describes plants and animals as having more than one type of tissue, whereas “protists, while showing multicellularity to varying degrees in certain groups […] fail to demonstrate the organization of cells into two or more clearly differentiated tissues”. Though, a criticism of this model is it “overlooks the fact that multicellular, differentiated organisms are known in all four eukaryotic kingdoms” such as cyanobacteria²¹.

In 1989, Michael Sleigh, in his second edition of Protozoa and other Protists opened the introduction with:

The position [regarding the origins of eukaryotes from prokaryotes] is now clearer, and there is much support for the view that eukaryotes are best divided into four kingdoms: Animalia or multicellular animals […], Plantae or green land plants […], Fungi […] and Protista, comprising eukaryoute groups formerly classified as algae, protozoa and flagellate fungi.

It appears that the scientific community were converging on an agreement that establishment of another kingdom was necessary. But there were different arguments as to how to organise the members of each kingdom and what criteria should be applied for classification.

Yet there was even a difference of opinion between Corliss and Copeland with respect to why a kingdom for protists should exist. Corliss believed that a Protista kingdom should exist only if “major uniqueness” can be determined, rather than classfication based on the absence of functions common in other kingdoms. Copeland argued that a shared lack of features or function is “not a detriment to classification as a coherent grouping”²².

Confusingly, Corliss advocated usage of the term protist to refer to any Protista, regards of cellularity. Yet the term protist was originally defined by C. Clifford Dobell in 1911 to specifically refer to Protista demonstrating a “unicellular type of organization'”²³. These sort of disagreements only further cloud my understanding of what the protozoa actually are.

A Very-Very Brief Introduction to Current Taxonomy

Domains and the Sequencing Revolution

With the development of chain-termination DNA sequencing in 1977 by Frederick Sanger, the field of molecular genetics was born. Taxonomy could now be based on differences expressed in the genetic sequences of organisms (particularly in highly conserved subsequences, such as those responsible for constructing ribosomal RNA molecules), rather than by subjective observations of morphology and function.

Indeed in 1990, Carl Woese described “textbook” definitions of the “basic organisation of life” reliant on classical phenotyping as “outmoded” and “misleading”²⁴. Referring to a result from Zuckerkandl and Pauling, Woese states it is clear that “it is at the level of molecules (particularly molecular sequences) that one really becomes privy to the workings of the evolutionary process”, such molecular methods reveal relationships that just cannot be inferred from an organism’s appearance or function²⁵. Certainly there have been many analyses of ribosomal RNA that provide clear evidence for phylogenetic separation of eubacterial, archaebacterial, and eukaryotic organisms.

Woese argued that a phylogenetic system must “first and foremost recognize the primacy of the three groupings, eubacteria, and archaebacteria and eukaryotes”²⁶ above the conventional five kingdoms that had developed over the past few decades that fail to represent an accurate view of the evolutionary relationship between the kingdoms. It was here that Woese proposed a radical change to taxonomy and added the taxonomic rank of Domain, superior to Kingdom.

Whilst Whittaker had previously introduced the concept of a Superkingdom to differentiate between the Monera and the rest of the kingdoms, this distinction merely noted a difference between “bacteria” and “everything else” and didn’t describe the relationship between the kingdoms within. And so, in his 1990 paper: Towards a natural system of organisms: Proposal for the domains Archaea, Bacteria, and Eucarya, Woese introduced the three domains of life²⁷:

Woese didn’t make suggestions for what kingdoms should exist beneath these domains (other than an outline for the archaea, which I will ignore here) anticipating that “analysis of the Eucarya will preserve the kingdoms Plantae, Animalia, and Fungi […] and will replace Protista with a series of kingdoms corresponding to the various ancient protistan lineages”.

The Rise and Fall of the Nine Kingdoms of Eukaryota

Nine Kingdoms for the eukaryotes,
Eighteen Phyla for the protists,
But we were all deceived, for a definition of the protozoa, still could not be agreed.

In 1981, a decade before the Domain, Thomas Cavalier-Smith published his own kingdom model. Initially sub-dividing the eukaryotes in to nine different groups and breaking Whittaker’s Protista kingdom into three: the Archezoa (without mitochondria), Chromista (featuring chloroplasts with particular chlorophyll in the lumen) and the Protozoa, defined by their phagotrophism²⁸ but still somewhat seeming as a bin for whatever couldn’t fit elsewhere. Just two years later, Cavalier-Smith culled his eukaryotic kingdoms to the Animalia, Plantae, Fungi, Chromista and Protozoa (which now contained the relegated subkingdom Archezoa). Bacteria still had their own kingdom but now also contained the archaebacteria²⁹.

In the following decades, Cavalier-Smith would revise his system multiple times as more genetic sequencing data became available for analysis. By 1993, Kingdom Protozoa had 18 phyla³⁰ and as recently as 2010, Cavalier-Smith significantly revised the ordering of subkingdoms between the Protozoa and Chromista. As you’ll see below, Cavalier-Smith’s work serves as a significant foundation of the organisation of taxonomy today.

Getting the Band Back Together: 21st Century Supergroups

With continuing innovation fueling rapidly moving research, especially in the fields of molecular biology and genetics, more sequences could be analysed than ever before. Scientists had been won over by Woese’s domain model and tried to maintain an underlying kingdom structure that maintained the intregrity of the “tree” model, ensuring branches are monophyletic (i.e. branches contain only descendants of that species).

In 2004, Simpson and Roger published a review article, outlining six Supergroups which I have attempted to condense in to the table below³¹. If, like me, however, you prefer pretty colour-coordinated diagrams, refer to the figure below the table instead.

Yet, even now there is no consensus on what supergroups definitely exist, how exactly they are related and what species belong where. As recently as 2007, Burki et al. proposed the SAR Supergroup (which as of this year is now Subkingdom Harosa³⁴), suggesting the Stramenopiles, Alveolata and Rhizaria should be organised beneath one branch together.

Woop Woop. I’m done.

A later paper by Burki introduces the term Megagroup³⁵. At this point, I realised I’ve spent the best part of a week digging for an answer to my classification conundrum that likely does not exist. This is yet another side-quest or interesting avenue that I must turn back on to get back to the research that I should be doing…

Ready or not, here I come!

UniProtKB

That was a lovely, confusing and borderline frustrating insight to the state of taxonomy Sam, but how does this help us get the protozoan-associated hydrolases? Who sets the taxonomic standard for TrEMBL and SwissProt? UniProt’s help documentation quickly offloads the responsibility to the NCBI:

The taxonomy database that is maintained by the UniProt group is based on the NCBI taxonomy database, which is supplemented with data specific to the UniProt Knowledgebase (UniProtKB). While the NCBI taxonomy is updated daily to be in sync with GenBank/EMBL-Bank/DDBJ […]

Accoding to Chapter 4 of the NCBI Handbook, The NCBI Taxonomy Project began in 1991 “to combine the many taxonomies that existed at the time into a single classification that would span all of the organisms represented in any of the GenBank sources databases”. The idea was to solve the problem of duplicated, conflicting and uncomparable taxonomies maintained by the three big sequencing databases: GenBank, EMBL and DDBJ, who were each keeping their own modified classification system originally derived from Los Alamos National Lab by unifying the terminology used.

Six years of curation later, both EMBL and DDBJ switched to adopt the NCBI taxonomic standard; SwissProt followed suit in 2001.

NCBI’s 21 Structures of the Eukaryotes

So, let’s take a look at NCBI’s taxonomy tree. As of May 2015 the following appear as entries beneath the Eukaryota domain superkingdom³⁶ top-level group³⁷:

† Parentheses indicate the entry was unranked by UniProtKB and a rank was interpreted from another source⁴¹
‡ There are no references for these classifications, I’ve just tried to make sense of where the most likely supergroup for each taxonomic entry lies, ? indicates Simpson and Roger were unable to fit this entry in their 2004 model.

Confusingly, most of the 21 entries are not even of the same taxonomic rank. The reasoning for so many “unorganised” entries is lost on me and I can’t seem to locate more information on the methods used to organise taxonomy from NCBI. For most of the entries, UniProt’s corresponding metadata does not even contain a taxonomic rank. I’ve thus lazily inferred missing ranks (in parentheses) from WikiSpecies⁴² Ruggiero et al.⁴³. Regardless, the footer of every page of the NCBI taxonomy database is adorned with:

Disclaimer: The NCBI taxonomy database is not an authoritative source for nomenclature or classification – please consult the relevant scientific literature for the most reliable information.

So perhaps any suggestion that the entries provide anything beyond an agreed set of boxes in which to place organisms in a database is invalid. Though the help pages confirm the schema attempts to maintain a phylogenetic taxonomy, my impression is that the list purposefully contains “lower” ranks such as Class, Order and even Family both to potentially avoid having to keep up with frequent re-shuffles and re-naming of the (Infra-, Sub-, Super-) Groups, Kingdoms and Phyla but also are left broken as a result of some of the changes that have already happened. For example, I can’t find a mention of either the Fornicata or Parabasalia in Ruggiero et al., but one cannot simply expunge records from a widely used taxonomy database without trouble⁴⁴!

So… What are protozoa?

I… I don’t know.

I think it is safe to conclude that the protozoa (or protozoans) consist of an incredibly diverse group of various micro-organisms. There appears to be wide ranging evidence to support phylogenetic separation of protozoans in to distinct groups (with whatever taxonomic rank one wishes to use), each exhibiting different evolutionary variation in their genomes. Whilst it appears that the term is a valid and widely accepted term for unicellular eukaryotes, given the current state of their broken-up taxonomy (and the ambiguous meaning of the terms in the past), I would discourage use of the terms when it is possible to substitute a more specialised term for organisms of interest.

For example Ciliates have their own phylum Phylum Ciliophora⁴⁵) and a quick search for Ciliophora-associated hydrolases returns 1,691 records.

…and where the hell are the virii?

Already far outside the scope of what I wanted to read up on, I’ll save you and myself the history of viral classification schemes and the varying opinions still around today. Suffice to say this is unsurprisingly another topic that the community at large has not reached an agreement on. Virii are typically excluded from the “tree of life” and to many are “non-cellular life” (often explained due to their inability to replicate on their own — they must hijack a cell’s machinery to do it for them) and thus have no place in the tree. Some argue that as evidence points to Viruses co-existing with organisms throughout evolutionary history they deserve representation as a supergroup or domain of their own⁴⁶, indeed viruses are a significant contributor to genetic diversity via their role in horizontal gene transfer (transferring genes from one organism to another (regardless of species) without ‘traditional’ reproduction).

Ultimately whether a virus is truly alive or not is somebody else’s problem, they still possess sequence data and that has to be organised somewhere. The NCBI database has chosen around this by adding a top-level taxonomic entry for “Viruses” with a structure beneath that appears to follow or emulate the Baltimore Classification system; where virii are organised in to one of seven classes based on how they store their genome and their mode of transcription.

Conclusion

The protozoa have had a long and muddled history and my bet is they will continue to do so as further comparative phylogenomics studies are completed. It’s worth noting that phylogenomics is still a field of scientific endeavour in itself, evolving and adapting to new evidence as other fields (including my own) move alongside it. For the purpose of getting on with the study of our own fields it may have been necessary to find a compromise between having the most up-to-date database and having a database that stays constant enough to know where things actually are.

Indeed, on the subject of compromise, Ruggiero et al. describe their work as a “consensus classification“; a “neither phylogenetic nor evolutionary” system that makes “practical compromises” to accomodate many wide ranging opinions and available evidence, primarily with the goal of providing a “backbone” for databases and collections⁴⁷.

In the end, I guess expecting the NCBI (or anyone else) to hold a concrete consensus on the organisation of life was naive and possibly reflects the fact that I’m still more of a Computer Scientist than Microbiologist. Here was me thinking we’d pretty much classified all of life already!

tl;dr

• Protozoa appears to have become a somewhat ambiguous and confusing term over the past few centuries and is applied to a vast number of different species which have now been split across the tree of life.
• I need to go back to original sample and find out what “protozoa” it might contain to try and conduct more specific searches.
• Even today, we still can’t agree on what to call things and where they belong in a taxonomy, or even how best to present that taxonomy. But that’s just how science works.

“No mouth. No respiration. No entry.”

⁶