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Review
Diversity and ecology of protists revealed
by metabarcoding
Fabien Burki1,2,*, Miguel M. Sandin1, and Mahwash Jamy1
1Department of Organismal Biology (Systematic Biology), Uppsala University, Norbyv. 18D, 75236 Uppsala, Sweden
2Science For Life Laboratory, Uppsala University, 75236 Uppsala, Sweden
*Correspondence: fabien.burki@ebc.uu.se
https://doi.org/10.1016/j.cub.2021.07.066

SUMMARY

Protists are the dominant eukaryotes in the biosphere where they play key functional roles. While protists
have been studied for over a century, it is the high-throughput sequencing of molecular markers from envi-
ronmental samples — the approach of metabarcoding — that has revealed just how diverse, and abundant,
these small organisms are. Metabarcoding is now routine to survey environmental diversity, so data have
rapidly accumulated from a multitude of environments and at different sampling scales. This mass of data
has provided unprecedented opportunities to study the taxonomic and functional diversity of protists, and
how this diversity is organised in space and time. Here, we use metabarcoding as a common thread to
discuss the state of knowledge in protist diversity research, from technical considerations of the approach
to important insights gained on diversity patterns and the processes that might have structured this diversity.
In addition to these insights, we conclude that metabarcoding is on the verge of an exciting added dimension
thanks to the maturation of high-throughput long-read sequencing, so that a robust eco-evolutionary frame-
work of protist diversity is within reach.

Introduction: Protists in a nutshell
For all of the importance of understanding biodiversity, and how
to preserve it in the face of rapid and global environmental
changes, there is a category of organisms that is rarely dis-
cussed. These often small organisms — called protists — are
much less studied than their macroscopic counterparts among
eukaryotes (animals, plants, and fungi), yet they are increasingly
recognised to play critical biogeochemical and ecological roles
in most ecosystems. As microbes, protists represent the vast
bulk of eukaryotic diversity, they live in virtually all environments
on Earth, and their biomass at global scales is immense (esti-
mated to be twice that of all animals)1–4. Understanding this
diversity, and how it interacts with other microbes and larger or-
ganisms to form functioning ecosystems in all biomes, is funda-
mental to provide a holistic view of biological communities.
Before we move to the core concepts of this review, it is
necessary to define what we actually mean by protists. This is
because there is not a single accepted definition of what protists
are and, as a fundamentally paraphyletic assemblage, protists
represent a working concept rather than a biological entity5. His-
torically, protists have been widely regarded as a grab bag
including anything eukaryote that is not an animal, land plant,
or dikaryon fungus6. Although this is a definition by exclusion,
it is useful because it clearly delineates — phylogenetically —
what does or does not go in this grab bag. But it also means
that the morphology, ecology, or more generally biology of pro-
tists encompass almost all of the broad spectrum of traits that
we otherwise associate with eukaryotes. Indeed, protists are
not only an essential component of today’s ecosystems, they
have also been the only eukaryotic component for most of life’s
history and as such have evolved into an incredible diversity of
forms and functions1,7.
Protists are generally microscopic, unicellular eukaryotes but
collectively they span more than six orders of magnitude in
size (from eukaryotes smaller than bacteria to seaweeds taller
than 10 story buildings). They can live solitary lives, preying on
other small eukaryotes or prokaryotes, or form large colonies
of a few meters. Protists can be purely phototrophic, in which
case they are referred to as algae, or purely heterotrophic if
they belong to the organisms traditionally called ‘protozoans’.
In-between these two modes of nutrition, there are also a wide
range of mixotrophic behaviours where phagotrophy and photo-
trophy co-exist in the same cells8. While some species can grow
to very high densities if environmental conditions are favourable,
most of the diversity remains regional and at low abundances;
this diversity is known as the ‘rare-biosphere’9. Sometimes,
blooms of microscopic protist algae are so widespread that
they can be seen from space and, when taken collectively, these
algae contribute as much to primary production as land plants10.
More generally, protists are essential to ecosystem functions as
partners in diverse symbioses (host and/or symbiont), as key
regulating parasites (or parasitoids) of natural populations, and
as intermediates in multiple trophic levels between small animals
that prey on them and other microbes they consume11.
In recent years, high-throughput methods and large-scale
sampling of different environments have provided unprece-
dented insights into the diversity and ecology of microbes. In
this review, we discuss how the environmental DNA sequencing
of protists has transformed our understanding of eukaryotic di-
versity as a whole, from revealing unprecedented lineage

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Figure 1. Environmental sequencing and
metabarcoding.
Overview of environmental sequencing with
emphasis on metabarcoding (see Box 1 for a
description of the main steps). Environmental
samples can be collected from any environment
such as soil, sediment, water, air. Aquatic samples
Collect can be size filtrated to study the different size
environmental fractions separately. Following total DNA and/or
sample cDNA extraction, and depending on the aim of the
study, metabarcoding and/or meta-genomics/
transcriptomics approaches can be carried out.
For metabarcoding, eukaryotic primers (taxo-
nomically broad or more restricted) are used to
Soil Sediment Water amplify targeted fragments (short or long) of the
rDNA operon which are then sequenced.
Sequence data are cleaned, clustered into oper-
ational taxonomic units (OTUs) or Amplicon
Sequence Variants (ASVs) and used for taxonomic
Total nucleotide and ecological analyses.
extraction

Metabarcoding Metagenomics and

Amplify rDNA
metatranscriptomics
fragment for
organisms that have not been isolated
metabarcoding,
or shear total or cultured. eDNA sequencing also per-
DNA/cDNA for mits the identification of species with
Short-read or Long-read metagenomics/ seemingly non-differentiable morphology
meta- (i.e., cryptic diversity)17, and even species
transcriptomics
that have gone extinct due to the preser-
Generate vation of DNA in sediments for thousands
High-throughput of years18.
sequencing sequencing
data Several options are available to molec-
ularly survey the environmental diversity,
including metagenomics (DNA) and meta-
Data analysis transcriptomics (RNA), which sequence
(shown for the total nucleic acids of environmental
metabarcoding samples, or the targeted metabarcoding
Cluster sequences into Identify taxa by only)
Community approach (Figure 1 and Box 1). While
OTUs / generate ASVs comparing to
reference database analyses meta-omics has been widely applied to
studying prokaryote diversity19,20, and
Current Biology
increasingly also for eukaryotes21,22,
metabarcoding is currently more routine
diversity in all parts of the eukaryotic tree, to organizational pat- for analyzing protistan diversity and distributions in nature. As
terns and processes of this diversity within and across major bi- such, metabarcoding has resulted in a very large body of exciting
omes and through evolutionary time, and to shedding light on the insights from all environments, which form the core of this review.
relative contribution of the different functional roles of protists in We will also focus on insights gained from the studies of the ‘total’
ecosystems. eukaryotic diversity of broadly defined ‘traditional’ environments
such as soil, sediments, and water. The word ‘total’ here is meant
Environmental DNA sequencing of protist diversity to indicate the intention rather than the certain output of environ-
Generally speaking, DNA (or RNA) sequencing can be done in mental sequencing, even when studies are designed to capture
two main ways depending on the source material: directed at in- all diversity. Indeed, an array of methodological and biological
dividual taxa, for example from a culture or isolated organisms, reasons typically prevent the recovery of the full diversity present
or aimed at sequencing the entire DNA pool of a community — in an environment, or can recover the DNA of organisms not
a process that is referred to as environmental sequencing. A necessarily present or alive at the time of sampling (Box 2).
community here represents many different entities within More than any other molecule(s), it is the sequencing of the ri-
different environments and scales, from entire ecosystems bosomal DNA operon genes (Figure 2), especially that of the
(e.g., oceanic scales12, or a lake13, or soil in a forest or a field14,15) gene coding for the small ribosomal subunit (18S rDNA), that
to host-associated microbiomes in a single specimen or a partic- revolutionised our understanding of protist diversity. Other mo-
ular tissue16. Although environmental DNA (eDNA) is typically lecular markers have been preferred to study more restricted
sampled with no other associated identifier than the environment taxonomic groups, including markers such as the internal
itself, the sequencing of eDNA allows the detection and charac- transcribed spacer (ITS) of the ribosomal operon for fungi23
terisation of natural communities without a priori knowledge of or oomycetes24, the chloroplastic ribulose-1,5 biphosphate
what members belong to these communities, in particular for carboxylase–oxygenase large subunit (rbcL) gene mainly for

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Box 1. Steps in metabarcoding analysis of protist communities.

Metabarcoding (also known as amplicon sequencing, or sometimes metagenetics) consists of several interconnected steps that
need to be critically assessed for correct interpretation. This box gives a brief overview of the main steps in a typical metabarcoding
analysis, but extensive literature exists for more details142,143. See Figure 1 for an illustration.
Study design. Arguably the most critical step when studying protist diversity is the scientific question. Depending on the scientific
question asked, the study might for example focus on one or several environments, on a specific taxa or functional group, on a
given time-frame or a temporal series. Consequently, the scientific question will determine the sampling strategy, i.e. the
sampling location and frequency, the sampling method, the sample volumes, biological replicates, storing, and the bioinformatic
analysis.
Laboratory. Metabarcoding requires the extraction of DNA (or RNA) from environmental samples. Commonly, DNA/RNA is ex-
tracted by breaking the cell, either chemically by detergents or physically by freezing and thawing. Thereafter, the targeted
DNA region undergoes PCR amplification using sets of primers that might be more or less specific for particular taxonomic
groups. Decisions on sequencing library preparation, reagents, multiplexing, or the choice of primers (Box 2) may all affect the
amplification process. After amplification, the amplicons are sequenced on a high-throughput sequencing platform, the choice
of which is typically based on a trade-off between three factors: the sequencing depth, the read length and the error rate.
Bioinformatics. After sequencing, the reads need thorough processing. This includes basecalling, removing low quality reads,
pairing pair-end reads, demultiplexing, dereplication, de novo chimera identification, etc. Probably the most debated procedure
of the read processing is read clustering or denoising into so-called Operational Taxonomic Units (OTUs) or Amplicon Sequence
Variant (ASVs), respectively (Box 2). To avoid clustering, it is possible to work with raw reads after applying stringent quality
and abundance filters. Extracting biologically meaningful reads is normally achieved by filtering based on arbitrary thresholds
of abundance and/or presence on different samples. This step attempts to remove further artifacts and errors not identified in
previous steps. Clean reads are also frequently assigned with taxonomy by comparison to reference databases, ideally curated
(Box 2).
Later, it is possible to conduct the data analysis in order to explore biological patterns such as multivariate analysis, beta/gamma
diversity, sequence similarity networks, etc.
Replicability. The last crucial step in metabarcoding analysis is the data deposition in public repositories or libraries. The data
deposited should contain, at the very least, everything needed for the reproduction of the given study, from associated metadata
to sequencing platform and technology. This step ensures that the analysis performed and the conclusions stated in the given
study can be replicated, confirmed with different approaches or improved with new methods/algorithms.

plants25 but also for example in diatoms26, and the mitochondrial
cytochrome oxidase c subunit I (COI) gene for a range of micro-
bial groups (e.g., red algae27, brown algae28, and dinoflagel-
lates29) and, especially, animals30. But it is the universality,
phylogenetic informativeness at different taxonomic levels, and
relative ease of amplification of the 18S rDNA gene that have
made this marker so widely used across the broad eukaryotic di-
versity31. Notably, the stark differences in evolutionary rates
along the rDNA operon result in conserved and more variable re-
gions (Figure 2) that can be advantageously used for the study of
the diversity at different hierarchical levels, by comparing
conserved regions for distantly related taxa and variable regions
for closely related taxa.
Initially, because sequencing methods such as Sanger allowed
the acquisition of relatively long sequences (up to 1000 bp),
environmental clone libraries of the full or near-full 18S rDNA
gene were sequenced. These environmental sequences were
long enough to be phylogenetically interpreted, leading to the
placement in trees of groups unseen before32–35. Rapidly, how-
ever, the development of high-throughput sequencing technolo-
gies (e.g., 454-pyrosequencing, Ion torrent, Illumina) took over
environmental sequencing, but in doing so introduced severe lim-
itations on the lengths of the fragments that could be targeted.
With a maximum length of 500 bp, these new metabarcodes
lacked the earlier phylogenetic signal of environmental clone li-
braries. Nevertheless, most of today’s metabarcoding datasets
are made of millions of short sequence reads (most often
produced by Illumina), which for protists often target either the
hypervariable regions V4 (frequently in combination with the
V5)36 or V937 of the 18S rDNA gene (Figure 2).
Despite their short lengths, the abundance and sequencing
depth of metabarcodes created exciting opportunities for dis-
coveries, enabling systematic diversity surveys of a multitude
of environments, including the detection of rarer and previously
overlooked taxa12,38–40. But this mass of short reads also pre-
sented new challenges, especially for their annotation with taxo-
nomic information. Currently, there are two main approaches for
annotating short metabarcoding datasets: using global or local
pairwise similarity searches against databases of reference se-
quences, such as PR241 or SILVA42, or ‘placing’ the short reads
onto a phylogeny of reference sequences43,44. A reference
sequence in these contexts is a verified — although with varying
levels of curation — sequence most often coming from identified
organisms45. While fast and reliable when similar references
exist, pairwise similarity methods are less accurate for divergent
sequences (such as for novel diversity or fast-evolving taxa), and
are heavily dependent on the taxonomic breadth and quality of
the reference databases. The phylogenetic placement methods
(e.g., EPA or pplacer) are better suited to identify sequences
distantly related to references (e.g., novel groups)14,43, yet they
still require the availability of high-quality and ideally long se-
quences to build a robust reference phylogeny.
As sequencing technologies continue to evolve, a new gener-
ation of environmental sequencing came to light that utilized

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Box 2. Limitations of metabarcoding, and solutions to deal with them.

Metabarcoding study design, execution, and analysis come with a range of potential technical and biological limitations. These
limitations have been identified and can be addressed to unravel the full potential of metabarcoding142–144. The most common
source of biological limitation comes from the multicopy nature of the rDNA operon and the variability in the number of copies
among different taxa. Copy number of the rDNA has been positively correlated to cell size145. Therefore, derivation of cell abun-
dance based on read counts should be interpreted with caution, especially for larger size fractions. For this reason (among others),
metabarcoding analysis provides only relative information, or semiquantitative information after correction, which requires prior
knowledge of the studied taxa. The multicopy nature of the rDNA operon not only affects abundance estimates, but also tends
to inflate diversity estimates because of intragenomic polymorphism among the copies146. Errors in DNA amplification or
sequencing, tag-jumping during library preparation147, cross-contamination during sequencing, or extracellular DNA may also
contribute to overestimate the diversity. A recently developed tool, LULU148, attempts to remove biologically redundant se-
quences (such as intra-genomic variability) by post-clustering reads based on similarity, abundances and co-occurrence.
In order to lessen the impact of these errors, as well as to reduce redundant diversity and decrease computational effort, reads are
traditionally clustered based on a similarity threshold, often 97–99% for protists, into OTUs. Several approaches exist to assemble
OTUs. One popular approach is to use single nucleotide differences and read abundances in order to cluster based on natural
thresholds (i.e., swarm149). Other approaches are based on the direct estimate and correction of the expected error rate (denoising)
of the given dataset, such as the Amplicon Sequence Variants (ASVs) produced by the DADA2 pipeline150.
At the same time as risks exist to inflate diversity estimates, the opposite problem — diversity underestimation — can also happen.
Environmental surveys typically lack technical replicates (i.e. multiple sequencing runs of the same sample), which means that it is
difficult to assess the true biological meaning of rare reads. Even though metabarcoding has become extremely high-throughput,
undetected taxa are not necessarily proof of absence because truly low abundant taxa may be lost (removed through physical
filtering), damaged (during storing) or not sequenced (stochastic factors). In addition, there are no truly universal primers covering
all eukaryotic diversity151. The presence of introns or insertions in the genetic marker can also hamper the sequencing of specific
diversity. The selection of the primers (and therefore of the genetic marker) will affect the taxa that are successfully amplified,
regardless of the natural abundance.
Lastly, reads are normally compared to a reference sequence (morphologically identified and annotated sequence) by either global
or local alignment, resulting in a similarity score and/or a confidence estimate of the assignment. However, this step strongly relies
on a comprehensive and carefully curated reference database, such as PR241 or SILVA42, of sequences previously sequenced and
annotated. The incompleteness of the reference database may lead to erroneous identifications of taxa. Therefore, the taxonomic
assignment of environmental diversity has to be taken with caution. Other methods implement phylogeny-aware annotations in
order to account for uncertainty and missing data in the reference database43,44,49.

long-read technologies such as Pacific Bioscience (PacBio) or
Oxford Nanopore Technologies (ONT). These technologies
have recently produced high-quality long-read metabarcoding
datasets of between 1,500 and 5,000 bp46–49, although in theory
the size limit is much higher than this range. Such datasets are
appealing because they can easily contain the entire 18S rDNA
gene, or even the full-length of the rDNA operon. This means
that it is possible, in a single molecule, to cover many classical
protist barcodes at once (e.g., V4, V9), as well as other markers
in the 28S rDNA50,51 or the highly variable ITS region23,24
(Figure 2), while allowing for direct comparison to the vast
body of knowledge provided by the 18S rDNA gene. The long
metabarcodes can also be used in addition to reference
sequences to produce denser and more robust reference phy-
logenies for the environmental diversity, allowing for direct
phylogenetic comparison of the environmental sequences them-
selves49. These are obvious advantages, yet it remains critical to
fully assess the strengths versus weaknesses of long-read meta-
barcoding because with enhanced sequence length comes po-
tential new biases. Among other issues, long insertions in
some protist groups may prevent amplification, and there is
potentially a higher risk of forming chimeric constructs for which
specific detection tools need to be developed. Still, we have
come a long way from the early days of low-accuracy long-
read sequencing technologies, as today millions of long
metabarcodes can be routinely produced with a quality similar
to that of Sanger sequencing52. In the future, it is likely that envi-
ronmental sequencing will greatly benefit from the improved
phylogenetic information contained in long metabarcoding
datasets, especially in hybrid approaches that combine the
current higher throughput and cheaper cost of short read
metabarcoding.
Diversity and abundance of protists derived from
environmental sequencing
Prior to the advent of environmental DNA sequencing, more than
a century of protistology studies based on the isolation of organ-
isms provided us with a rich account of what many of the main
groups of protists are. This seminal and extensive body of
work was first based on the morphology and functional charac-
teristics of the isolated organisms (e.g., algae, small hetero-
trophs, absorptive nutrition)6,53, but was quickly complemented
by molecular phylogenies when the sequencing of DNA became
practicable54. Nowadays, the study of the evolutionary relation-
ships among the main groups of eukaryotes at the deepest
phylogenetic levels almost always involves multiple genes, often
hundreds of genes in a practice known as phylogenomics7.
These deep phylogenetic relationships have considerably
changed over the decades, and continue to be challenged espe-
cially by the continuous flow of new genomic data for isolated

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18S 5.8S 28S

ITS1 ITS2
V4 V9 D1 D2

Sanger
Short read
Long read
Entropy

Alignment position
Current Biology

Figure 2. Ribosomal DNA genetic structure.

A schematic representation of the ribosomal DNA (rDNA) operon genetic structure, including the genes coding for ribosomal RNAs (18S, 5.8S and 28S) and
transcribed spacers (ITS 1-2). The rRNAs assemble together with a number of ribosomal proteins to form the two subunits of the ribosomes. The 18S rRNA is a
component of the small subunit (SSU) of the ribosomes; the 28S rRNA along with the 5.8S rRNA are components of the large subunit (LSU). In most studied
eukaryotes, the rDNA operon is arranged in tandems repeated in several copies in the genome. The arrow lines below the coloured boxes show comparison of the
typical lengths of Sanger, short read and long read metabarcoding. Note that long read sequencing is potentially able to cover the (near) complete length of the
rDNA operon (dotted arrow). The line plot shows the different conserved and variable regions of the 18S and 28S rDNA genes as measured by Shannon Entropy.
The higher the entropy value is, the more sequence variability among eukaryotes is found. Shannon entropy values were smoothed by calculating at every given
position the average of a 100 bp range (± 50 bp), based on the alignment from Bernier et al.152 after removing positions with less than 30% of the sequences. The
highlighted hypervariable regions V4 and V9 are the main metabarcodes used for protists at large.

species but also increasingly from uncultured taxa55,56; a
description of the major eukaryotic groups, and their evolving re-
lationships, have been recently discussed elsewhere1,7.
Remarkably, however, it is worth noting that the catalogue of
these major protist groups has not been fundamentally altered
since environmental sequencing, even high-throughput meta-
barcoding, came into play. In fact, less than a handful of high-
ranking environmental lineages branching outside of established
major groups have been discovered since the beginning of envi-
ronmental sequencing (e.g., Picozoa57, Rappemonads58, or a
novel group related to animals called MASHOL59), some of which
have now been assimilated to known groups (e.g., Rappemo-
nads belong to haptophytes60).
We can identify a few reasons as to why, perhaps against our
initial expectations, high-throughput metabarcoding has not
yielded the discovery of independent major groups of eukary-
otes. After all, this is a method which by its sheer sequencing
power could in theory sequence up to the last cells in any envi-
ronment (but see Box 2). Firstly, as said above, the long history
of microscopic descriptions and molecular phylogenetics of iso-
lated taxa did a really good job at establishing the overall picture
of protist diversity, so the emergence of metabarcoding built on
an already richly populated diversity framework especially for the
most abundant, economically important (e.g., pathogens), or
easily identifiable (e.g., large cells, algae) taxa. But this rich his-
torical account only partially explains the absence of novel
high-ranking groups in metabarcoding datasets, because we
know that such undescribed diversity exists from regular discov-
eries based on much lower-throughput, classical culturing ap-
proaches. For example, in the last five years, at least four major
new branches in the tree of eukaryotes have been discovered or
re-described, all from cultivation (Rigifilids61, Ancoracysta62,
Hemimastigophora63, Rhodelphids64).
We argue that the contrast between metabarcoding and culti-
vation in our ability to identify novel high-ranked diversity primar-
ily stems from the current lack of phylogenetic signal in short
read metabarcodes, and from the lack of reference sequences
for groups that have not been discovered yet (logically). Indeed,
most metabarcoding datasets do contain a proportion, often sig-
nificant (e.g., about 30% in the oceans), of sequence diversity
that cannot be reliably assigned to known groups due to high
dissimilarity to references12. This unassignable diversity mainly
corresponds to the smallest and most rare protists12, which
coincidentally are the organisms that have historically been the
least studied. It is therefore reasonable to assume that among
this unassignable diversity lie novel major eukaryotic groups
awaiting characterization. Furthermore, there are other issues
that prevent environmental sequences from being generated
from taxa even if they exist. For example, some lifestyles are
more likely to be underrepresented in environmental DNA sur-
veys, notably obligate parasites of animals or endosymbionts
in general, simply because those organisms are filtered out
from common sampling practices that remove the larger size
fractions corresponding to the hosts. So unless specifically tar-
geted, host-associated organisms are often missed65. Other
inherent biases of metabarcoding, most importantly the choice
of primers or the presence of insertions (Box 2), will select
against species that are too divergent, resulting in some diversity
remaining hidden unless specifically targeted66,67. Lastly, while
some environments have been heavily sampled (most notably
the surface oceans), other environments remain severely under-
explored and thus constitute an untapped reservoir of potential
new diversity. These poorly studied environments not only corre-
spond to far and difficult places to reach, such as the deep-sea
or dense tropical forests, but also environments closer to us like
lakes, ponds, rivers and soil, as well as whole hosts or specific
tissues, that should all be the target of future sampling effort if
we are to more fully grasp protist diversity68.
So if not the discovery of novel high-ranked diversity, and amid
the issues mentioned above, what have we learned about the di-
versity of protists from nearly 15 years of metabarcoding? The
short answer is: a great deal, obviously. First and foremost, it
is the expansion, generally massive, of the molecular diversity
within already established groups that jumps to mind (Figure 3).
Virtually every known group of protists saw its diversity soar with
the addition of environmental lineages we had little suspicion

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Environment
Marine
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Figure 3. Protist diversity across three environments.

A simplified representation of protist diversity across three main environments (marine, freshwater and soils) adapted from Singer et al.68. Briefly, bars represent a
prediction of the V9 rDNA OTU richness by 100 bootstraps after rarefaction (further details in original reference). Red dots indicate the main groups of parasites
discussed in the text (but note that parasites are found in many other groups). Note that Embryophyceae (within Chloroplastida), Fungi, Metazoa and Rhodophyta
were not considered in the original study. Groups that are environmentally rare can escape detection (i.e., Glaucophytes, Malawimonads), as can groups that are
specific to certain environments (e.g., Metamonads and Breviatea have anoxic or micro-oxic preferences), while other groups that were only recently described
lack reference sequences (e.g., Hemimastigophora). The tree is based on a consensus of recent literature7.

existed. In the oceans, this is true for some of the better studied known to harbor the richest protist communities on Earth, with
groups, such as diatoms69 and dinoflagellates70, but also for lin- rarefaction curves showing no sign of plateauing14,68. Soil eco-
eages with only few named species like MALVs and MASTs (Ma- systems are not only diverse, they also contain more unknown
rine Alveolates and Stramenopiles, respectively). MALVs and diversity (i.e., OTUs with lower similarity to reference sequences)
MASTs are particularly significant because they were among than freshwater, and especially marine environments14. Interest-
the first marine environmental diversities to be described32,33,35, ingly, comparisons across these ecosystems for the major pro-
and together represent dozens of clades that contain some of tist groups have started to reveal extensive composition shifts,
the most diverse and widespread lineages in both surface and with terrestrial communities being much more similar to each
deep waters12,71. These hyperdiverse lineages, including others other than to marine communities in terms of both diversity
like radiolarians (e.g., acanthareans, collodarians) or hapto- and abundance68. As a result, some of the richest and most
phytes, have been recognized to play key roles in marine abundant groups in the oceans (e.g., radiolarians, diplonemids)
plankton biodiversity and ecology for a long time12. But other lin- are almost absent from terrestrial environments, while rare and
eages, although apparently also extraordinarily diversified, had low-diversity marine groups (most notably cercozoans and
remained tucked away in the oceans until global metabarcoding some amoebozoans) appear dominant in soils (Figure 3).
efforts. This is most strikingly the case of diplonemids (Figure 3), While most studies have thus far focused on the most abun-
a group with just a few described species but that was shown to dant taxa in the environment, rare taxa (i.e., those with low local
contain over 12,000 Operational Taxonomic Units (OTUs) in the relative abundance) have also taken on a new significance as a
Tara Oceans dataset12,72 (although a subsequent single-cell result of metabarcoding9. After a rocky start trying to weed out
genome analysis indicated that some of this diversity at least noise in the data (e.g., sequencing errors) and DNA present in
may be due to intragenomic diversity of the 18S rDNA gene73). the environment but not representative of active cells (e.g., mori-
Many of the hyperdiverse taxa also appear hyperabundant, bund or dead cells, resting stages, or extracellular DNA), studies
with sequence read counts reaching unexpected heights for lin- have shown that a metabolically active but rare protist biosphere
eages like diplonemids, collodarians, MALVs, or diatoms12. is a common occurrence9. In fact, this rare biosphere seems sta-
The diversity and abundance of marine protists is thus ble across communities, thus possibly representing an equilib-
revealing, but the total species richness of protists becomes rium shaped by biotic and abiotic interactions (although the
even more staggering after looking at terrestrial environments ecological functions of the rare biosphere remain hypothetical)9.
(freshwater, soil). Although less studied, soils in particular are Strikingly, this rare biosphere contains even more diversity than

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A B Ordination of marine and Figure 4. Protist diversity patterns.

Everything is not everywhere terrestrial communities A simplified representation of some of the most
important diversity patterns of protists at global
scale. (A) The vast majority of taxa have restricted
Freshwater biogeography, and only a few hyperdominant taxa
Marine are cosmopolitan. Figure adapted from de Vargas
euphotic et al.12. (B) Marine and terrestrial (freshwater and
soil) communities form distinct clusters68. Within
Total abundance

the marine habitat, communities from the euphotic

OTU
No. of sites
C Ordination of sunlit marine
communities (size > 20 µm)
and aphotic layers are also distinct71. (C,D) Marine
surface waters are the best studied ecosystem,
and size filtration of water samples makes it
possible to investigate patterns of diversity across
different body sizes. Large protists tend to cluster
by ocean basin, while small protists display more
fine-grained structure, consistent with Longhurst
Marine biogeochemical provinces12,87. (E) Global soil
aphotic Soil
studies have found that the mean annual precipi-
tation is the best predictor of soil commu-
nities39,82. (F) A trend of decreasing diversity
towards the poles has only been detected in
global marine surface so far, not in the marine
aphotic100 or soil39, and has not been investigated
D Ordination of sunlit marine in freshwater.
communities (size < 20 µm)

rare OTUs seems to be evolutionarily

related mainly to other rare OTUs, sug-
gesting that some groups at least repre-
sent permanently rare taxa (whereas
other taxa might increase in relative
abundance in response to environmental
changes)74. Because these rare taxa are
among the least studied protists, and
almost none have been characterised
Ocean basin Longhurst province phylogenetically, it is likely that the rare
biosphere includes the greatest potential
for new lineage discoveries.
Taking all these results together, it is
E Ordination of soil communities F Latitudinal diversity gradient
evident that the unprecedented breadth
Marine Marine of metabarcoding data for protists has
Dry soil Humid soil surface aphotic Soil
transformed our understanding of eu-
communities communities
karyotic diversity in general, removing
High

any possible doubts that these tiny or-

ganisms are the most diverse and abun-
dant eukaryotes in the environment.
Latitude

0 Global and local patterns of protist

diversity, and the processes
governing them
The breadth of metabarcoding data has
High

not only given a better sense of the vast

Low
more frequently encountered taxa, suggesting that abundant
OTUs are always a minority in nature, while rare OTUs encom-
pass the highest protist richness, up to a staggering 99% of all
reported OTUs in some cases9. Moreover, the majority of these
taxonomic diversity of protists, it has
High Low High Low High also provided new opportunities to effec-
Diversity tively study how this diversity is struc-
Current Biology tured within and across environments.
This is a vital task to better understand
the factors governing the assembly of
protist communities, but also to predict
how these communities will respond to global climate change75.
As often, a background of knowledge is available for animals and
plants76, but protist communities are structured following
different patterns (described below; Figure 4). While the

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OPEN ACCESS
dominant processes structuring these microbial communities
are still largely unknown, a range of mechanisms has been pro-
posed, including: biogeography driven by abiotic factors (e.g.,
salinity, pH, temperature, etc.) and biotic factors (interactions
with other protist and non-protist members of the community),
which can strongly influence which species are found in a habitat
by filtering out ill-adapted ones; new species can be introduced
into communities through dispersal, as well as speciation; and
community composition can also change simply due to stochas-
ticity (or sometimes referred to as ecological drift)77. These pro-
cesses all act in conjunction but the relative importance of each
varies depending on the scale of sampling and the environment.
For example, protist communities separated by one meter will be
less (or not at all) influenced by dispersal limitation than commu-
nities separated by hundreds of kilometers, and this effect will be
less pronounced in marine environments than in more heteroge-
neous soil systems78. Below we briefly describe some of the
main findings from protist ecology studies, namely endemicity
of protist taxa, structuring of protist communities at global and
local scales, and global patterns of species richness.
One of the main long-standing questions related to the organi-
sation of microbial communities is how much of microbial diver-
sity is cosmopolitan. A popular idea in the 1990s and early 2000s
was formulated in the Baas Becking hypothesis, which stated
that ‘‘Everything is everywhere, but the environment selects’’79.
This hypothesis proposed that the same microbial taxa will be
found anywhere a suitable habitat exists, with no geographical
limits, because microbes can disperse with no limitation due to
their small size and high abundance. The availability of global-
scale metabarcoding studies provided excellent opportunities
to put this hypothesis to test, and it turned out that the vast ma-
jority of protists are in fact not cosmopolitan but instead have
restricted geographical distributions (Figure 4), in part due to
dispersal limitation12,40,80–82. For example, in the surface ocean
only 0.35% of all OTUs (381 out of 110,000 OTUs) were
deemed cosmopolitan12, and this was even less the case in
soil (0.15% of cosmopolitan taxa)82. But what these cosmopol-
itan taxa lack in diversity, they make up for in abundance: these
few OTUs are indeed typically hyperdominant in all locations,
making their role in ecosystem functioning likely critical83.
The biogeographical structuring of protist communities hap-
pens at different scales (i.e., global or local) and seems largely
dependent on cell size and the environment (e.g., marine or
terrestrial) (Figure 4). As previously mentioned, marine and
terrestrial communities have been found to be compositionally
distinct, indicating that salinity is perhaps one of the strongest
factors shaping protist communities68,84. In the global ocean,
protists form separate clusters corresponding to the sunlit and
deeper dark regions, reflecting different physico-chemical con-
ditions along the water column, as well as different trophic
modes (see next section)71,85. On the surface, marine protists
in the larger size fractions (>20 mm), such as foraminiferans
and radiolarians, are more similar to each other within the
same ocean basin than communities across different basins, re-
flecting the role of major oceanic circulation patterns12,86,87.
Converesely, smaller protists (<20 mm) display biogeographic
patterns at finer scales that are consistent with more limited
Longhurst biogeochemical provinces, each of which has a
unique set of ecological conditions88. These biogeographic
R1274 Current Biology 31, R1267–R1280, October 11, 2021
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patterns thus suggest that smaller marine protists (especially
phototrophs, for which these patterns are more pronounced)
are more intimately linked with abiotic conditions such as
nutrient availability and temperature than larger protists86,87. In
contrast, soil and freshwater environments represent more
discrete habitats than marine systems with abiotic factors
showing a patchy distribution even at small scales. As a result,
protist communities in these environments are more heteroge-
neous than in marine systems and are overall less predicted by
geographic distance39,68,82. At a global scale, soil protist com-
munities are better explained by environmental conditions, in
particular mean annual precipitation, but other abiotic factors
such as pH, temperature, and to some extent geographic dis-
tance also shape communities39,80,82,89. Co-occurrence pat-
terns between protist and bacterial species have also been
observed in soils and marine systems globally, highlighting the
general importance of biotic interactions39,90.
At smaller spatial scales, both dispersal limitation and environ-
mental conditions (such as temperature and salinity) contribute
to protist community assembly in marine waters91,92. Thus, local
oceanic features such as hydrothermal vents93 and anticyclonic
gyres94 can lead to shifts in the communities even over short
distances. For terrestrial ecosystems, the effects of dispersal
limitation, which is typically not detected globally, become
more manifest locally, for example in soils from different
Neotropical forests81, lakes across Europe40, and paddy fields
in East Asia95. However, the relative impact of dispersal limita-
tion is variable, perhaps depending on the scale of the study,
and environmental factors such as nutrient content, tempera-
ture, elevation, bacterial community, etc. also contribute to pro-
tist community assembly96–98. Furthermore, dispersal limitation
and selection are not the only factors influencing protist commu-
nities; for instance, the protist communities in lakes in Eastern
Antarctica seemed to have been assembled primarily through
stochasticity99.
Finally, metabarcoding studies have also investigated pat-
terns of species richness39,100. For instance, a large study of a
marine macroclimatic latitudinal gradient from tropic to poles re-
vealed a trend of diminishing diversity towards the poles, paral-
leling the poleward decline of local diversity known for larger or-
ganisms100 (Figure 4). The causes of this pattern are unclear,
but it could be that protists in general are less tolerant of colder
temperatures, and/or the warm tropics might be the sites of
faster speciation rates due to higher kinetic energy and thereby
faster mutation rates101,102. A similar pattern was not detected
in the deeper ocean layer, probably because of a lower range
of temperature differences between the poles and equator and
increased nutrient availability100, nor has it been found in soils39.
The elevational diversity gradient has been less studied, but
there is some mixed evidence that protist species richness de-
creases with increasing elevation, as is the case for plants and
animals103,104.
Functional diversity of protists inferred from
metabarcoding
Beyond cataloguing protist diversity, and explaining organisa-
tional patterns of this diversity, one burning question raised by
metabarcoding studies is: what does all this diversity actually
do in the environment? Simply put, protists play the same broad

Review
functional roles as the rest of cellular life; that is they produce,
degrade, and consume. While this is nothing new, the
actual functional diversity of protists remains in many ways
underappreciated3,105. In reality, the enormous size range
and taxonomic diversity of protists, their frequent abundance,
their many nutritional modes, their complex morphology and
behaviours, and their rapid metabolic rates result in key and
complex functional roles in the first several trophic links of food
webs. These roles have been best studied in marine sys-
tems3,11,105, but they are also increasingly scrutinized in terres-
trial environments2.
The importance of primary production in the oceans for global
carbon fixation, with marine unicellular algae accounting for
about as much as terrestrial plants, is well established10. More
surprisingly, however, is an appreciation coming from compari-
sons of metabarcoding datasets across environments showing
that the relative abundance of phototrophy in soil is not much
lower than for marine plankton68. If confirmed, this unexpectedly
high proportion of soil phototrophy indicates a remarkable role of
microbial photosynthesis outside of marine systems. Fixed car-
bon from primary production, in turn, becomes available for con-
sumer herbivorous protists and small animals at several entry
points in the food chains due to a vast array of pico- to micro-
sized algal diversity3. These heterotrophic consumers take a
range of nutritional strategies, the best-known being predation
by exclusive phagotrophs but mixotrophy (which are also pro-
ducers, thus shifting the boundaries between trophic modes),
parasitism, osmotrophy, and saprotrophy are all crucially inter-
acting to structure ecosystems105. In fact, heterotrophy is the
over-dominant nutritional mode of protists12,39,68,106, these or-
ganisms greatly contributing to control the population dynamics
and community assembly of bacterial and eukaryotic preys
alike107,108. Among these heterotrophic protists, phagotrophic
consumers account for at least half of all sequences in the ocean
and on land12,39,68. Parasites are also consistently shown to be
extremely abundant (Figure 3): ballpark estimates show that in
marine and soil systems, parasites represent up to 20% of all
metabarcoding reads, supporting the idea that different parasite
species likely infect every animal species on Earth14,68. In marine
environments, many of these parasites (or rather parasitoids, in
which the host is always killed) belong to the syndiniales, a sub-
group of the hyperdiverse MALVs, which are known to control
blooms of dinoflagellates but can also infect other protists
(e.g., cilliates, radiolarians), as well as animals109,110. Land plants
and fungi are also hosts of scores of parasites, as are other pro-
tists. In soil, the gregarine parasites of invertebrates (member of
apicomplexans, which include for example the malaria agent
Plasmodium) were shown to be the most abundant OTUs in
neotropical forests14, and similar observations will probably be
made everywhere else we look.
Protistan producers and consumers are thus not only taxo-
nomically highly diverse and from all places in the eukaryotic
tree, but they also represent a great functional diversity that is
essential to ecosystem services. Deciphering the complexity of
behaviours and lifestyles of these protists, who they are, and
how exactly they interact not only among themselves but
also with prokaryotes and larger organisms will be key to deep-
ening our understanding of the functional roles of microbial
eukaryotes.
ll
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An eco-evolutionary approach to understand protist
diversity
The great taxonomic and functional diversity of protists outlined
above is a result of hundreds of millions of years of evolu-
tion111,112, perhaps more than two billion years since the origin
of eukaryotes according to a recent estimate based on molecular
clocks112. Therefore, to study how this diversity is generated and
maintained not only requires looking at the current patterns of di-
versity, but also turning to the past to look at diversification pro-
cesses throughout geological times. This diversification — the
balance between speciation and lineage extinction — is, how-
ever, difficult to study because direct organismal observations
are typically lacking for most of life’s history. Fossils have been
used for many decades as a direct means to estimate diversifica-
tion dynamics in a multitude of eukaryotic groups, including
protist groups using their continuous and well-documented
microfossil records113–115. But while we have gained some major
insights from these paleontological studies116,117, the vast ma-
jority of eukaryote diversity is non-fossilized and existing fossils
can be hard to interpret, particularly those very ancient microfos-
sils from the Proterozoic Era (ca. 2000–1400 to 720 Ma)118,119.
However, phylogenetic comparative methods implementing sto-
chastic models of diversification based on extant diversity have
flourished in recent years120. These methods use lineage rela-
tionships and contemporary trait information to look at how
these traits have evolved and which factors (e.g., paleoenviron-
mental) have influenced diversification121. No method is perfect,
however, and purely phylogenetic-based studies of diversifica-
tion have raised concerns that the extrapolation of extinction
rates from only living taxa cannot be reliably made122,123.
As often, it is a combination of approaches and data that have
helped to gain a deeper understanding of how ecological and
evolutionary processes have generated the biodiversity we
observe today. Indeed, joint interpretations of both molecular
data from extant specimens and the paleontological record
have shown increasingly coherent diversification histories and
represent an exciting avenue of research, including the diversifi-
cation of important fossilizable protist groups such as dia-
toms124,125, dinoflagellates126, foraminifera127, radiolaria128,129,
and testate amoebae55,130. The diversification of these ecologi-
cally dominant groups appears to have occurred relatively sud-
denly in the late Neoproterozoic (ca. 650 Ma) fossil record, but
before this time early eukaryote evolution seems to have been
much calmer. Eukaryotes during the Proterozoic showed a
generally low standing diversity in unfavorable conditions while
most of the biological activity was likely governed by prokary-
otes131. This period is in fact sometimes referred to as the ‘boring
billion’ (ca. 1800–800 Ma) to reflect the rarity of eukaryotic fossils
and the constantly low oxygen concentration132.
It was, however, during this period of low eukaryote activity
that the stage was set for the later expansion to hyperdominance
of some protist groups, notably by the establishment of plastid
endosymbioses that gave rise to a variety of algae (e.g., dinofla-
gellates, diatoms) that profoundly altered the global geochem-
ical and ecological conditions of the Earth112,133. More generally,
species interactions of many types (i.e., symbiosis such as para-
sitism or predation) have likely played critical roles in the overall
diversification of eukaryotes during the late Proterozoic134. For
example, it was suggested that predation from large unicellular
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rhizarians (e.g., Foraminifera and Radiolaria) contributed to
important ecological changes, from the continuous proliferation
of algae134 to perhaps an arm race with animals leading to the
development of multicellularity in this group135. Conversely, the
predatory pressure from animals may have led to the develop-
ment of extracellular skeletons in many protist groups136,137.
During the Jurassic (ca. 200–145 Ma), dinoflagellates estab-
lished photosymbiosis with corals138. In this period, Foraminifera
and Radiolaria also developed endosymbiotic relationships with
photosynthetic dinoflagellates and haptophytes likely in
response to oligotrophic oceans, bringing these associations
to the planktonic realm139,140. On land, organismal interactions
involving protists also critically contributed to the diversification
processes. For example, it has been suggested that interactions
between bacteria, plants and testate amoebae led to the devel-
opment of a complex rhizosphere and the necessary biogeo-
chemical changes for the expansion of land plants during the
early Phanerozoic141.
Metabarcoding data have much to contribute to these eco-
evolutionary studies of protist diversification, because their
throughput currently represents the best approximation of
OTU-level diversity in many environments. This is important
because a near-full sampling of extant diversity offers the best
possibilities for fitting diversification models using phylogenetic
approaches120. The use of metabarcoding data in eco-evolu-
tionary studies is in its infancy, but it has already shown great
promise in spite of the limited phylogenetic signal in short-read
metabarcodes. For example, combining phylogenetic models
of diversification with palaeoenvironmental data, a recent study
showed a strong effect of specific environmental conditions
(variation in carbon dioxide partial pressure) on early diatom evo-
lution124. With the current development of long-read metabar-
coding, more robust phylogenies including large proportions of
the environmental diversity will become available, therefore al-
lowing more detailed analyses of diversification dynamics
across the diversity of eukaryotes.
Concluding remarks
The last 15 years of research on protist diversity have seen a
great deal of inspiring insights. Our understanding of the vast-
ness of this diversity, how and by which processes it is struc-
tured through space and time, and what its functional roles
are, is changing so rapidly that it can be challenging to keep
pace. Excitingly, there is no sign that this pace is slowing
down. Metabarcoding has massively contributed to this new
perspective on protist diversity, allowing deep sequencing of
an always broader range of environments. While this approach
will likely remain dominant in diversity studies, it will also evolve
together with new sequencing technologies, as it has in the past.
Notably, we anticipate that long-read sequencing will lead to a
better integration of the different molecular markers commonly
used, greatly facilitating the comparison of all the data we have
produced and will continue to produce. Other methods will
play key roles too, such as metagenomics that not only allows re-
covery of taxonomic markers without potential amplification
biases, but also offers a powerful way to integrate more param-
eters (e.g., metabolism). Beyond molecules, however, it is impor-
tant to emphasize that we actually have no idea what the vast
majority of this protistan diversity looks like, and how it lives,
R1276 Current Biology 31, R1267–R1280, October 11, 2021
Review
from lack of direct observations. Therefore, a critical next step
in the study of protist diversity will be to devise strategies to
link this catalogue of environmental sequences to images and
genomes, and ideally cultures, in order to increase the predictive
power of metabarcoding data and in turn more fully grasp the
ecological functions of protists.
ACKNOWLEDGEMENTS
F.B.’s research is supported by a Fellowship grant from SciLifeLab, a research
project grant from the Swedish Research Council (VR-2017-04563), and a
Future Research Leader grant from Formas (201701197). We would like to
thank Christophe Seppey and the rest of the authors from the study
‘Protist taxonomic and functional diversity in soil, freshwater and marine eco-
systems’ 68 for kindly sharing their data and helpful scripts to produce Figure 3,
and for being very responsive to our emails.
DECLARATION OF INTERESTS
The authors declare no competing interests.
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