The origins of malaria: There are more things in heaven and earth .

Abstract

SUMMARY Malaria remains one of the most significant global public health burdens, with nearly half of the world's population at risk of infection. Malaria is not however a monolithic disease - it can be caused by multiple different parasite species of the Plasmodium genus, each of which can induce different symptoms and pathology, and which pose quite different challenges for control. Furthermore, malaria is in no way restricted to humans. There are Plasmodium species that have adapted to infect most warm-blooded vertebrate species, and the genus as a whole is both highly successful and highly diverse. How, where and when human malaria parasites originated from within this diversity has long been a subject of fascination and sometimes also controversy. The past decade has seen the publication of a number of important discoveries about malaria parasite origins, all based on the application of molecular diagnostic tools to new sources of samples. This review summarizes some of those recent discoveries and discusses their implication for our current understanding of the origin and evolution of the Plasmodium genus. The nature of these discoveries and the manner in which they are made are then used to lay out a series of opportunities and challenges for the next wave of parasite hunters.

Full text

The origins of malaria: there are more things in heaven
and earth ...
P. J. KE EL ING
1
and J. C. RAYNER
2
*
1
Department of Botany, Canadian Institute for Advanced Research, Evolutionary Biology Program, University of British
Columbia, Vancouver, BC V6T 1Z4, Canada
2
Malaria Programme, Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton,
Cambridge CB10 1SA, UK
(Received 9 December 2013; revised 14 April 2014; accepted 15 April 2014; first published online 25 June 2014)
SUMMARY
Malaria remains one of the most significant global public health burdens, with nearly half of the world’s population at risk
of infection. Malaria is not however a monolithic disease – it can be caused by multiple different parasite species of the
Plasmodium genus, each of which can induce different symptoms and pathology, and which pose quite different challenges
for control. Furthermore, malaria is in no way restricted to humans. There are Plasmodium species that have adapted
to infect most warm-blooded vertebrate species, and the genus as a whole is both highly successful and highly diverse.
How, where and when human malaria parasites originated from within this diversity has long been a subject of fascination
and sometimes also controversy. The past decade has seen the publication of a number of important discoveries about
malaria parasite origins, all based on the application of molecular diagnostic tools to new sources of samples. This review
summarizes some of those recent discoveries and discusses their implication for our current understanding of the origin and
evolution of the Plasmodium genus. The nature of these discoveries and the manner in which they are made are then used
to lay out a series of opportunities and challenges for the next wave of parasite hunters.
Key words: Apicomplexa, Plasmodium, evolution, origin, phylogeny.
A NEW AGE OF PARASITE DISCOVERY
Scientists in general, and perhaps parasitologists
in particular, have always been driven by a need to
define the landscape in which they work. Almost as
soon as Charles Laveran first glimpsed the wink of a
hemozoin crystal down his microscope there was a
rush to categorize these strange new organisms – to
give labels to the phases of their extraordinarily
complex life cycle, and to group them into species.
After a period of occasionally fractious dispute, with
the distinction between Plasmodium vivax and
Plasmodium ovale proving the most difficult to
resolve, by 1922 the four major human Plasmodium
species had been defined and named. Discovery of
new Plasmodium species in other hosts continued for
some time, with a particularly golden era of discovery
in Asian monkeys in the early 1960s. However by the
late 1960s some of the world’s most eminent
malariologists felt that they had a secure enough
grip on the Plasmodium genus that they could define
it in elegant and definitive textbooks (Garnham,
1966; Coatney et al. 1971), complete with the
beautifully detailed illustrations of each species that
are still widely used in teaching and research
presentations to this day. At the same time, ultra-
structure and molecular phylogeny combined to give
us an equally well-defined view of the broader place
of Plasmodium in the tree of life, and amongst its close
relatives in the phylum Apicomplexa.
In the past 5 years, however, the clear and
definitive worldview desc ribed in these textbooks
has been radically overhauled by successive waves
of new discoveries, powered by a combination of
molecular diagnostic technologies and extensive
sampling efforts. Some of these searches involve
painstaking observations of blood smears from wild-
caught animals and would be instantly recognizable
to the original Plasmodi um pioneers. Others would
appear completely alien, involving ape faeces col-
lected off forest floors in West Africa and massive
molecular surveys of the world’s greatest coral reef
ecosystems. Together these studies have forced a
re-evaluation of the origin of the most deadly human
malaria species, Plasmodium falciparum, the source of
the rodent malaria parasite species and even the very
origins of the phylum Apicomplexa itself.
UNEXPECTED ROLES FOR CORAL AND
PHOTOSYNTHESIS IN APICOMPLEXAN
EVOLUTION
Tracing the evolutionary history from Plasmodium
back through its apicomplexan relatives and beyond,
* Corresponding author: Malaria Programme, Wellcome
Trust Sanger Institute, Wellcome Trust Genome Campus,
Hinxton, Cambridge CB10 1SA, UK. E-mail: julian.
rayner@sanger.ac.uk
S16
SUPPLEMENT ARTICLE
Parasitology (2015), 142, S16–S25. © Cambridge University Press 2014. The online version of this article is published within an Open
Access environment subject to the conditions of the Creative Commons Attribution licence http://creativecommons.org/licenses/by/3.0/
doi:10.1017/S0031182014000766

there was a point at which the parasitic lifestyle that
characterizes the entire apicomplexan lineage origi-
nated. Finding this point in time and explaining
how and why this massive transition took place have
long been difficult questions. Over a decade ago the
answers took an unexpected turn with the discovery
that Plasmodium and other apicomplexans contained
a plastid (generally called the ‘apicoplast’; McFadden
et al. 1996; Wilson et al. 1996; Kohler et al. 1997), an
organelle usually used by plants and algae for
photosynthesis. So the question became more pre-
cise, but stranger: how did a presumably photosyn-
thetic ancestor turn into an obligate intracellular
parasite of animals? Recently, thanks to some good
old-fashioned organism hunting, these two questions
are merging into a new way to look at the deep origins
of apicomplexans.
Critical to this new understanding was the dis-
covery of living descendants of a recent ancestor
of the apicomplexans – in effect photosynthetic
members of the apicomplexan lineage (Moore et al.
2008). Chromera and Vitrella are two new genera of
fully photosynthetic algae that were isolated from
coral reefs, and branch near the base of the phylum
Apicomplexa in molecular phylogenetic trees (Moore
et al. 2008; Janouskovec et al. 2010)(Fig. 1). The
plastid genomes of both have been fully sequenced,
and demonstrated that apicomplexan plastids are
derived from the same red algal endosymbiont
that also gave rise to plastids of dinoflagellate and
stramenopile algae (Janouskovec et al. 2010). The
Chromera and Vitrella plastid sequence data also
revealed another unexpected fi nding. When they
were compared with bacterial environmental sequen-
cing data, it become apparent that environmental
microbial populations are heavily contaminated with
sequences from eukaryotic plastid genomes, presum-
ably derived from cyanobacteria. Comparing these
inadvertent but vast surveys of plastid sequences to
known plastid genomes revealed that Chromera and
Vitrella are just the tip of the iceberg: there is a brace
of new and unknown plastid lineages, all clustering at
the base of the apicomplexans. Remarkably, all of
these are specifically associated with coral reef
environments: coral samples consistently contain
sequences from apicomplexan-related plastids, and
a variety of other environments, many of which are
far more thoroughly sampled, do not. Some of these
are related to Chromera and Vitrella, most are new
and independent lineages (Janouskovec et al. 2012,
2013). Indeed, the most common apicomplexan
relative from coral is a new lineage known only as
Apicomplexan Related Lineage-5 (ARL-V: Fig. 1).
ARL-V is the closest known relative of apicomplex-
ans, but its biology is totally unknown: it is as yet
defined only by DNA sequences (Janouskovec et al.
2012, 2013).
Coral has never been regarded as a particularly
important habitat for apicomplexans (only one as yet
unidentified coral parasite known as Genoty pe N has
been investigated at all), but these new data suggest
something much more – that coral may be the cradle
of apicomplexan origins (Toller et al. 2002). It is
possible that the association between the ancestor of
apicomplexans and animals began as a mutually
beneficial one based on photosynthesis with an
ancient and likely now extinct lineage of corals,
similar to the association between modern corals and
zooxanthellae. Later this association soured and
became one-sided, perhaps when the lineage leading
to apicomplexans lost photosynthesis but retained
their ability to invade coral cells. This would tip the
balance to an association more like the parasitic ones
we see today. This story sounds appealing, and may
even be partly true, but the truth is almost certainly
more complex. For a start, another lineage is known
to branch at the base of apicomplexans, and its
members are neither parasitic nor photosynthetic.
Instead, the colpodellids are free-living heterotrophs
that seem to specialize in attacking and eating other
eukaryotes using a feeding apparatus homologous to
the apical complex (Kuvardina et al. 2002; Leander
and Keeling, 2003). The exact relationship between
colpodellids, Chromera, Vitrella and apicomplexans
remains unclear, so it is too early to make firm
conclusions about which, if either, kind of lineage
made the transition to obligate parasitism. However,
the nearly perfect correlation between the photo-
synthetic members of the lineage and corals gives
us a number of new intriguing leads to follow
(Janouskovec et al. 2010, 2012, 2013). Whether
these resolve the ultimate origin of apicomplexan
parasitism remains to be seen, but at the very least
they have provided us with a completely new context
with which to examine the question and new
perspectives from which to view the parasites.
FINDING CONNECTIONS IN UNEXPECTED PLACES
– BAT PLASMODIUM REVEAL NEW INSIGHTS INTO
THE ORIGINS OF RODENT MALARIA
Just as surveys of coral reefs have revolutionized
our understanding of the origins of the phylum
Apicomplexa, an even more recent survey of apicom-
plexan parasites of bats has begun a new revolution,
this time in our understanding of the origins of
rodent parasites. Plasmodium parasites infecting
rodents were first observed in 1948 by two Belgian
scientists, Vincke and Lips, in what is now the
Democratic Republic of Congo (Vincke and Lips,
1948). This initial description, of Plasmodium spor-
ozoites in an infected mosquito, led to a series of
expeditions that defined a number of Plasmodium
species that infected African thicket rats (primarily
Grammomys and Thamnomys species). Four species,
Plasmodium berghei, Plasmodium yoelii, Plasmodium
chabaudi and Plasmodium vinckei were all trans-
ferred to laboratory mice, where they have been
S17Apicomplexan parasite origins

extraordinarily useful tools for understanding ma-
laria biol ogy. These rodent species are non-infectious
to humans, which makes working with them in a
laboratory setting straightforward. While they have
proven controversial models for specific aspects of
malaria pathology (Craig et al. 2012), there is no doubt
that they also offer many completel y unique advan-
tages, and have enabled experiments and approaches
that would simply not have been possible without
them. Most notably, rodent Plasmodium species have
allowed systematic analysis of liver and mosquito
stages, which are technically demanding or even
inaccessible when working with human parasites
(Lindner et al. 2012), and are proving amenable to
high-throughput experimental genetics and system-
atic immunology. While transferring findings from
rodent to human Plasmodium will always be critical for
validation, and for certain questions such as studying
host-parasite interactions which may evolve rapidly
between species, in general these African thicket rat
parasites have proven invaluable for studies of basic
Plasmodium metabolism and biology.
Psammosa
Coccidia
Piroplasms
Haemosporidians
Voromonas
Acavamonas
Colpodella
Chromera
Vitrella
Alphamonas
Perkinsids
Dinoflagellates
Gregarines
Gregarines
ARL-V
Plastid
?
?
?
?
Fig. 1. Schematic representation of relationships between apicomplexan parasites and their closest relatives and
the evolution of their plastids. The closest known branch to the ‘true’ apicomplexans (at top, including Coccidia,
Piroplasms, Haemosporidians and the paraphyletic Gregarines) is a biologically undescribed lineage known only
from plastid environmental surveys, the so-called ARL-V lineage. The nearest relatives that have been biologically
characterized include a diverse array of predatory flagellates (Colpodella, Voromonas and Alphamonas), photosynthetic
coral symbionts (Chromera and Vitrella) and a large number of unknown environmental lineages (many from coral, but
also many from other environments). These are all in turn related to a large group including dinoflagellates and their
closest relatives, the Perkinsids and Psammosa, both of which possess structures homologous to the apical complex, and
the enigmatic predator Acavamons. The column to the right summarizes what we know about plastids in each lineage:
red plastids indicate photosynthesis, colourless plastids indicates plastids that are known but non-photosynthetic.
ARL-V is hypothesized to be photosynthetic but this has not been tested, and dinoflagellates contain about 50%
photosynthetic and non-photosynthetic species. Lineages for which no plastid has been detected are indicated
by a question mark.
S18P. J. Keeling and J. C. Rayner

However, while they may have proven an exper-
imental boon, where these rodent parasites fit in the
overall picture of Plasmodium phylogeny has always
been somewhat of an anomaly, lying clearly outsid e
the primate Plasmodium radiation that includes
human parasites, but with no other close relatives
(Escalante et al. 1998). This is largely because only
a very few rodent Plasmodium samples exist – no new
rodent Plasmodium isolates have been obtained since
those initial expeditions in the 1940s and 50s,
although samples from these initial expeditions
have recently again become available to researchers
through a new repository, from which new species
may emerge (http://www.malariaresearch.eu). It has
taken a new expedition in West Africa, similar
in pioneering spirit to those performed more than
50 years ago, to provide some context. Surveying
bats in remote forests of Guinea, Liberia and Cote
d’Ivoire revealed multiple haemosporidian parasites,
including two Plasmodium species (Schaer et al.
2013). These species, Plasmodium voltaicum and
Plasmodium cyclopsi, had been previously identified
but classified only based on morphology. Molecular
phylogeny of the new samples, using a combination
of mitochondrial, apicoplast and nuclear genes,
revealed the surprising finding that these bat parasites
fall within the rodent Plasmodium clade (summarized
in Fig. 2), despite the distant evolutionary relation-
ship between their mammalian hosts (Schaer et al.
2013). Host switching in Plasmodium species is well-
established through work in avian parasites (Ricklefs
and Fallon, 2002; Ricklefs et al. 2004; Beadell et al.
2009), although it has not been as frequently
described for mammalian species. In this case,
switching may be facilitated by the fact that African
thicket rats are arboreal, and therefore are presum-
ably exposed to the same Anopheles vectors as bat
species. Further work is clearly needed, incl uding
the exciting prospect of attempting to transfer some
of these bat Plasmodium species to laboratory rodents,
but again this discovery emphasizes that systematic
screening of a wide range of biological specimens,
P. cyclops
Laverania sp.
P. falciparum
P. knowlesi
P. fieldi (etc.)
P. cynomolgi
P. vivax
P. ovale
P. berghei (etc.)
P. cyclops (etc.)
P. gallinaceum
(etc.)
P. mexicanum
(etc.)
P. malariae
African Apes
Humans
Asian Monkeys
Asian Monkeys
Humans, African Apes
Asian Monkeys
Humans
Humans
Rodents
Bats
Birds
Lizards
Host
Fig. 2. Schematic representation of the major radiations amongst Plasmodium species. Since becoming parasites of
vertebrates, the genus Plasmodium has expanded to infect a wide variety of hosts. Only a handful of these species, those
referred to specifically in the text as well as some other major groupings, are represented here. The precise relationships
between the species are not always known, so branch positioning is indicative rather than definitive – more detailed
analyses are available elsewhere (Martinsen and Perkins, 2013). Human parasites originate from Plasmodium clades that
have expanded in related groups of hosts, including the Laverania radiation in African apes, which includes the most
deadly form of human malaria, P. falciparum , and an expansion of P. vivax-related parasites in African apes and
Southeast Asian monkeys. The other two major human malaria parasites, P. ovale and P. malariae, also have relatives in
African apes, but the full diversity and relationship between these species is not currently known. Recent works indicates
that rodent and bat Plasmodium parasites are closely related, with possible host switching occurring on more than one
occasion (Schaer et al. 2013).
S19Apicomplexan parasite origins

an approach that had fallen somewhat out of favour in
the Plasmodium field, can yield new and completely
unexpected insights.
FAECAL SAMPLES LEAD TO A NEW
UNDERSTANDING OF THE ORIGINS OF
P. FALCIPARUM
Perhaps the most widely reported example of this
new wave of Plasmodium discovery lies in a series of
papers investigating the origins of P. falciparum,
the species that causes almost all human malaria
mortality. From the very earliest days of Plasmodium
discovery it was established that African apes were
naturally infected with Plasmodium reichenowi,a
parasite that was morphologically nearly identical to
human P. falciparum, but appeared to be a distinct
species (Reichenow, 1920). Only a single isolate of
P. reichenowi was ever obtained for study, from a wild
chimpanzee that had been transported to the USA
in the 1950s (Collins et al. 1986). There the matter
rested for decades, with P. falciparum and P. reich-
enowi thought to be sister species completely isolated
from all other known Plasmodium. In the past five
years this view has been comprehensively overhauled
by a variety of means (Rayner et al. 2011), including
harnessing the power of modern molecular techni-
ques to study samples that our parasite-prospecting
forebears would never have dreamed of – ape faeces.
Such unusual samples were required because unlike
bats, and even more so than coral reefs, wild-living
African apes are highly protected, and invasive
capture and collection approaches for wild primates
are clearly inconceivable. However, ape blood is
available in very restricted circumstances from
samples taken for health surveillance of captive
animals, and in 2009 a study of samples from two
pet chimpanzees revealed what appeared to be
a new species of P. falciparum-related parasite,
Plasmodium gaboni (Ollomo et al. 2009). A series of
similar studies using small numbers of samples from
captive apes followed, with somewhat contradictory
results or interpretation, although all agreed that
there appeared to be a much greater diversity of
P. falciparum-related parasites in great apes than was
previously realized, and potentially more than could
be defined as a single species (Rich et al. 2009; Duval
et al. 2010; Prugnolle et al. 2010).
It was the discovery that Plasmodium DNA could
be amplifi ed from faecal samples (Prugnolle et al.
2010), which can be non-invasively collected in large
numbers from multiple sites, that generated a
sufficient sampl e size to clarify the matter, just as
similar studies of HIV-related virus RNA from ape
faecal samples had clarified the origin of human
HIV (Keele et al. 2006). A survey of nearly 3000 such
samples revealed six clades of P. falciparum-related
parasites, now collectively referred to as sub-genus
Laverania (Liu et al. 2010). All six Laverania clades
appeared to be host-species specific, at least in wild-
living animals, with some species only infecting
chimpanzees and others only infecting gorillas, even
when the ape species are sympatric. Most sign-
ificantly all human P. falciparum sequences cluster
within the radiation of a gorilla-specific parasite,
implying that the human P. falciparum epidemic is
the result of a single transmission event from this
gorilla parasite species, which is now referred to as
Plasmodium praefalciparum. While the definition of
these P. falciparum-related clades as individual
species might not fit classical definitions due to the
current lack of morphological data, the impossibility
of obtaining blood samples from wild-living apes,
coupled with the frequent occurrence of mixed
infections within the same animal will make this
more rigorous naming barrier a hard, if not im-
possible, hurdle to clear. However, these studies
do clearly show that P. falciparum is not the
orphan that it was once thought to be, but, much
like the rodent Plasmodium species, it nestles within a
much broader radiation of related species in related
hosts, with host switching occurring in some
branches and not others (summarized in Fig. 2).
MOVING FROM DIAGNOSTICS TO DEFINITIONS –
THE POWER OF GENOMES
These three discoveries are far from the only
examples of recent changes to our understanding of
how and where apicomplexan parasites come from –
the establishment of frequent zoonotic transmission
of Plasmodium knowlesi from macaques to humans in
Malaysia (Singh et al. 2004), or the subdivision of P.
ovale into two sub-species (Sutherland et al. 2010)
both stand as further evidence that we are in the
process of a radical realignment of Plasmodium
phylogeny. Why, decades after the initial age of
Plasmodium discovery, is this occurring? Part of the
answer is simply that we are now looking in new
places – all of the studies discussed in this review rely
on surveying samples that would not have previously
been considered for systematic study, either because
of logistical difficulties in obtaining them (such as
trapping large numbers of bats), or because they were
not previously realized to contain useable infor-
mation (such as ape faeces or samples from coral
reefs). However, a critical part of the answer, as with
most scientific breakthroughs, lies in advances in
technology – in this case the systematic application of
DNA sequencing. The first wave of parasite hunters
could only define what they saw based on microscopy
and morphological characteristics. The new wave, led
by researchers studying avian malaria where samples
have always been limiting, can use highly sensitive
PCR for detection, an array of related genome
sequences to provide potential targets for amplifica-
tion, and, in the case of the origins of Apicomplexa,
S20P. J. Keeling and J. C. Rayner

massive molecular surveys of whole microbial eco-
systems from which to pluck sequences.
The success of these approaches means that the
question of morphology has in fact become somewhat
a controversial one, with some researchers question-
ing of the validity of species identification based
entirely on molecular means and emphasizing that
morphology will always play a critical role in defining
apicomplexan species (Perkins et al. 2011; Valkiunas
et al. 2011). Although there may be differences of
emphasis, most researchers would agree that mi-
croscopy and morphology will always play a key part
in our understanding of Plasmodium and
Apicomplexan spec ies and should remain the gold
standard for species identification (Perkins, 2014),
just as microscopy remains the gold standard for
clinical diagnosis. However, there is equally no doubt
that in some circumstances, such as the case of
African ape Plasmodium species, or the coral-dwell-
ing ARL-V, morphological definitions will be slow if
not impossible to perform. Samples from wild-living
apes, for example, will quite right ly never be possible
to obtain because of ethical restrictions, and while
opportunistic investigation of samples taken for
health-related reasons from captive apes is sometimes
possible, the frequency of multiple infections with
different Laverania species will make interpretation
difficult in most circumstances. For the more deep
ancestors of apicomplexans, it may simply never be
possible to obtain samples from which morphological
identification can be performed. Until such a time
that morphological data become available, if ever,
the scientific community clearly needs some sort
of framework to discuss findings and definitions,
and that framework currently relies on molecular
phylogeny. While such an approach might be less
palatable to some, it is far from a unique approach –
research in bacterial ecology, for example, has been
coping with large numbers of unidentified ‘molecular
taxa’ for over a decade.
While molecular diagnostics are clearly here to
stay, and in some cases may never be replaced by
other diagnostic methods, it is critical that for these
new species we move beyond simple diagnosis to clear
definition. An important element of this will be the
generation of complete genome sequences for all
these new parasites. Not many years ago that would
have seemed a very distant prospect, but the advent of
next-generation sequencing technology means that
we should now expect fairly rapid progress towards
full genomic definitions. In some cases, where
samples are limited or are of limited quality,
generating complete genomes will be challenging
even using these new technologies. However, these
challenges are not radically different to those facing
environmental microbiologists, who often must deal
with uncultivated organisms, or population genetic
studies of human Plasmodium species, where the
drive is to extract the maximum genomic information
from the smallest possible volume of clinical samples
such as dried blood spots. Developments in the
field of human Plasmodium population genetics,
such as methods to digest human DNA and leave
Plasmodium DNA untouched (Oyola et al. 2013), or
hybrid capture approaches to pull out Plasmodium
material from mixed samples (Melnikov et al. 2011),
will clearly be applicable to even the most challenging
samples such as small volumes of bat blood.
Once genomes of these new species are generated,
comparative genomics will hopefully lead to specific
hypotheses that can be tested experimentally. In the
case of the Laverania, genomes exist for only two
members of the sub-genus, P. falciparum and P.
reichenowi (Otto, Newbold & Berriman, manuscript
submitted), and the as yet unreported genome of P.
praefalciparum will clearly be of great interest in
understanding the origin of human P. falciparum and
of severe malaria pathogenesis. Comparative geno-
mics will also enlighten one of the most interesting
aspects of Laverania parasites, their apparent strict
host restriction. Recent in vitro studies suggest that in
the case of P. falciparum, host specificity may be due
at least in part to specificity in a critical protein-
protein interaction that mediates erythrocyte in-
vasion (Wanaguru et al. 2013 ). Complete genomes
from multiple Laverania species will allow a much
more comprehensive test of this hypothesis.
Similarly, genomes of P. voltaicum and P. cyclopsi
will be of great interest to compare with the genomes
of rodent parasites. The fact that the entire
Plasmodium life cycle can be replicated in these
rodent models will allow for a much more systematic
analysis of the factors that control switching between
bat and rodent hosts, including analysis of mosquito
and liver stages. As for the deeper evolutionary origin
of apicomplexans as a whole, complete genome
sequences for both the cultured relatives, Chromera
and Vitrella, is a relatively straightforward problem,
and both are currently underway. These genomes
should allow some insights into a number of
interesting questions about the ancient transition
from free-living phototroph to obligate parasite, and
perhaps even generate more specific hypotheses
about the possible role of coral in the origin of
parasitism. However, a major piece of the puzzle for
understanding the origin of apicomplexans remains
the uncultured, unidentified and generally unknown
lineages, especially ARL-V, which is the closest
relative of apicomplexans that we currently know
of, but which has never even been seen under the
microscope as yet.
CONCLUSIONS AND A WAY FORWARD: THE NEED
FOR SYSTEMATICS AND AGREEMENT
There is no question that many new discoveries
await, particularly as genome sequencing becomes
applicable to ever smaller and more complex samples.
S21Apicomplexan parasite origins

In this concluding section, we highlight a series of
challenges that have sometimes bedevilled the field in
the past, and suggest ways in which the community
can move forward in a more systematic and rigorous
manner.
Discovery
The simple message here is that new sample surveys
are urgently needed, and that no sample should be
overlooked. What is missing in the current surveys?
In terms of the broader context of apicomplexan
origins, the link between coral reefs and photosyn-
thetic relatives of apicomplexans is helpful, but our
understanding of these coral communities remains
rudimentary. Moreover, we have not even begun to
sample the diversity of non-photosynthetic api-
complexan relatives, colpodellid predators, which
inhabit a range of environments, so we should not
be restricted to focusing on coral. In terms of
Plasmodium specifically, there is clearly much more
to discover in African apes, with P. vivax, P. ovale
and Plasmodium malariae-related parasites all de-
tected, but their relationship to the human counter-
parts not yet understood (Duval et al. 2009;
Hayakawa et al. 2009; Prugnolle et al. 2013). A
systematic revisiting of Plasmodium species in Asian
monkeys is also long overdue, especially because
type specimens of a number of species, including
Plasmodium cynomolgi, Plasmodium fieldi, P. knowlesi,
Plasmodium coatneyi and Plasmodium simiovale now
exist in public repositories. New World monkeys
also represent a relatively untapped field. Plasmodium
vivax and P. malariae-related species have been
identified in multiple New World monkey species,
and are widely assumed to be anthroponotic (Tazi
and Ayala, 2011), but the range of host species for
these simian parasites, and how far they are diverg-
ing in these new hosts, is not known. The list
of fascinating specimens is long, with reptile
Plasmodium a particularly untapped field. Many
exciting discoveries await.
Diagnostics
It is clear that several Plasmodium species frequently
co-exist in the same hosts. It will therefore be im-
portant to develop molecular diagnostic approaches
that are both broad and deep to ensure that rarer co-
infecting species are not missed. An excellent
example is non-Laverania parasites in African apes.
Plasmodium vivax, P. malariae and P. ovale-related
species clearly exist in apes, but relatively few
sequences have been generated so far when compared
with Laverania sequences. This is likely to be because
the prevalence, and presumably parasitaemia, of
Laverania parasites is so high that it swamps the
signal from non-Laverania parasites when broad
pan-Plasmodium primers are used for screening, in
much the same way that P. ovale and P. malariae
in Africa are likely under-estimated because they
occur at much lower densities than the dominant
P. falciparum. It will therefore be essen tial to use both
broad genus spanning and targeted species-specific
approaches for screening, and resurveying the same
sample set with targeted primers once the extent of
diversity is established with broad primers will be an
important approach.
Standardization
There is an urgent need for consistency in which gene
fragments are used for diagnosis in order to maximize
comparison between studies. While there are some
general standards in the field, primers and specific
gene coordinates vary widely between studies and
even the genome being targeted can di ffer – for
Plasmodium species mitochondrial gene fragments
are widely used, while for coral-dwelling apicom-
plexan relatives plastid sequences are used, and for
apicomplexan relatives in other environments nuclear
sequences are used. Agreement on such matters is
never easy, but where it can be made the results are
incredibly powerful. The most striking examples are
DNA diversity barcodes now used by multiple
scientific communities such as the iBol community
attempting to catalogue all multicellular eukaryotes
(ibol.org), and the protist barcode group who are
using a two-tiered barcoding approach (Pawlowski
et al. 2012). In both of these cases large research
communities have agreed to co-ordinate efforts and
standardize markers, and in doing so have allowed the
systematic identification and comparison of literally
thousands of species. The Plasmodium and apicom-
plexan communities would do well to follow suit and
agree on standard gene fragments for amplification
wherever possible.
Definition
As noted above, while diagnosis is important and
useful, it needs to be followed rapidly by definition,
which means whole genome sequences. Rapid
advances in sequencing technology, coupled with
methods to increase sample quantity (for example by
whole genome amplification) and decrease contami-
nation from non-Plasmodium sequences (by hybrid
selection, or digestion of non-target sequences) offer
hope that we can rapidly move to complete genomes
from even small and difficult samples. In parallel,
advances in single cell genomics offer even greater
promise. The field is moving towards the ability to
physically isolate a single parasite and generate data
from the whole genome or whole transcriptome,
which will quickly open newly identified species
to functional analysis, as well as rapid population
genomics analysis. The advantage of genomic defini-
tion is that there is no need for agreement on specific
S22P. J. Keeling and J. C. Rayner

fragments to focus on, and the principles of open
data access and sharing are deeply embedded in the
genomics community, which will facilitate com-
parisons between studies.
Taxonomy fit for purpose
By pushing our sampling and identification efforts
to the limits of new technology, we will inevitably
surpass current constraints of taxonomic regulations.
Specifically, this means finding ways to deal with new
taxonomic units that are defined in non-traditional
ways. For example, it is now conceivable to have
complete genomes from organisms we have never
seen, but these cannot be formally ‘described’.
Indeed, some journals refuse to allow descriptions
even with comprehensive microscopy, if the organ-
ism is not available in culture. Luckily this is not
a new problem, and we can examine how bacterial
ecology has dealt with the taxonomic turmoil created
by metageno mics and environmental tag sampling
for some hints as to how we migh t proceed.
Caution
As we discover more and more species, as a
community we need to set our bar higher for what
actually constitutes a discovery. In recent years there
have been publications that have taken even a single
sample of a new species or a new host as the only proof
required (Prugnolle et al. 2011a, b; Sharp et al. 2011).
While discoveries based on small sample numbers
can be later borne out, such as the initial discovery of
P. gaboni based on only two samples, it is also true
that large-scale surveys of multiple samples using
highly sensitive molecular diagnostics present very
real risks of sample mix up and contamination, even
in the most careful labs. Indeed, as genome sequen-
cing has scaled up, so too has the problem of cross
contamination, so now low-level contaminants ap-
pear in many or all large-scale sequence projects. In
studies aiming to produce fully assembled genomes
this is not a great problem, but in environmental
surveys seeking rare species it can be a very serious
problem indeed. Independent validation and caution
in interpretation are scientific watchwords, but
should be doubly respected where discovery of new
species is concerned.
Nomenclature
When multiple groups are working in the same
scientific area, differences in nomenclature inevitably
arise. While these are unavoidable to some extent,
respect for the scientific principle of deferring to the
first names given to a new species would go a long way
to resolving some of the conflicts (just as the same
principle would be helpful for Plasmodium gene
and protein names). When the literature becomes
too entangled, as a field we should consider the
old-fashioned approach of getting the relevant people
in the same room until a consistent nomenclature can
be agreed upon. Consistency in naming will only help
drive the field forward.
These are exciting times for those of us working
on the evolution of and new species discovery in
apicomplexans, and the combination of new sample
collections, molecular diagnostics and new genomic
technologies make it likely that more breakthroughs
will follow. By using consistent and sensitive
approaches, applying them to every sample set that
can be collected, and adhering to the most rigorous
principles of scientific proof, the breakthroughs of
the last few years are likely to be only the tip of the
iceberg.
ACKNOWLEDGEMENTS
The authors thank Andrew Jackson and James Cotton for
organizing the Wellcome Trust Retreat on the Evolution of
Parasitism, which stimulated this review.
FINANCIAL SUPPORT
JCR was supported by the Wellcome Trust (grant number
098051) and the National Institutes of Health (grant
number R01 AI58715). PJK was supported by the
Canadian Institutes for Health Research (grant number
MOP-42517) and a Fellowship from the John Simon
Guggenheim Foundation. PJK is a Senior Fellow of the
Canadian Institute for Advanced Research.
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