TREPAR-1338; No.

of Pages 8

Review

A primer for Leishmania population

genetic studies
V. Rougeron1,2*, T. De Meeus3*, and A-L. Banuls1
1

MIVEGEC (Laboratoire Maladies Infectieuses et Vecteurs: Ecologie, Genetique, Evolution et Controle), Centre National de la
Recherche Scientifique (CNRS) Unite Mixte de Recherche (UMR) 5290 Institut de Recherche pour le Developpement (IRD)
224 Universites Montpellier 1 et 2, Montpellier, France
2
Centre International de Recherches Medicales de Franceville, Franceville, Gabon
3
IRD/Centre International de Recherche-Developpement sur lElevage en zone Subhumide (CIRDES), UMR 177,
INTERTRYP IRDCentre de Cooperation Internationale en Recherche Agronomique pour le Developpement (CIRAD),
CIRDES 01, BP 454 Bobo-Dioulasso 01, Burkina Faso

Leishmaniases remain a major public health problem.

Despite the development of elaborate experimental
techniques and sophisticated statistical tools, how these
parasites evolve, adapt themselves to new environmental compartments and hosts, and develop resistance to
new drugs remains unclear. Leishmania parasites constitute a complex model from a biological, ecological,
and epidemiological point of view but also with respect
to their genetics and phylogenetics. With this in view,
we seek to outline the criteria, caveats, and confounding
factors to be considered for Leishmania population
genetic studies. We examine how the taxonomic complexity, heterozygosity, intraspecific and interspecific
recombination, aneuploidy, and ameiotic recombination
of Leishmania intersect with population genetic studies
of this parasite.
State of the art of Leishmania population genetics
Protozoan parasites of the Leishmania genus (Kinetoplastida: Trypanosomatidae) are responsible for human leishmaniases which remain neglected diseases despite the
serious public health problem they represent worldwide.
Leishmaniases are vector-borne diseases in humans and
animals with approximately 350 million persons at risk
and 2 357 000 new cases per year [1]. Leishmania parasites
are characterized by a complex digenetic life cycle
(Figure 1). They are present in extremely diverse ecosystems and are able to infect a wide range of mammals [2]. In
human populations, the majority of Leishmania infections
lead to asymptomatic cases. However, when the disease is
declared, it is expressed in a variety of more or less serious
clinical forms: cutaneous, mucocutaneous and visceral
[3]. For these reasons, Leishmania constitute a complex
model from biological, ecological, and epidemiological point
of view but with further important genetic and phylogenetic aspects [2,3].

Corresponding author: Rougeron, V. (rougeron.virginie@gmail.com).

Keywords: Leishmania; population genetic; sex; mix mating; aneuploidy..
*
These two authors contributed equally to this work.
1471-4922/
2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.pt.2014.12.001

Recently, many studies have dealt with the reproductive

strategies, population structure, and biosystematics in the
genus Leishmania [412]. Most of the literature focusing
on these topics uses the terminology in different ways,
sometimes mixing notions or less-than-optimal sampling
strategies (regarding the objectives being targeted), and
also sometimes without questioning the taxonomic status
of the entities being under study.
In this context, this review aims to draw an updated
picture of Leishmania population genetic studies and thus
on the rules used to infer the structure of natural parasite
populations, and then to discuss the evolution of these
microorganisms in considering sex, mix-mating, taxonomic
issues, and aneuploidy.
Rules for Leishmania population genetics
Population genetics can be used to infer the structure of
natural populations and thus to answer the questions: how
are individuals distributed within and among natural
sites? What dispersal pattern is followed by juveniles?
What is the reproductive strategy used to propagate
and/or recombine? In this respect, population genetic studies have been proved to be very useful, in particular for
organisms that are difficult to observe directly [13]. Nevertheless, for such inferences to be reasonably accurate, some
simple rules are required.
The first rule is that the individuals studied must belong
to the same species. Even in purely sexually propagating
organisms, genetic trajectories will necessarily evolve independently for the different lineages (or species or subspecies) without demographically (or sexually, if any)
interference with the other entities. Thus, if population
genetics analyses are performed without taking into account the species entity level, it will inevitably produce
unpredictable (erroneous) conclusions because they are
dependent of the relative proportion of the different entities and on how sampling of each is representative of the
population. Given its complex genetic and subspecies
structures, this is a recurrent challenge in studying Leishmania at the population level.
The second rule is that individuals identified as belonging
to the same subpopulation are able to interact ecologically.
Pooling individuals from different cohorts and from remote
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Review
Glossary
Gene: a coding sequence of DNA.
Gene conversion: ameiotic correction of an allelic variant rendering it
homozygous for the correct allele. Consequently, this phenomenon should
only involve coding DNA. In particular gene conversion of microsatellite,
given the particular structure of those, should usually result in another
mutation and more rarely to a homozygous state. Given the probable cost of
this phenomenon, we may expect gene conversion to be restricted to coding
sequences (hence the term gene conversion).
Genetic diversity: often known as (but rather misleadingly so) expected
heterozygosity. It reflects the probability that two randomly chosen alleles, in a
defined group of individuals, are different. In samples, it is generally estimated
by the unbiased Neis estimators Hs for diversity within subsamples and HT
across subsamples (total population).
Genetic marker: a locus used for genetic and/or ecological inferences. It must
be variable (polymorphic) to be useful. If ecological inferences are desired, the
marker must be reasonably neutral such that it reflects predominantly (if not
uniquely) demographic processes (and not selective ones). Coding sequences
are thus not ideal because these might reflect selective in addition to
demographic processes, even if only silent (synonymous) mutations are
studied (because of hitch-hiking). In this respect approaches based on
isoenzymes or MLST (multilocus sequence typing) are not ideal. SNPs from
non-coding sequences are better, but the choice of SNPs must not bias these
markers toward excessively heterozygous sites. Junk DNA markers, in
particular dinucleotide microsatellite loci, which presumably are non-coding,
are probably the best markers (when available).
Genetic recombination: the process of DNA exchange that produces new
combination of alleles, either between individuals of the same species
(intraspecific recombination) or between individuals of different species
(interspecific recombination). Crossing-over and gene conversion are considered to be genetic recombination processes.
HardyWeinberg equilibrium: a state reached by a population that is not
subject to genetic drift (very large population size), selection, mutation, or
migration, and which is panmictic with non-overlapping generations. Under
these conditions the population reaches allelic and genotypic equilibrium of
the form pi2 and 2 p i 1  p i for all alleles i and in all generations provided that
all conditions remain fulfilled.
Heterozygosity: in the strict sense this corresponds to the proportion of
heterozygous individuals found in a population (sample). This terminology is
also often used to describe genetic diversity (see above). Further, it is
sometimes used to describe the proportion of heterozygous sites in a
sequenced individual. This last use is unsafe because it provides statistics
that bear no relationship to the biology of the targeted population.
Hitch-hiking: a process under which a neutral variant is driven to a different
frequency in a population by selective processes affecting a linked gene. This
can be because the neutral locus is close to a region of DNA that is under
positive or negative selection, or may in some cases be part of it (e.g., a silent
SNP or a locus within an intron).
Isolate: an individual sample of a parasite taken from a host and which is
intended to represent a single individual (or group of individuals) of a single
genotype.
Linkage disequilibrium (LD): a state of LD exists between two (or more) loci
when their combined haplotypes are not present at frequencies equal to the
product of the corresponding allelic frequencies. For example, for two loci A
and B, each with alleles 1 and 2 at frequencies p1 and p2, or q1 and q2,
respectively, the loci are in linkage equilibrium when the haplotype A1_B1 is
present in the population at frequency p1  q1. Departure from this frequency
indicates that the two loci are in LD. Linkage equilibrium between all loci can
only be achieved after several generations under HardyWeinberg conditions,
even for physically independent loci. This is a big difference from Hardy
Weinberg equilibrium that is reached in a single generation. All evolutionary
forces produce and maintain LD, in particular drift, migration, and reproductive
systems. Hence, no natural population is expected to display linkage
equilibrium. Panmictic populations are only expected to display reasonably
low LD. Strongly structured populations, even if locally panmictic, are expected
to display local LD.
Locus: a defined section of DNA (not necessarily coding) that is located on a
precise portion of a chromosome.
Meta-population: a group of populations, as defined above, that are connected
by a regular pattern of migration and are submitted to the same rules of
extinction/recolonization.
Multilocus genotype (MLG): the sequence of genotypes occurring at each of
the genotyped loci. If enough loci are genotyped, and if those loci are
reasonably polymorphic, sexually reproducing populations should display no
or very low (e.g., highly inbred populations) levels of repetition of MLGs. In
pure clones, such repeated MLGs are expected to be occur frequently.
Neighbor joining trees: phylogenetic trees constructed based on a bottom-up
clustering method.
Panmixia: random union of gametes to form zygotes (fertilized eggs). In terms
of population genetics, this typically results in a genotypic distribution for each

Trends in Parasitology xxx xxxx, Vol. xxx, No. x

allele of frequency pi of the form pi2 and 2 p i 1  p i for homozygotes and

heterozygotes, respectively, in a given generation, for that allele.
Parasexual recombination: recombination that occurs without meiosis and that
involves parts of the whole genome.
Population: a group of individuals of the same species (sharing the same
ecological niche) that share similar demographic pressures (i.e., the same
chance of escaping or of being eliminated after regulating processes).
Sample of individuals: a random sample of individuals from a population. This
sample must target actual populations as precisely as possible.
Sexual recombination: this corresponds to meiotic sex and involves the whole
genome.
Strain: a collection of clonally propagating individuals that are thus presumed
to be genetically highly homogeneous (a single lineage of closely related
individuals).
Subpopulation: a subpart of a population. When a meta-population is itself
referred to as the population, then the term subpopulation becomes
synonymous to population as generally used. The term is useful when more
than three hierarchical levels (individual, subpopulation, population) are
discussed. In this case the subpopulation should correspond to the smallest
possible ecological scale that encompasses individual units.
Subsamples: as above, but targeting the smallest possible demographic unit in
the zone studied.
Wahlund effect: the consequences of biased sampling on population genetic
parameter estimates. When individuals from different cohorts and/or different
subpopulations that are genetically differentiated are gathered into a single
subsample, this will increase the relative proportion of homozygous individuals compared to the proportion expected under panmixia (HardyWeinberg
genotypic proportions). Depending on the genetic structure of subpopulations,
the individuals of which are pooled, the Wahlund effect may increase or
decrease LD, which represents another difference from HardyWeinberg
genotypic proportions. If LD is locally low, the Wahlund effect will increase
LD. If LD is locally strong, the Wahlund effect will decrease LD. Wahlund effects
will be typically met for populations of unknown structure, hence with an
unpredictable effect on LD.

geographic sites will have unpredictable consequences on

the distribution of genetic variation within and across what
are defined as subsamples. This will produce what is called
Wahlund effects (see Glossary). Depending on the reproductive system, and on how inaccurately real demographic
units subpopulations are sampled, subsamples may display
more or less important heterozygote deficits and increased
or decreased linkage disequilibrium (LD) [14]. Furthermore,
temporal and/or spatial Wahlund effect will strongly modify
the picture given by the spatial distribution of genetic
variability across subpopulations and will bias (in an unpredictable direction) dispersal inferences. This is an important point because Leishmania species are potentially
structured at small scales [4,7,8] and have (very) short
generation times [7]. It is worth noting that most population
genetic studies on Leishmania sample strains from very
remote places and dates. It is thus fundamental for population genetic studies to base the analysis on individuals from
one subsample sampled at the same date (or reasonably so in
terms of generation time) and at a geographical scale that
reasonably fits what is expected for the species under study
(e.g., the focus of a disease).
The third rule is that concepts used in empirical population genetics must be fully assimilated. These concepts
are described in the Glossary.
Accounting for the complex taxonomy of Leishmania
Microscopic observations rarely display obvious external
features that would allow easy discrimination of microbes
at the subspecies level. Indeed, several studies have
reported that unexplained genetic heterogeneity can be
found within a single taxon, even at a very local scale.
In the complex L. donovani, most studies have described
genetic heterogeneity across the whole sample, even for

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Trends in Parasitology xxx xxxx, Vol. xxx, No. x

Sandy takes a blood meal

and injects metacyclic
promasgotes
into the skin
Nectomonad
promasgotes
migrate
to proboscis

Transformaon of
amasgotes into
procyclic
promasgotes
in midgut
and divide

Promasgotes are
phagocyzed
by macrophages
Transformaon
of promasgotes
into
amasgotes

Mulplicaon
of amasgotes

Ingeson
of parasized
cells
Sandy takes a blood meal
and ingests macrophages
infected with amasgotes
TRENDS in Parasitology

Figure 1. The Leishmania life cycle displays an obligatory passage through a vertebrate host, that can be a human or various other mammalian species (wild or domestic
mammals for medically relevant species) and an obligate vector, a sandfly (Diptera: Psychodidae, subfamily Phlebotominae). Arrows in pink indicate where sexual
recombination can take place (in the vector). This figure was freely inspired by a figure from the Centers for Disease Control and Prevention (CDC; http://www.dpd.cdc.gov/
dpdx) and Figure 1 of Lipoldova and Demant [75].

geographically close isolates. Two studies, based on a

larges samples of L. donovani, L. infantum, and L. chagasi
(species of the New World), analyzed the population structure based on microsatellites loci [15,16]. Phylogenetic
results of these two studies showed that L. infantum is
heterogeneous, and this may be a consequence of the
geographical heterogeneity of the samples. L. chagasi
appears to be a case of spurious naming, as previously
reported in many studies [15,16]. L. donovani is composed
of fairly distant but more homogeneous lineages (especially
in Indian subsamples). Moreover, in one of the studies of
Khuls et al. [16], some multilocus genotypes (MLG) seem to
coexist in the same region for very long periods of time,
which is in line with the coexistence of separated ecological
entities (species) because otherwise genetic drift would
favor the replacement of different MLGs by others. A very
small sample of Turkish L. infantum and L. donovani was
added to a bigger dataset of European isolates [17] and
analyzed based on 14 microsatellite loci. Results showed
four clear lineages (and probably more within each): two
were classified as L. infantum and two as L. donovani, but
none of these could be grouped accordingly. Hide et al. [18]
also found, based on microsatellite markers, divergent
L. infantum isolates in the South of France. Finally, a
recent study based on multilocus sequence analysis of 222
Leishmania strains from Africa and Eurasia questioned
the current classification, specifically for species that cause
visceral forms, based on the heterogeneity observed
between these species (L. infantum, L. donovani, and
L. archibaldi) [19]. Currently, it is assumed that L. donovani should be found more on humans than on dogs, while
L. infantum and L. archibaldi (if both species are correctly

determined) should be found on both humans and dogs

[8,20]. However, and obviously, criteria used to study the
diversity through zymodemes, within species complexes, or
within species, have weak (at best) a genetic basis.
For L. major in Iran, a relatively small sample was
analyzed through nine microsatellite markers [21].
Authors found that overall the available isolates from Asia
and Africa distribute in three (at least) fairly distinct
lineages, some of which coexist in the same region. This
is in line with the coexistence of several distinct taxa.
For New World Leishmania (sub-genus Viannia), studies also tend to highlight the problems linked to species
determination and genetic heterogeneity. Multilocus sequence typing of Brazilian Leishmania [22] indicated that
it is difficult to differentiate between lineages and species:
L. naiffi and L. lainsoni were confirmed as monophyletic,
while L. guyanensis appeared to be more complex and
contained the highly differentiated L. shawi. In a larger
sample, 14 amplified fragment length polymorphism
(AFLP) markers suggested the coexistence of divergent
lineages in L. peruviana, as already demonstrated by
Dujardin et al. [23], and, above all, in L. braziliensis from
across central and South America [24]. In Rougeron et al.
[25], the majority of the genetic variance at 12 microsatellite loci of L. braziliensis from Bolivia and Peru occurs
within the two countries and not between: this could reflect
ecologically divergent entities [25]. Another study, which
used 12 microsatellites in L. guyanensis and L. panamensis, revealed again strong genetic heterogeneity, at least in
Ecuador [25]. Moreover, L. guyanensis appeared to be
much more homogeneous in Peru [25] than in French
Guyana [7]. These studies also suggest the potential
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coexistence of different genetic taxa that remain to be
identified.
The sampling of recent studies discussed above strongly
suggests the existence and coexistence of multiple genetically divergent lineages. Accurate population genetic studies of such lineages would require them to be investigated
separately using adequate samples sizes. This makes the
study of Leishmania population structure and reproductive strategy very difficult, if not impossible. Moreover, it
might be very interesting and fundamental in the future to
further explore the parasite reservoir at the vector species
level because this might reflect differences in ecology and
provide a better understanding of particular epidemiological and/or clinical situations.
Factors that confound population structure analysis
In most of the literature cited in the section above, sampling appeared to be heterogeneous both in space and time.
The small size of the subsamples (a subsample should be
reasonably homogeneous in time and space) is also a
caveat. Such sampling will necessarily create strong (if
not huge) Wahlund effects, which, combined with the
probable taxonomic challenges highlighted above, will provide an unpredictable picture as far as population genetic
analyses are concerned.
Using STRUCTURE software [26] (which looks for a
simple population structure in randomly mating clusters
in linkage equilibrium), or computing the fixation index
F ST between subsamples that are obviously composed of
many different entities is not sufficient to draw reliable
conclusions. STRUCTURE assumes that the clusters being
looked for are in HardyWeinberg equilibrium (genotypes
distributed according to panmictic expectations) and that
loci are in linkage equilibrium or display weak LD. The
effects of strong deviations from panmixia on clustering by
STRUCTURE (or by any other Bayesian software) will
need to be investigated. Nevertheless, it is known that
substantial LD can lead to spurious results [27]. For this
reason, results using STRUCTURE should be interpreted
extremely cautiously in populations experiencing high
levels of LD as is the case for Leishmania. Moreover,
because the procedure (as undertaken by STRUCTURE)
necessarily leads to maximum allele frequency variance
across the resulting clusters, computing F ST between these
clusters will trivially lead to the maximum possible F ST
and is thus also meaningless. In such situations, parameter inferences cannot be reasonably linked to any biological
reality. The clusters found by STRUCTURE were poorly
informative as compared to those obtained by neighbor
joining tree methods. However, given the spatial and
temporal heterogeneity of sampling, it is currently difficult
to assess why some lineages appear to be more homogeneous, or why some branches of the tree are so long.
Factors to consider in defining strains: clonality and
heterozygosity
Discussion is essential before any Leishmania isolates (or
group of isolates) can qualify as strains. To a non-specialist,
classification as a strain indicates clonality, and is thus
analyzed as such, without bearing in mind that more or
less frequent recombination might affect some or most of
4

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the data. In some lineages (and possibly most lineages) of

Leishmania, sexual recombination is frequent. Under
these conditions, the terminology of strain no longer
applies. Ultra-clonality and accurate taxonomy are both
highly debatable issues where referring to Leishmania
populations.
In population genetics, the term heterozygosity refers to
the probability that two alleles from one individual and at
one locus (all randomly drawn) in a population (or subsample) are different [13]. This is different from the expected
heterozygosity, which should properly be referred to as
genetic diversity, and is also different from the proportion
of heterozygous sites found in a single individual (or
strain). Heterozygosity is also different from the index,
Wrights F IS, that classically measures relative homozygosity, and which is a very useful tool to infer reproductive
systems [13]. Heterozygosity and F IS are both influenced
by reproductive systems, but heterozygosity will also be
dependent on polymorphism (hence population size in the
wild) except in pure clones where it is fixed to 1. Wrights
F IS, by contrast, is independent of the degree of polymorphism, except in pure clones where it can be used as a
measure of population size [28]. Confounding these notions
can lead to important misinterpretations. Population genetic notions are often mixed with individual genomics. For
Leishmania, an apparent excess of heterozygous sites
(rather than an excess of heterozygosity) was seen in a
laboratory strain of L. braziliensis, and this excess was not
commonly observed other natural clinical strains (Hideo
Imamura, personal communication). Nevertheless, the
proportion of heterozygous sites in one laboratory strain
of one species is not sufficient to provide information about
the F IS that could be found in natural populations. Another
study showed high levels of heterozygosity (heterozygous
sites) in one hybrid Giardia individual [29], but this does
not provide any information about either the extent of
heterozygosity at the population level or about the frequency of recombination in that species.
LD and clonality are often referred to synonymously.
Indeed, in their reviews Tibayrenc and Ayala propose an
unambiguous definition for clonal evolution that does not
take into account the cytological mechanism [11,30]. They
propose instead to diagnose clonality as the absence or
near-absence of recombination. Of course, one of the consequences of clonality is a restriction on recombination (as
it is for segregation). Nevertheless, restricted recombination is not a hallmark of clonal propagators. In particular,
small and strongly isolated populations can lack recombination, and, depending on the sampling strategy used, a
purely biparentally sexually reproducing species could
present a clonal structure in accordance with Tibayrenc
and Ayalas criterion. Moreover, sampling may not always
perfectly fit the real structure of populations, especially for
microbes where signatures of a lack of recombination may
be far from what occurs in reality [31]. Based on the
sampling strategies where isolates are sampled from different places and at different dates (sometimes decades
apart), we cannot expect any accuracy regarding estimates
of recombination rate. Even different species are probably
pooled together, as in the case of Trypanosoma brucei in
the pioneering paper of Smith et al. [32], which turned out

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to be a complex of at least three different species [33] and
was erroneously interpreted to fit an epidemic-like population structure. Small rates of sexual recombination are
enough for populations to globally display sexual evolutionary ecology trajectories. LD can of course help to
maintain newly created favorable gene combinations,
and this might provide an advantage to all organisms
using clonal propagation. Nevertheless, strongly or purely
sexual subdivided populations can also do that. Prokaryotic recombination was selected for acquiring new functionalities, whereas meiosis was selected to repair
damaged DNA and protect it against alien (parasitic)
DNA [34]. In that respect, selfing and endogamy (not
homogamy, which is locus-specific) represent more perfect
mechanisms for meiotic sex, and not particular clonal
modes.
Intraspecific and interspecific genetic recombination in
Leishmania
Leishmania parasites are characterized by extremely complex breeding strategies and can thus implement various
evolutionary processes [2,35]. As introduced above, for
decades there was debate concerning the reproductive
strategies of Leishmania parasites: asexual versus sexual
reproduction [3638]. The disagreement between these
two positions relied on the frequency of genetic exchanges,
specifically low versus high. Irrespective of the theory
defended, there was agreement about the occurrence of
genetic exchanges. Currently, the existence of genetic
exchanges is supported by five points: (i) the demonstration
of hybrid isolates between and within Leishmania species,
(ii) the observation of nuclear fusion in Leishmania, (iii) the
existence of conserved meiotic orthologous genes whose
role is still unknown in Leishmania, (iv) high homozygosity
theoretically compatible with the recurrent occurrence of
sexual reproduction events, and (v) the lack of correlation
between genetic dendrograms drawn from DNA genetic
markers and zymodeme (allozyme-based) classification.
The existence of interspecific hybrids among Leishmania has been known for decades. For example, in the New
World, hybrids between L. braziliensis and L. peruviana,
L. guyanensis and L. braziliensis, L. braziliensis and
L. panamensis, and L. panamensis and L. guyanensis have
been described [3944], and in the Old World hybrids have
been reported between L. major and L. arabica, L. infantum and L. donovani, L. donovani and L. major, L. infantum and L. major [4552]. Recently, three experimental
studies provided evidence of intraspecific crosses in
Leishmania. In the first study, Akopyants et al. [53]
infected natural sandflies using transgenic L. major parasites resistant to different selective drugs, and then isolated parasites resistant to both drugs. The study succeeded
in producing hybrid progenies demonstrating that genetic
recombination occurs only in the vector, and that hybrid
progeny are transmitted to the mammalian vertebrate
host by sandfly bites. In the second study, using transgenic
parasites and fluorescent microscopy, Sadlova et al. [54]
co-infected two sandfly species with promastigotes of
L. donovani. Results revealed L. donovani hybrids in
the early stage of development in the vector [54]. The
third study confirmed the results of Akopyants et al. by

Trends in Parasitology xxx xxxx, Vol. xxx, No. x

demonstrating the sexual competency of L. major strains

based on pairwise mating of different strains [55]. Indeed,
consistent with the process of meiosis, they obtained hybrid progeny which inherited both parental alleles at the
major chromosome marker loci. Nevertheless, even if
the majority of hybrids displayed 2n DNA content, the
authors observed a significant number of 3n and one 4n
offspring. These data suggest that the mating process in
Leishmania does not correspond to classical meiosis (see
below). Finally, another recent study showed the existence
of interspecific hybrids, which should be stable over time,
based on phylogenetic analysis and congruence tests
[19]. It must be noted that the same type of experimental
study, based on fluorescent clones, has been performed on
Trypanosoma brucei, the causative agent of African sleeping sickness. Indeed, Gibson et al. [56] described the existence of mating and the utility of this approach to gain a
better understanding of mating systems. Another recent
work studied the compatibility mating system, by analyzing fluorescent cloned cell lines, and showed that cell lines
were characterized by variable numbers of crosses [57]. In
summary, these two papers showed that meiotic sex takes
place between different strains of T. brucei and that cell
fusion is preceded by meiosis. The observation of naturally
occurring hybrid isolates and of experimentally produced
recombinants provide biological proof of the existence of
sexuality and genetic recombination.
The direct observation of nuclear fusion in Leishmania
parasites could also be considered as evidence of genetic
recombination. The first observation was made by Lanotte
and Rioux in 1990 [58]. The authors presented a video
depicting spontaneous fusion between two promastigotes
(corresponding to the vector stage) of L. tropica isolated
from Phlebotomus sergenti and cultivated until the formation of a zygomastigote [58]. Subsequently, Kreutzer et al.
[59] demonstrated by quantitative microspectrophotometry that nuclear fusion occurred in the intracellular amastigote form within the mammalian host. This was
confirmed by Youssef et al. [60] who stained amastigotes
of the Old World Leishmania parasites, using the Feulgen
procedure followed by determination of nuclear DNA content using computerized image analysis. These three studies strongly suggest the existence of genetic exchange
through sexual events. However, the location of sexual
events was still questionable. Indeed, mating was shown
to occur either at the amastigote stage (in the mammalian
host) [59,60] or in the promastigote stages, thus within the
vector [58].
Analyses of Leishmania genomes showed the presence
of conserved meiotic orthologous genes [6163]. However,
the presence of these meiotic genes does not obligatorily
suggest the existence of sexual recombination, but could
instead be considered as clue pointing to genetic recombination. For instance, a study demonstrated that genetic
recombination in Candida albicans was dependent on the
presence of Spo11p, a conserved protein that in other
eukaryotes initiates meiotic recombination [64]. The
authors suggested that this meiotic gene has been conserved and reprogrammed to mediate an alternative pathway, here genetic recombination [64]. For Leishmania
parasites, even if meiotic genes have been identified in
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their genomes, currently the proteins have not been shown
to be expressed during the life cycle of Leishmania parasites. However, we could hypothesize that these meiotic
genes have been reprogrammed to generate genetic
exchanges, as for C. albicans. In future studies it would
be interesting to identify if these meiotic proteins such as
MND1 or/and DMC1 are expressed during the life cycle of
Leishmania parasites, and if these gene products have
been reprogrammed to mediate a type of genetic recombination distinct from classical meiosis as has been demonstrated for C. albicans [64].
Recent population genetic studies support the existence
of sexual recombination in Leishmania [710,15,25,65,
66]. Indeed, all these studies showed heterozygote deficits
at all loci in Leishmania irrespective of species. As described
above, theoretically, heterozygote deficit is incompatible
with a predominant clonal mode of reproduction at ecological time-scale, but is in agreement with the occurrence of
frequent mating between related individuals (endogamy)
and more rarely between non-related ones (allogamy)
[67]. For example, analysis of 125 human isolates of
L. braziliensis from Peru and Bolivia with 12 microsatellite
loci revealed very high levels of homozygosity within samples [4]. From this population genetic study the authors
suggested that L. braziliensis parasites alternate between
different modes of reproduction, in other words a mixmating model clonality in both vertebrate host and insect
vector, endogamy within the insect vector, and rare recombination events within both hosts [4]. Significant homozygosity has been recurrently found for all Leishmania species
in the literature, combined with a generalized rarity of
MLGs (except for L. infantum [16,68] and for L. major
[69]). In the work of Adaui et al. [70] an absence of correlation
was reported between 15 microsatellites randomly dispersed in the genome and antimony resistance associated
with two different forms of treatments for L. braziliensis in
Peru [70]. If clonality explained most of genetic structure of
this species, a correlation should have emerged. A genomic
SNP approach, applied to a very small and timely heterogeneous sample from India and Nepal, also suggested substantial redistribution of resistance variants across lineages
of L. donovani, which is in line with a substantial role of
recombination in the history of those isolates [63]. In a
recent study, the 12 strains of L. donovani/infantum were
shown to be derived from a recent hybridization event
between two divergent species, followed by clonal propagation, with the rate of sexual reproduction being as rare as
2.105, but they reported an F IS as large as 0.98 (almost
equating to fixed homozygosity) [71].
Finally, the lack of correspondence between zymodeme
classification within species (allozyme-based) and dendrograms built from DNA data (e.g., microsatellites), which
both depend on many physically independent loci, as
reported in many studies, is more a hallmark of recurrent
recombination than evidence for clonal propagation
[16,17,68,72,73].
Aneuploidy and ameiotic recombination
Aneuploidy is defined as an aberrant number of chromosomes, and is generally considered to represent dysfunction of chromosomal segregation. In pathogens, aneuploidy
6

Trends in Parasitology xxx xxxx, Vol. xxx, No. x

may be associated with drug resistance and may appear in

vitro under drug pressure. Aneuploidy aneuploidy seems to
be the rule for Leishmania parasites. This topic was
reviewed recently in Sterkers et al. (2014) and is not
discussed here in detail.
Regarding the population genetic studies, aneuploidy
should have an impact because the tests used have assumed diploidy. Indeed, co-dominant markers (microsatellite markers) are traditionally used. Nevertheless, in all
microsatellite data published in Leishmania, three, four, or
five alleles were never reported. Furthermore, in our three
population genetic analyses, results revealed low levels of
heterozygosity [4,7,8]. Theoretically, this low heterozygosity could be due to reproductive strategies, to null alleles,
or to fluctuating chromosome copy number (aneuploidy
and especially haploidy). Another assumption to consider,
to explain the absence of more than two alleles in these
studies, is that aneuploidy might be a transitory state.
Such transient aneuploidy would alter DNA quantities but
not the extent of intra-individual polymorphism. This
means that, from a population genetic perspective, these
parasites are not or are barely aneuploid. If aneuploidy
was indeed a permanent state, then triploid or tetraploid
loci would finally experience mutations and three or four
picked (or banded) individuals should appear in subsamples. This might be the case in several organisms such as
yeasts (e.g., [74]). However, without appropriate sequencing it is always hard to distinguish such cases from locus
duplication. To summarize, the discrepancy between population genetic results and cytological observations might
indicate that the aneuploidies observed are only transitory
states, and that during a life cycle some type of regulation
occurs, preventing the observation of three- and four-allele
microsatellite profiles.
Concluding remarks and future perspectives
Advances in the discovery of mixed-mating reproduction in
Leishmania parasites represent a fundamental step towards a new consideration in the population genetics of
these complex parasites. These pathogens present different
proportions of reproductive modes in relation to their diversity in terms of hosts. This suggests a continuum of mixedmating reproductive strategies for parasites across different
species and environments. Aneuploidy seems to be the rule
in Leishmania parasites, but appears to undergo some type
of cyclic turnover that thus weakly alters genotypic distributions. Even if we are far from understanding how these
organisms evolve in natural populations, when performed
correctly population genetic studies can provide key clues to
the ecology and transmission patterns of Leishmania parasites. Understanding the precise roles of clonality, selfing,
and outcrossing, and the exact contribution of aneuploidy,
represents a key objective if we are to use population genetic
tools to infer ecologically relevant parameters defining these
organisms. These pathogens have unsuspected resources to
survive, adapt, and propagate. In this context, discovering
how and why these mechanisms operate is of primary
relevance in understanding the epidemiology, ecology,
and evolutionary biology of these parasites. Four major
future research axes should be developed: (i) testing
experimentally the hypothesis of endogamy in one isolate

TREPAR-1338; No. of Pages 8

Review
(the population of parasites that constitutes the isolate is
characterized cell by cell, and genotyping is followed over
several generations), (ii) exploring why the meiotic genes
have been conserved in the genome of Leishmania (have
these genes been reprogrammed as for C. albicans?), (iii)
demonstrating if the parasexual hypothesis is coherent in
this complex system, and (iv) confirming that aneuploidy is a
cyclic phenomenon, probably linked to parasexual cycles,
with no consequences on the genotypic distributions in
natural populations of Leishmania.
Acknowledgments
The authors acknowledge the assistance of Dr Hideo Imamura who
provided very helpful advice regarding genomics and aneuploidy. We are
grateful to the IRD and the CNRS for financial support. This work was
also supported by the French National Project ANR 06- SEST-20 IAEL.

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