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The population genetics of Trypanosoma cruzi revisited in the light of the predominant clonal evolution model
Accepted Manuscript Title: The population genetics of Trypanosoma cruzi revisited in the light of the predominant clonal evolution model Author: Michel Tibayrenc Francisco J. Ayala PII: DOI: Reference:
S0001-706X(15)00140-0 http://dx.doi.org/doi:10.1016/j.actatropica.2015.05.006 ACTROP 3618
To appear in:
Acta Tropica
Received date: Revised date: Accepted date:
9-3-2015 2-5-2015 6-5-2015
Please cite this article as: Tibayrenc, M., Ayala, F.J.,The population genetics of Trypanosoma cruzi revisited in the light of the predominant clonal evolution model, Acta Tropica (2015), http://dx.doi.org/10.1016/j.actatropica.2015.05.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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The population genetics of Trypanosoma cruzi revisited in the light of the
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predominant clonal evolution model.
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Michel Tibayrenc1 and Francisco J. Ayala2
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Montpellier Cedex 5, France
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92697, USA
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MIVEGEC (IRD 224-CNRS 5290-UM1-UM2), IRD Center, BP 64501, 34394
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Department Ecology and Evolutionary Biology, University of California, Irvine, CA
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Corresponding author: Tibayrenc, M. ([email protected]).
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Comparing the population structure of Trypanosoma cruzi with that of other
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pathogens, including parasitic protozoa, fungi, bacteria and viruses, shows that the
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agent of Chagas disease shares typical traits with many other species, related to a
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predominant
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disequilibrium,
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subdivisions somewhat blurred by occasional genetic exchange/hybridization) and
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―Russian doll‖ patterns (PCE is observed, not only at the level of the whole species,
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but also, within the near-clades). Moreover, T. cruzi population structure exhibits
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linkage with the diversity of several strongly selected genes, with gene expression
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profiles, and with some major phenotypic traits. We discuss the evolutionary
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significance of these results, and their implications in terms of applied research
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(molecular epidemiology/strain typing, analysis of genes of interest, vaccine and drug
evolution
(PCE)
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multilocus
statistically significant
genotypes,
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linkage (genetic
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overrepresented
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design, immunological diagnosis) and of experimental evolution. Lastly, we revisit the
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long-term debate of describing new species within the T. cruzi taxon.
27 Key-words: predominant clonal evolution, near-clade, discrete typing unit,
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recombinational load, drug resistance
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30 * see glossary of specialized terms.
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Although much progress has been made in vector control, Chagas disease
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remains a major health problem in Latin America, and is now threatening industrial
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countries, in particular Spain and the USA. With the hope of developing radical control means for Chagas disease, extensive
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studies have been conducted on Trypanosoma cruzi population genetics, and have
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made this parasite probably one of the micropathogens whose intraspecific diversity
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is best known.
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A comparative population genetic analysis of T. cruzi and of many other
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pathogens, including parasitic protozoa, fungi, bacteria and viruses, has broaden our
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view of the agent of Chagas disease’ population structure and has made it possible
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to reveal the evolutionary strategies used by this pathogen. Moreover, such a
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comparative population genetics has brought relevant considerations about the
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possibility to describe new species within the T. cruzi taxon.
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The predominant clonal evolution (PCE) model of pathogens
The model will be recalled only briefly here, since it has been exposed at length in several recents articles (Tibayrenc & Ayala, 2012, 2013, 2014 a and b).
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PCE is not defined by any precise mating system or cytological mechanism, but
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only by strongly restrained genetic recombination* on an evolutionary scale. It
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therefore deals with population structure of the whole species in the long run only.
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This definition is accepted by most authors working on pathogen population genetics,
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including parasitic protozoa, fungi and bacteria (Tibayrenc & Ayala, 2012) and is well
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accepted in the T. cruzi literature (Barnabé et al., 2013; Flores-López & Machado,
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4 2011; Minning et al., 2011). According to our definition, PCE includes, not only mitotic
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reproduction, but also selfing*/strong homogamy*, several cases of parthenogenesis*
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(Tibayrenc & Ayala, 1991, 2002), and all cases of ―unisex‖ (Feretzaki & Heitman,
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2013). PCE would group all means used by micropathogens to escape the
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―recombinational load‖ (disrupting favorable multilocus genotypes by genetic
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recombination) (Agrawal, 2006).
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Equating selfing/strong homogamy and many cases of parthenogenesis to
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clonality is not confined to microbiologists and extends to evolutionists working on
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higher organisms too (Avise, 2008). Some authors, working on Leishmania
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(Rougeron et al., 2009) or on T. cruzi (Llewellyn et al., 2009a) recommend to
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distinguish selfing/homogamy from ―strict‖ clonality (= mitotic propagation). This is a
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matter of definition. The problem deals with the means used for distinguishing
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selfing/homogamy from ―strict‖ clonality.
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The main consequences of PCE are: (i) statistically significant linkage
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disequilibrium, or nonrandom association of genotypes occuring at diffferent loci
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(LD); (ii) overrepresented multilocus genotypes (MLGs), which can be lacking if the
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mutation rate of the marker is high (all strains have different genotypes); (iii) the
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presence of ―near-clades‖ (genetic subdivisions somewhat blurred by occasional
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genetic exchange/hybridization); ―Russian doll‖ patterns (RDPs- PCE is observed,
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not only at the level of the whole species, but also, within the near-clades) (Tibayrenc
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& Ayala, 2013).
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We have coined the term ―near-clade‖ (Tibayrenc & Ayala, 2012) because it can
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be suspected that virtually all micropathogen species undergo some bouts of
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recombination, including among different species. This makes the demands of a strict
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cladistic* analysis impossible. We have recommended to evidence the presence of
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5 near-clades with the tools of population genetics analysis (LD), then by a flexible
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phylogenetic approach relying on the ―congruence principle‖ (Avise, 2004). The use
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of more reliable information supports more and more the working hypothesis (here:
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the presence of near-clades). For example, in the case of MLST*, the phylogenies of
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individual genes may exhibit some discrepancies. However, the use of the whole set
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of genes evidences a strong phylogenetic signal (Lauther et al., 2012). Or different
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clustering/phylogenetic approaches, relying on different working hypotheses, give
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convergent results, which confirm the robustness of the branchings (fig.1). Or the
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phylogenies designed from different molecular markers corroborate each other
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(Brisse, Barnabé & Tibayrenc, 2000; Tibayrenc et al., 1993). Identification of PCE
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relies on the concept of a ―clonality threshold‖, beyond which the impact of clonal
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evolution overcomes that of recombination and leads to the inescapable evolutionary
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divergence of the near-clades. This concept of a ―PCE border‖ makes it possible to
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replace vague and subjectives assertions (―substantial‖ recombination; de Paula
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Baptista et al., 2014; "Gross incongruences", "multiple introgressions‖: Messenger et
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al., 2012) with a clear-cut criterion. The alternative hypothesis to PCE is that
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recombination is abundant enough to erase the effects of clonal evolution in the long
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run. This corresponds to the ―epidemic clonality‖ model (Maynard Smith et al., 1993)
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or ―semiclonal model‖ (Maiden, 2006)).
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Some important points of the model
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- As recalled many times, the PCE model does not state that recombination is
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absent, but rather, that it is not frequent enough to break the prevalent PCE pattern.
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The model considers that recombination/hybridization could play a major role in
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pathogens’ evolution, but only on an evolutionary scale. It does not state by any
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means that recombination is ―inconsequential‖ (Ramírez & Llewellyn, 2014) or ―of
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little consequence‖ (Miles et al., 2009). - In a first step, PCE should be explored at the level of the entire species. The
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species concept, as we will see further, is a matter of debate in the case of
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micropathogens. However, the unit of analysis when PCE is explored should be the
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presently described species. It has been argued that ―it makes little sense to address
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each parasite species as a whole‖, because these ―genetic subdivisions (i.e.: the
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near-clades) act as reproductive barriers‖ (Ramírez & Llewellyn, 2014). However,
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only the PCE approach is able to evidence the existence of reproductive barriers
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among the near-clades. Claiming that such reproductive barriers are self-evident
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(Ramírez & Llewellyn, 2014) is quite untrue. Evidencing them is the very goal of the
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PCE approach.
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- To explore PCE, sampling should be twofold: (i) in a classical population genetics
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strategy, it should be conducted in close sympatry* on very limited spans of time, in
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order to ascertain that the organism under study has actual opportunity for mating
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(de Meeûs et al., 2007). However, it is extremely difficult to ascertain a strict
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sympatry when micropathogens are concerned. Even sampling at the level of
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individual hosts is not a guarantee for it. If different genotypes of the species
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concerned do not have the same tropism for different tissues of the host, they could
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have little opportunity for mating. (ii) Since the PCE pattern is definitely a ―bird’s eye
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view‖ of the overall genetic variability of the whole species in the long term, we
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recommend a global sampling of the entire species on its complete ecogeographical
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range, including all known hosts, and relying on retrospective studies to evaluate the
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7 stability of the PCE pattern. Retrospective studies can concern the analysis of
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ancient culture collections, or of ancient publications, or both. Such a sampling
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strategy makes it possible to ascertain that ubiquitous MLGs, near-clades and RDPs
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are stable in space and time, are specific properties of the species under study, and
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cannot be explained by a Wahlund effect* (Rougeron et al., 2014). If inhibition of
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recombination were due to trivial physical obstacles (Wahlund), overrepresented
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MLGs, near-clades and RDPs would be strongly linked to space and/or time.
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- The PCE model predicts that different clonal genotypes, due to inhibition of
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recombination among them, will tend to accumulate divergent mutations, which could
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concern genes governing relevant phenotypes (virulence, resistance to drugs). This
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should be especially true when the evolutionary divergence among clones is high.
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However, the model does not predict that all phenotypes should be strictly linked to
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the clonal population structure. This should be only a general tendency, and could be
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not verified at microevolutionary levels, and when strongly selected phenotypes (drug
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resistance) are concerned. Such selected phenotypes are bound to emerge
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independently in distinct lineages. This is not an evidence for recombination in itself
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(Rougeron, de Meeûs & Bañuls, 2015), although it can be consistent with
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recombination events. Chloroquine resistance is observed in different Plasmodium
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species. This is not the result of genetic exchange between these different species,
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but only of a common selective pressure.
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- The clonet concept: Identical MLG is a relative notion, and is highly dependent
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on the resolution power/mutation rate/molecular clock* of the marker employed. A set
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of strains that appears as identical for a given marker could reveal genetic
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heterogeneity when a more discriminative marker is used. If PCE obtains, this set
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should be considered as a family of closely related clones, rather than a true clone.
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8 The term ―clonet‖ has been coined by us to refer to those sets of strains that appear
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to be identical with a given set of genetic markers in a species that undergoes PCE
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(Tibayrenc, Kjellberg & Ayala, 1991). A typical example of clonet is the zymodeme*
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MON1 of Leishmania infantum. This MLEE* MLG is widespread in the ancient and
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new world. The use of a more discriminating marker (microsatellites) shows that
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MON1 is genetically heterogeneous (Amro et al., 2009; Chargui et al., 2009). One
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important point with the clonet concept is that the population genetic tests and
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phylogenetic analyses made on the basis of a given marker remain entirely valid
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when more discriminating markers evidence additional genetic variability by
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comparison with this marker. A given marker addresses a given level of resolution
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and evolutionary divergence and is better adapted for given studies.
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PCE and Trypanosoma cruzi:
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T. cruzi is a paradigmatic case of PCE. It is actually the parasitic model used by
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us for long to design the PCE approach (Tibayrenc et al., 1981, 1986), and to extend
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it later to various other pathogens (Tibayrenc, Kjellberg & Ayala, 1990; Tibayrenc et
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al., 1991, 2012).
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Apart from our own studies, many data are available in the literature, back to the
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70s (the pioneering studies by Miles et al., 1977, 1978, 1981), relying on various
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markers, which makes comparative and retrospective studies easy (Zingales et al.,
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2012). Retrospective studies (ancient strain collections and publications) are highly
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relevant to evaluate the persistence of T. cruzi PCE features (widespread MLGs,
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near-clades, RDPs). Many of the ancient studies dealing with the population genetics
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and strain typing of T. cruzi and various other micropathogens rely on MLEE. It is
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9 remarkable that MLEE results as a rule have been fully confirmed by modern DNA
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markers. So ancient MLEE data, when properly interpreted, give a reliable basis for
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retrospective studies. Lastly, the analysis of paleo DNA (Araujo, Reinhard & Ferreira,
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2008) constitutes a promising approach to evaluate the antiquity of T. cruzi
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population structure.
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The PCE indices rely on many raw data sets that can be easily verified by anybody. They are as follows:
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- LD: We do not consider LD and PCE as ―synonymous‖ (Rougeron, de Meeûs &
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Bañuls, 2015). However, we and many authors working on micropathogen population
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genetics (Tibayrenc & Ayala, 2012) consider LD as a valid circumstantial evidence
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for clonal evolution, as long as the bias due to Wahlund effect is excluded. LD in T.
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cruzi is observed: (i) among MLEE loci (Tibayrenc et al., 1981, 1986); (ii) among
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different markers (―test g‖ of LD; Tibayrenc, Kjellberg & Ayala, 1990): between MLEE
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and RAPD* (Tibayrenc et al., 1993; Brisse, Barnabé & Tibayrenc, 2000); between
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microsatellites and ribosomal DNA restriction fragment length polymorphism (Oliveira
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et al., 1998); between MLST*, MLEE and RAPD (Lauthier et al., 2012); between
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microsatellites and the sequence of the Gpi gene (Lewis et al., 2011). LD in T. cruzi
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is not limited to classical markers, and is observed between genetic markers and
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copy number variants (Minning et al., 2011); microsatellites and DNA content (Lewis
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et al., 2009); between MLEE and RAPD on one hand, 12 antigen genes (which are
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under strong selection) on the other hand (Rozas et al., 2007); between classical
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markers ad the cruzipain antigen gene (which is under strong selection too) (Lima et
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al., 2012). Lastly, a statistical association is observed between the variability
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10 208
revealed by molecular markers and the polymorphism of expressed genes (Telleria
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et al., 2004, 2010, 2013).
210 - Overrepresented, widespread genotypes: we have called them: ―major clones‖
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(Tibayrenc & Brenière, 1988). This is the case for example of the MLEE MLGs n° 19,
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20, 32 and 39 in Tibayrenc et al. (1986), which have been observed in diverse
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countries, years apart. For example; MLG 19 has been sampled in Brazil, Venezuela,
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Colombia, Bolivia, in Chagasic patients, as well as in various mammalian hosts and
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triatomine bug species, from 1977 to 1983. With more discriminating MLEE typing,
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ubiquitous MLGs still are observed. One MLG repeated 31 times has been observed
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in four different countries (Barnabé, Brisse & Tibayrenc, 2000). The remarks made
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about the clonet concept (see above) should be recalled here. MLEE reveals
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ubiquitous MLGs. However, when more discriminating markers (microsatellites) are
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used, each strain may have a different genotype, due to the fast mutation rate of this
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marker (Oliveira et al., 1998).
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- Near-clades. The near-clade concept (Tibayrenc & Ayala, 2012) gives a clear
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evolutionary definition to the descriptive term DTU* (discrete typing unit) coined by us
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(Tibayrenc, 1998). On the basis of both MLEE and RAPD typing, it has been
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proposed that T. cruzi is subdivided into 6 discrete genetic lineages, or DTUs
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(Barnabé, Brisse and Tibayrenc, 2000; Brisse, Barnabé & Tibayrenc, 2000). T. cruzi
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DTUs perfectly fit the definition of near-clades: well-individualized genetic lineages
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that are somewhat blurred up by occasional genetic exchange and/or hybridization.
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Hybridization events have been described since long in T. cruzi (Machado & Ayala,
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2001; Brisse et al., 2003), although their precise evolutionary history still is a matter
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11 of debate (de Freitas et al., 2006; Ferreira & Briones, 2012; Subileau et al., 2009;
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Westenberger et al., 2005, 2006). The classification into 6 DTU/near-clades has
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been recently validated by a panel of international experts (Zingales et al., 2012). To
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a certain extent, T. cruzi near-clades correspond to the ―principal zymodemes‖ early
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described by Miles et al. (1977, 1978). This illustrates their permanency in time. T.
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cruzi near-clades have been corroborated by: (i) MLEE, RAPD (Brisse, Barnabé &
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Tibayrenc, 2000); (ii) MLST (Lauthier et al., 2012; see fig. 1); (iii) fluorescent
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fragment length barcoding (Hamilton et al., 2011); (iv) microsatellites, 3 mitochondrial
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genes and the 24Sα rRNA gene (de Freitas et al., 2006); (v) the SSU rDNA,
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Cytochrome B, Histone 2B genes, ITS1rDNA genotyping, RAPD (5 primers) and
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PFGE* (Marcili et al., 2009); (vi) the sequences of 32 genes (Flores-López &
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Machado, 2011). All T. cruzi near-clades are stable in time and widespread, although
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their geographical distribution is not the same (Barnabé, Brisse & Tibayrenc, 2000;
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Zingales et al., 2012). Although the precise history of hybridization still is questioned
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(see above), it is considered that near-clades V and VI have a hybrid origin.
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- An additional near-clade, named ―TC-Bat‖ (for it has been isolated from bats only
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until now) has been recently described. It has been recorded in Brazil and Panama
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years apart, in different species of bats (Marcili et al. 2009; Pinto et al. 2012). This is
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a striking illustration of the stability in space and time of the near-clades.
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- Russian doll patterns (RDPs). It is frequently inferred that genetic exchange is
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inhibited among the genetic clusters (= near-clades) that subdivide a species, but not
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within each of them (Campbell et al., 2005). When exploring this hypothesis, it is
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important to be conscious that a lower evolutionary scale is under stidy. The
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molecular markers used at the level of the whole species may lack resolution at such
Page 11 of 52
12 lesser levels. This, together with the fact that the sample size is generally much
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lowered when considering the near-clades separately, increases by far the risk of
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statistical type II error (impossibility to reject the null hypothesis of random genetic
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exchange, not because it is verified, but because the test lacks power). This bias
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being discarded, contrary to what has been recently stated (Ramírez & Llewellyn,
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2014), RDP evidence is abundant in T. cruzi, as well as in other parasites
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(Leishmania, Giardia). The near-clade ―TC I‖ (Zingalez et al., 2012) has never been
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considered ―homogeneous‖ (Guhl & Ramírez, 2011). On the contrary, MLEE as well
266
as RAPD studies have always shown that it exhibits a high variability (Tibayrenc et
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al., 1993; Barnabé, Brisse & Tibayrenc, 2000; Brisse, Barnabé & Tibayrenc, 2000).
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However, until recently, it had been not possible to evidence LD and a clear
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structuring within it (RDP). This is now done, thanks to the availability of more
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discriminating markers and ampler samplings. Geographical distance has an obvious
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role in the distribution of TCI genetic structuring (Llewellyn et al., 2009b). However, it
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cannot account for the data cited thereafter. Within TCI selvatic strains, strong LD
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evidences ―widespread clonality, infrequent recombination‖ (Llewellyn et al., 2011).
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Contrary to what Ramírez et al. (2013b) state, within Colombian TCI strains, there is
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LD too. As a matter of fact, the p values for the LD test and the Ia index of
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association (another LD test) are 4 X 10
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2013). As noted by Tomasini et al. (2014), this is evidence for LD, and not for the
278
opposite, as wrongly concluded by Ramírez et al. (2013). Moreover, absence of LD
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would be inconsistent with the presence of 2 genetic subdivisions, clearly
280
corroborated by bootstrap analysis, within this sample. This last result is evidence for
281
RDP. As a matter of fact, not only TCI does exhibit LD, but it is also subdivided into
282
lesser near-clades that are widespread throughout different countries and exhibit
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-4
and 0.037, respectively (Ramírez et al.,
Page 12 of 52
13 host specificities (fig. 2; Guhl & Ramírez, 2011; Herrera et al., 2007). TCI lesser near-
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clades have been corroborated by various markers, including the Cytochrome b gene
285
sequence (a mitochondrial gene) and the mini-exon gene (Ramírez et al., 2011),
286
PCR-RFLP of 5 coding regions (Ramírez et al., 2012), mini-exon and MLST
287
(Tomasini et al., 2011, 2014).
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Population genetics within other T. cruzi near-clades has been less explored.
289
However, in the near-clade TCIII, a strongly significant (p<0.001) LD (measured
290
using the Index of Association (IA)) was detected in all populations except one,
291
where only marginal significance was observed (p=0.032) (Llewellyn et al., 2009a).
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This supports the hypothesis that TCIII exhibits also a RDP.
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- Indices of frequent recombination within near-clades?: A lack of congruence
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between mitochondrial and nuclear phylogenies (Ramírez et al., 2012) indicates at
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best limited introgression, as it can be observed even between different biological
296
species. The null hypothesis of the RDP model is not occasional bouts of genetic
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exchange, which is consistent with PCE. It is random or quasi-random
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recombination, as it can be observed in human populations for example. In humans,
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the only obstacles to genetic exchange are geographical or cultural ones (quasi-
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random recombination). TCI populations isolated from selvatic triatomine bugs in
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Bolivia show data that are compatible with such frequent genetic exchange.
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However, according to the authors themselves, these data are prone to type II error
303
and should be corroborated by stronger sampling (Barnabé et al., 2013). A survey in
304
Ecuador (Ocaña et al., 2010) is consistent with recombination in a small TCI
305
subpopulation. However, as we have shown (Tibayrenc & Ayala, 2013), (i) the
306
―domestic‖ subpopulation is poorly defined (33% of selvatic strains are included in it,
307
while the ―selvatic‖ clade comprises 12% of domestic strains); (ii) the risk of type II
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14 error is high, due to very limited sampling (only 12 truly ―domestic‖ strains).
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Additionally, the ―selvatic‖ subpopulation exhibits strong LD and clonality, which fits a
310
RDP. De Paula Baptista et al. (2014) have proposed that Brazilian strains of the TCII
311
near-clade undergo substantial recombination. However, this study is based also on
312
limited sampling and is subject to type II error. Moreover, there is a strong
313
contradiction between the hypothesis of substantial recombination (with lack of LD)
314
and the evidence for clear structuration of these populations, even in sympatry, as
315
shown by STRUCTURE analysis. In conclusion: it is quite possible that some
316
populations within T. cruzi near-clades undergo frequent recombination. However,
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the limited evidence presently available obviously has to be completed by far more
318
extensive studies. On the contrary, the evidence above exposed for RDPs is robust.
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From the results above exposed, it is hardly questionable that T. cruzi at the level
321
of the whole species undergoes PCE. Moreover, when enough data are available, it
322
is apparent that PCE operates also within the near-clades (RDP). Genetic
323
recombination/hybridization in the agent of Chagas disease has probably an
324
important impact at an evolutionary scale. However, it is unable to counter the effects
325
of clonal evolution in the long run (―clonality threshold‖) and to prevent the
326
evolutionary divergence of the near-clades. When looking for recombination in a
327
given species amounts to ―searching for a needle in a haystack‖ (Ramírez &
328
Llewellyn, 2014), it does show that recombination is strongly restrained, in other
329
words, that the PCE model is corroborated. When it is not, like in the highly
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recombining bacterium Helicobacter pylori, one finds easily clear indications for
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recombination without searching a haystack.
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Page 14 of 52
15 In the framework of PCE, it is quite possible that local populations of genetically
333
related strains undergo more genetic exchange than clonal propagation (Barnabé et
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al., 2013; Ocaña et al., 2010; de Paula Baptista et al., 2014). However, this has to be
335
better explored on more extensive samples. It is possible that in T. cruzi,
336
recombination is less restrained among identical genotypes (= selfing) or similar
337
genotypes (tending to selfing) than among more distantly related genotypes. This
338
happens in bacteria, where recombination is more frequent among ―sister cells‖ than
339
among cells that have radically different genotypes (Caugant & Maiden, 2009). This
340
leads to ―invisible sex‖ (Balloux, 2010) and is explained by the fact that the higher the
341
genetic divergence, the more the restriction systems are different (Caugant &
342
Maiden, 2009). Moreover, local introgressions of mitochondrial genomes are quite
343
possible in T. cruzi (Messenger et al., 2012; Ramírez et al., 2012). All this does not
344
challenge the PCE model. As exposed earlier, the alternative hypothesis to PCE is
345
that recombination is abundant enough to overcome the effects of clonal evolution in
346
the long run.
ce pt
347
ed
M
an
us
cr
ip t
332
Lastly, restrained recombination in T. cruzi cannot be attributed to a lack of
349
opportunity for mating. As a matter of fact, multiclonal infections in the same host,
350
either vectors (Tibayrenc et al., 1985) or chagasic patients (Brenière et al., 1985) are
351
very frequent.
352
Ac
348
353 354
Aneuploidy and the selfing/homogamy debate
355
Page 15 of 52
16 We recall here that selfing/homogamy is included in our PCE concept, a view
357
shared by many, if not most, specialists of pathogen population genetics (Tibayrenc
358
& Ayala, 2012) as well as by evolutionist working on metazoa undergoing uniparental
359
reproduction (Avise, 2008). However, other scientists recommend distinguishing
360
selfing/homogamy from ―strict‖ (= mitotic) clonality (Rougeron et al., 2009; Rougeron,
361
de Meeûs & Bañuls, 2015). When the macroevolution scale recommended by the
362
PCE approach is considered, the distinction in terms of population structure has
363
limited relevance, since both ―strict‖ clonality and selfing/homogamy lead to
364
restrained recombination and LD. Now selfing as a mode of restrained recombination
365
has the evolutionary advantage of permitting DNA repair, which is not the case of a
366
strictly mitotic mode of propagation. If one wants to distinguish selfing from ―strict‖
367
clonality, the problem lies on the way to do it. When bacteria are considered, since
368
they are haploid, selfing, which is certainly very frequent in these pathogens (Maiden
369
2008; Caugant & Maiden 2009), is impossible to distinguish from mitotic propagation
370
(―invisible sex‖; Balloux, 2010). When protozoa are concerned, the evidence
371
proposed for selfing relies on heterozygote deficit (Rougeron et al., 2009). However,
372
apart from selfing, many phenomena could cause heterozygote deficit, such as null
373
alleles, allelic drop-out, homoplasy (a strong concern in the case of microsatellites)
374
(Alam et al., 2009; Lehmann et al., 2004, Pearson et al., 2009), and genome-wide
375
mitotic gene conversion (Llewellyn et al., 2009a, 211). Experiments in the strictly
376
asexual species Daphnia pulex have shown that a loss of heterozygosity by mitotic
377
recombination is 1,000 times more frequent than an accumulation of divergent
378
mutations (Omilian et al. 2006). The probability of the possible causes of
379
heterozygote deficit should not be evaluated separately (Rougeron et al., 2009),
380
since they are not mutually exclusive. Moreover, heterozygote deficit inferred from
Ac
ce pt
ed
M
an
us
cr
ip t
356
Page 16 of 52
17 microsatellite analysis clashes with SNP* data for Leishmania mexicana and L.
382
braziliensis (Rogers et al., 2011) and T. cruzi (Yeo et al., 2011), where an excess of
383
heterozygotes is recorded. However, selfing should cause a heterozygote deficit for
384
all loci, including the ones involved in SNPs. The main problem with the way to
385
explore heterozygote deficit is that all tests proposed for it (de Meeûs et al., 2007)
386
are based on the hypothesis that the organism under survey is diploid*. Now strong
387
convergent results suggest that Leishmania undergoes widespread aneuploidy
388
(Downing et al. 2011; Lachaud et al. 2014; Mannaert et al. 2012; Rogers et al. 2011;
389
Sterkers et al. 2011, 2012, 2014), which renders the tests invalid, and at the same
390
time, is an explanation for apparent heterozygote deficit. As a matter of fact,
391
widespread aneuploidy should cause a rapid elimination of heterozygosity by
392
frequent passage through a haploid state (Sterkers et al. 2012). This should happen
393
even if aneuploidy is transitory in Leishmania, as proposed by Rougeron, de Meeûs
394
& Bañuls (2015). The evidence for aneuploidy in T. cruzi still is more limited than for
395
Leishmania, although progress is rapid (Minning et al. 2011; Souza et al. 2011).
396
When population genetic data are concerned, contrary to what has been claimed
397
(Ramírez et al., 2013a), in a survey dealing with more than 200 cloned stocks from
398
the same host, in mammals from Venezuela and Bolivia, ―multiple aneuploid clones
399
were isolated across those infrapopulations studied‖ (Llewellyn et al., 2011). It has
400
been proposed that microsatellite data do not suggest aneuploidy in Leishmania
401
(Rougeron, de Meeûs & Bañuls, 2015). However, Lachaud et al. (2014) disagree with
402
this statement. The conclusion of all this is that selfing in Leishmania and T. cruzi is a
403
reasonable hypothesis. However, the tests proposed to explore it may not be reliable.
404
As stated by Ramírez et al. (2012): ―In general, we urge caution in the interpretation
405
of heterozygosity statistics at STR loci in the context of parasite sexuality, especially
Ac
ce pt
ed
M
an
us
cr
ip t
381
Page 17 of 52
18 406
given that strong evidence for linkage disequilibrium often accompanies both
407
negative and positive values for FIS‖. Such difficulties in evidencing selfing in
408
parasitic protozoa gives additional ground to our proposal of including it in the PCE
409
model. It should be noted that LD tests do not suffer from this problem. As a matter of
411
fact, they can be practiced whatever be the ploidy of the organism under study, and
412
even, when this ploidy level is not known (Tibayrenc, Kjellberg & Ayala, 1990).
cr
ip t
410
414
us
413
Evolutionary features that could mimic recombination
an
415
Recombination can be confounded with several evolutionary features. They
417
include: (i) homoplasy; (ii) lack of phylogenetic signal; (iii) natural selection; (iv)
418
different molecular clocks; (v) long branch attraction; (vi) different modes of
419
inheritance.
ed
M
416
Homoplasy is a concern for many, if not most, molecular markers. Microsatellites
421
are considered as especially prone to it (Alam et al., 2009; Lehmann et al., 2004,
422
Pearson et al., 2009). This could lead to overestimate both recombination and
423
heterozygote deficit.
ce pt
420
Lack of phylogenetic signal: if the population is close to genetic monomorphism,
425
or if the molecular marker has too low a resolution power, it is impossible to evidence
426
LD and the presence of near-clades. This should not be taken as evidence for
427
recombination. A lack of phylogenetic signal could be due also to a saturation of the
428
molecular marker. As an example, microsatellites are not adapted to survey higher
429
levels of evolutionary divergence, since their molecular clock is too fast. MLSTs and
430
MLEE could reveal a strong phylogenetic signal where microsatellites cannot.
Ac
424
Page 18 of 52
19 Natural selection is difficult to distinguish from recombination (Didelot & Maiden,
432
2010). Strongly selected characters, such as drug resistance, should be especially
433
prone to this bias. Resistance to the same antibiotics can be present in different
434
bacterial lines, and even in different species. This can be the result of genetic
435
exchange, but it is also easily explained by an identical selective pressure. Such
436
convergent selected characters should be therefore considered as evidence for
437
recombination only carefully (Rougeron, de Meeûs & Bañuls, 2015).
cr
ip t
431
Differences in molecular clocks could lead to discrepancies between trees
439
designed from different genes. In Giardia duodenalis, Wielinga & Thompson (2007)
440
have attributed the discrepancies observed among trees built from different genes to
441
both homoplasy and molecular clock differences, not to recombination. Similarly, in
442
the case of the SARS coronavirus, phylogenetic incongruence among trees has been
443
attributed to different molecular clocks (Moya et al., 2004).
M
an
us
438
Long branch attraction: analyzing the phylogeny of the SARS coronavirus, Yip et
445
al. (2009) have recommended to not confound recombination with both homoplasy
446
and long branch attraction.
ce pt
ed
444
Markers that have a different mode of inheritance will exhibit different evolutionary
448
patterns. For example, in humans, the population structures inferred from autosomal
449
genes, mitochondrial genes and Y chromosome genes, respectively, are quite
450
different (Zoossmann-Diskin, 2010). In T. cruzi, experimental mating has shown that
451
nuclear genes are inherited from both parental cells, while the kinetoplast is inherited
452
from only one parental cell (Gaunt et al., 2003). In the framework of the PCE model,
453
in case of occasional bouts of genetic exchange, this phenomenon is bound to
454
maximize the discrepancies between mitochondrial and nuclear phylogenies.
Ac
447
Page 19 of 52
20 Lastly, these different phenomena are not exclusive of each other and could
456
combine their effects. In the case of T. cruzi, microsatellites are subject to
457
homoplasy. They have a fast molecular clock, they are explored at the level of the
458
nucleus, whose inheritance is biparental, and they are considered as selectively
459
neutral. Mitochondrial maxicircle genes correspond to coding sequences, which are
460
subject to natural selection. They have a slower molecular clock than microsatellites,
461
and their mode of inheritance is uniparental. So, although the discrepancies between
462
microsatellite and mitochondrial gene phylogenies are consistent with recombination
463
(Ramírez et al., 2012), alternative explanations are possible.
us
cr
ip t
455
an
464 465
A brief comparison with other micropathogens
468
We have performed a compared population genetic approach dealing with viruses
469
(22 species and categories), bacteria (43 species), parasitic protozoa (30 species)
470
and fungi (13 species) (Tibayrenc & Ayala, 2012 and in preparation). This shows that
471
T. cruzi population structure is not unique and, on the contrary, is very similar to that
472
of many major pathogen species. Each species exhibits specific traits. However, the
473
common denominator among many of them is strong, and suggests the existence of
474
common evolutionary strategies, which could be either ancestral or the result of
475
convergence.
Ac
ce pt
ed
467
M
466
476
Well-established T. cruzi ―siblings‖ in terms of population structure are the species
477
for which the PCE features (LD, widespread MLGs, near-clades and RDPs) have
478
been recorded. It is assumed that these species have crossed the ―clonality
479
threshold‖ beyond which the impact of clonal evolution will overcome the effects of
Page 20 of 52
21 recombination. Such species include: (i) parasitic protozoa: the Leishmania infantum
481
complex (Tibayrenc & Ayala, 2013), Giardia duodenalis, (Tibayrenc & Ayala, 2014b),
482
Toxoplasma gondii (Tibayrenc & Ayala, 2014a); (ii) fungi: Candida albicans, the
483
Cryptococcus neoformans complex; (iii) bacteria: Escherichia coli, Mycobacterium
484
tuberculosis, Neisseria meningitidis, Staphylococcus aureus. Many other species
485
exhibit clonality traits, but the PCE pattern is not complete, either because these
486
species undergo more recombination, or because the studies are less advanced than
487
for the species above listed, or both. When parasitic protozoa are concerned, such
488
examples include: Leishmania braziliensis/peruviana, the Trypanosoma brucei
489
complex, T. congolense, Cryptosporidium spp.
an
us
cr
ip t
480
Plasmodium falciparum and P. vivax constitute distinctive cases. Since P.
491
falciparum undergoes meiosis in the vector, it has been long assumed that the agent
492
of the most malignant form of malaria was a panmictic* organism. Our cautious
493
proposal that ―uniparental and biparental lineages could coexist within this species‖,
494
based on the observation of departures from panmixia in some populations
495
(Tibayrenc, Kjellberg & Ayala, 1990), had been rejected but it was subsequently
496
confirmed (Anderson et al., 2000). Many P. falciparum and P. vivax populations
497
exhibit clonality traits. Selfing is probably at the origin of them. However, the
498
hypothesis that selfing is strictly correlated to the level of transmission is at odds with
499
many data (Tibayrenc & Ayala, 2014a). This is epidemiologically relevant, since LD
500
and the degree of clonality in P. falciparum cannot be taken as an indication for the
501
transmission rate (Tibayrenc & Ayala, 2014a), as it had been proposed (Volkman et
502
al., 2012). Now P. falciparum and P. vivax definitely do not fit the PCE model.
503
Clonality traits (LD, widespread MLGs and near-clades) that are observed in these
504
species are labile and change over time, due to frequent genetic recombination. Still
Ac
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ed
M
490
Page 21 of 52
22 505
the fact remains that clonal propagation introduces a major stratification effect in P.
506
falciparum and P. vivax natural populations, that should be taken into account in
507
studies dealing with malaria epidemiology, drug resistance and the analysis of genes
508
of interest. When considering the various species that fit the PCE model, two facts should be
510
underlined: (i) for several of them, PCE was not expected. Neisseria meningitidis was
511
considered a ―highly recombining‖ bacterium (Maiden, 2008). However, widespread
512
MLGs and near-clades in this species are extremely stable, as verified by
513
retrospective studies. Moreover, whole genome sequencing has revealed the
514
existence of deep phylogenies (Budroni et al., 2011), which is incompatible with the
515
―epidemic clonality model‖ (occasional bouts of clonal propagation in an otherwise
516
recombining species; Maynard Smith et al., 1993). Toxoplasma gondii constitutes
517
another unexpected case of PCE, since this parasite undergoes meiosis in the
518
mammalian host. We have proposed that T. gondii undergoes frequent, and possibly
519
predominant clonal evolution (Tibayrenc et al., 1991). This has been fully confirmed
520
by further studies (Sibley & Boothroyd, 1992). High resolution typing with many
521
different markers (Su et al., 2012) has shown the existence of deep phylogenies, with
522
6 major ―clades‖ (= near-clades), which definitely includes T. gondii in the PCE model
523
(Tibayrenc & Ayala, 2014a). (ii) the species listed pertain to phylogenetically highly
524
diverse categories (bacteria, fungi, and parasitic protozoa), and exhibit various host
525
ranges, modes of transmission (air-, vector-, food- and water-borne) and tissue
526
tropisms. This strongly suggests that PCE represents a common evolutionary
527
strategy of many pathogens, probably linked to parasitism.
Ac
ce pt
ed
M
an
us
cr
ip t
509
528 529
Experimental mating, meiosis genes and PCE
Page 22 of 52
23 530 The seminal work by Gaunt et al. (2003) has demonstrated that the potential for
532
recombination is present in T. cruzi. However, these experiments dealt only with
533
related strains pertaining to the near-clade TCI. It remains to be established whether
534
mating can also be obtained between strains pertaining to different near-clades.
535
More important: these experiments provide no indication about the frequency of
536
genetic exchange in natural populations, which matters when population structure is
537
concerned. Successful mating experiments cannot therefore be used to challenge
538
the PCE model.
us
cr
ip t
531
Genes homologous to meiosis genes are observed in many micropathogen
540
species, including T. cruzi (El-Sayed et al., 2005). However, this does not imply that
541
T. cruzi undergoes meiosis (Ramírez & Llewellyn, 2014), and if it did, the presence of
542
these genes does not give indications about the frequency of it. Moreover, genes
543
analogous to meiosis genes may have acquired totally different functions through
544
evolution. ―Evolution is constantly re-using old genes for new purposes‖ (Birky 2009).
545
We have suggested that, in micropathogens undergoing PCE, the meiosis kit could
546
actually
547
micropathogens to use either one or the other mode of propagation when facing
548
different evolutionary challenges (Tibayrenc & Ayala, 2012). In conclusion: like for
549
mating experiments, the presence of genes homologous to meiosis genes is
550
therefore not evidence against the PE model.
ce pt
ed
M
an
539
to
a
―sexuality/clonality
machinery
that
could
allow
Ac
correspond
551 552
The PCE model and the problem of pathogen species definition
553
Page 23 of 52
24 Defining species in micropathogens has been always a challenge, since the
555
mixiological species concept (Dobzhansky, 1937), which addresses only organisms
556
with obligatory mating, cannot be applied to them. Moreover, the phylogenetic
557
species concept (Cracraft, 1983) is difficult to apply, since it is most probable that
558
some genetic exchange always occurs in micropathogens natural populations, even
559
between different species. The near-clade concept, which relaxes the cladistic
560
demands, makes it possible to revisit the phylogenetic species concept for
561
micropathogens (Tibayrenc and Ayala, 2014b). Two situations occur. The first case is
562
when presently described species can be equated to near-clades in an evolutionary
563
point of view. Many, if not most, Leishmania species are in this case. As a matter of
564
fact, in general, they are clearly discriminated by molecular markers. However,
565
hybridizations are frequent among them. Actually, many Leishmania species are
566
perfectly equivalent to T. cruzi near-clades from an evolutionary point of view. The
567
second case is when presently observed near-clades have not been described as
568
Linnean species yet. This is the case for the Giardia duodenalis ―assemblages‖ (=
569
near-clades) (Tibayrenc & Ayala, 2014b). T. cruzi near-clades could constitute the
570
basis for splitting the agent of Chagas disease into several species. We do not
571
recommend it for several reasons. First, the inventory of T. cruzi genetic diversity
572
could still evidence new features and unknown near-clades. Second, the advent of
573
whole genome sequencing could lead to reconsider the presently accepted near-
574
clade classification (Zingales et al., 2012). Third, the epidemiological and
575
pathological specificity of T. cruzi near-clades is not clear-cut. The Chagas disease
576
scientific community will decide whether the description of these new species is
577
desirable. The near-clade concept may constitute a necessary, but not sufficient,
578
criterion for it. Since near-clading is widespread among micropathogens, it would be
Ac
ce pt
ed
M
an
us
cr
ip t
554
Page 24 of 52
25 579
misleading to equate all of them to Linnean species. This should be done only when
580
a given near-clade exhibits strong phenotypic specificities dealing with relevant
581
characters (host range, drug resistance, pathogenicity for example).
582
584
ip t
583 The PCE model and applied research.
cr
585
The PCE model provides applied research with relevant units of analysis that are
587
based on simple concepts, and are sharply defined. Their stability in space and time
588
has been fully ascertained by retrospective studies, as well as by population genetics
589
and phylogenetic analysis. Clonal MLGs (clonets) can be used for epidemiological
590
tracking. To characterize them, markers with different mutation rates and level of
591
resolution can be selected according to the study concerned: microsatellites for
592
microepidemiology, MLST and whole genome sequencing plus SNPs (when these
593
two last tools become more accessible) for epidemiological surveys at broader
594
scales. Near-clades and their subdivisions (RDPs) can, and should be used as units
595
of analysis for epidemiological tracking, drug design (Zingales et al., 2014), clinical
596
studies and experimental evolution. Taking the near-clades as units of analysis, we
597
have conducted long-term experimental evolution studies, which have shown that
598
many phenotypic characters (including drug sensitivity, virulence in mice and cell
599
infectivity), as well as gene expression patterns, were linked to the near-clades
600
(Revollo et al., 1998; Telleria et al., 2004, 2010, 2013).
Ac
ce pt
ed
M
an
us
586
601
A major application of the PCE model for strain typing/molecular epidemiology is
602
indirect typing, which is a remarkable property of LD. Since all genes are linked
603
together (―block of genes‖), the near-clades can be identified by only one or two
Page 25 of 52
26 markers (―tags‖; Tibayrenc, 1998). Several RAPD and isoenzyme tags have been
605
identified for the 6 near-clades, including near-clade TCI, (Brisse et al., 2000). By the
606
amplification of a unique gene (TcSC5D), Cosentino & Agüero (2012) were able to
607
identify the T. cruzi near-clades I, II, II, IV, V+VI, TC-Bat, and the subspecies T. cruzi
608
marinkellei. We recommend this ―phylogenetic character mapping‖ (PCM) approach
609
(Avise, 2004). A convenient population genetics framework is first designed through
610
the PCE approach. Once the relevant units of analysis (near-clades) are clearly
611
identified, phenotypes of interest (drug resistance, virulence) are mapped onto this
612
framework. This makes it possible to see the links between the overall evolution of
613
the organism under study and its phenotypes, and at the same time, to show the
614
specific phenotypic properties of the near-clades.
an
us
cr
ip t
604
616
M
615 Conclusion
ed
617
Due to the dynamism of many research groups throughout the world, with a major
619
contribution from Latin American teams, the intraspecific genetic variability of T. cruzi
620
is probably one of the best known among micropathogens. The agent of Chagas
621
disease could be considered a star model for evolution, like Drosophila
622
melanogaster, Mus musculus, Caenorabditis elegans and Escherichia coli.
Ac
623
ce pt
618
The PCE model, which has been
elucidated and enriched by wide surveys
624
dealing with many pathogens (Tibayrenc & Ayala, 2012), sets in perspective the
625
particular case of T. cruzi population genetics. It shows that T. cruzi shares many
626
features with other major pathogens, including parasitic protozoa, fungi and bacteria.
627
Promising avenues of evolutionary research are represented by experimental
628
mating (less advanced for T. cruzi than for T. brucei), within near-clade population
Page 26 of 52
27 genetics, experimental evolution, and the development of more powerful tools of
630
analysis (whole genome sequencing, SNP analysis). These tools constitute a
631
challenge for analysis, since the computing power required for treating these data is
632
huge. However, the use of these tools becomes routine for viruses and bacteria, and
633
should be more and more accessible when parasitic protozoa are concerned, making
634
it possible to develop a population genomics of these pathogens.
Ac
ce pt
ed
M
an
us
cr
635
ip t
629
Page 27 of 52
28 636 637
Legends of the figures
638 Figure 1: The six Trypanosoma cruzi near-clades evidenced by MLST analysis (after
640
Lauthier et al., 2012). Left: neighbor-joining tree; right: split decomposition analysis.
641
The two modes of analysis, which rely on different computing methods and different
642
working hypotheses, show the same near-clading pattern. This is an indication that
643
the clustering of the near-clades is robust, according to the congruence principle
644
(Avise, 2004).
us
cr
ip t
639
an
645
Figure 2: Geographic distribution of the five lesser near-clades evidenced within the
647
Trypanosoma cruzi near-clade TCI (see fig. 1) (after Gull & Ramírez, 2011). The
648
lesser near-clades are widespread and occur sympatrically, which is an evidence of a
649
―Russian doll Pattern‖ (Tibayrenc & Ayala, 2013).
ed
M
646
Ac
ce pt
650
Page 28 of 52
29 651 652
Glossary of specialized terms:
653 Allopatry: a situation where different populations occur at different locations; cf
655
sympatry*
ip t
654
656
Aneuploidy: a situation in which the chromosome number of an individual is not an
658
exact multiple of the haploid set of the species. Copy numbers may vary among
659
chromosomes.
us
cr
657
an
660
Clade: evolutionary line defined by cladistic analysis. A clade is monophyletic* and is
662
genetically differentiated (i.e., evolves independently) from other clades.
M
661
663
Cladistic analysis: phylogenetic analysis based on the distribution of characters that
665
are
666
characteristics. Only apomorphic characters shared by all members of a given clade*
667
(synapomorphic character) convey relevant phylogenetic information.
670
ancestral
(plesiomorphic)
and
derived
(apomorphic)
ce pt
669
between
Diploid: possessing 2 copies of each chromosome.
Ac
668
shared
ed
664
671
DTU = Discrete Typing Unit: sets of strains that are genetically closer to each other
672
than to any other stock and are identifiable by common genetic, molecular or
673
immunological markers called tags (Tibayrenc, 1998).
674 675
Haploid: possessing only one copy of each chromosome.
Page 29 of 52
30 676 677
Homogamy: preferential mating between individuals that are genetically identical or
678
extremely similar to each other.
679 Homoplasy: distinct evolutionary units possess similar characters that do not
681
originate from a common ancestor.
ip t
680
cr
682
MLEE: = Multilocus Enzyme Electrophoresis. Analysis by electrophoresis of protein
684
extracts from biological samples. MLEE banding reflects the aminoacid sequence of
685
the proteins, coded by the DNA sequences. It is an efficient genetic typing method.
686
However, identical MLEE bands may correspond to different DNA sequences
687
(homoplasy*).
M
an
us
683
688
MLST = Multilocus sequence typing : a highly standardized typing technique
690
based on the sequencing of 450-bp segments of a set of housekeeping genes
691
(usually seven). It has been widely used for a high number of bacterial species and
692
some eukaryotic pathogens as well.
693
Molecular clock : in its strict, original sense (more correctly named: ―DNA clock
694
hypothesis‖), the concept that the rate of nucleotide substitutions in DNA remains
695
constant. In a broader sense, simply the rate of evolution of the genomic part that
696
codes for the variability of a given marker. This fastness is driven by the rate of
697
substitution/mutation. It may be regular or irregular.
Ac
ce pt
ed
689
698 699
Monophyletic: the case where a given phylogenetic lineage has only one ancestor
700
(see clade*).
Page 30 of 52
31 701 702
Panmixia, panmictic: a situation where genetic exchange occurs at random.
703 Parthenogenesis: Reproduction by the development of a single gamete without
705
fertilization by a gamete of the opposite sex.
ip t
704
706
PFGE = Pulse Field Gel Electrophoresis: separation of large DNA fragments by a
708
particular electrophoresis technique using alternately pulsed, perpendicularly
709
oriented electrical fields. Strains that share the same PFGE are referred to as pulse
710
types. In the case of bacteria, the large DNA fragments result from the action of a
711
―low-frequency cutter‖ (a bacterial endonuclease which restriction action has a low
712
frequency) on the bacterial chromosome. . In the case of parasitic protozoa
713
(Trypanosoma and Leishmania) and of yeasts, the large DNA fragments correspond
714
to entire chromosomes (―molecular karyotype‖).
ed
M
an
us
cr
707
715
RAPD = Random Amplified Polymorphic DNA: In the classical PCR method, the
717
primers used are known DNA sequences, whereas the RAPD technique relies on
718
primers whose sequence is arbitrarily determined.
719
Ac
ce pt
716
720
Recombination, recombining : Reassortment of genotypes occurring at different
721
loci.
722 723
Selfing : self-fertilization of an organism. If the organism is haploid (bacteria), or if all
724
loci are homozygous, selfing is undistiguishable from mitotic clonality. In diploid
Page 31 of 52
32 725
organisms, selfing leads to a deficit of heterozygotes, and to a lack of genetic
726
recombination
727 SNP = Single Nucleotide Polymorphism : a DNA sequence variation occurring
729
commonly within a population, in which a single nucleotide — A, T, C or G differs
730
between individuals.
ip t
728
cr
731
Sympatry: a situation where different populations are present in the same place; cf
733
allopatry*.
us
732
an
734 735
Wahlund
736
subpopulations (isolated in time or space or both) that have different genotype
737
compositions, population genetic tests performed at the level of the whole population
738
show departures from panmictic expectations, even if each subpopulation is
739
panmictic*.
742
sampled
population is composed
of
several
ed
M
the
ce pt
741
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Ac
740
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Highlight
We expose our view on Trypanosoma cruzi population genetics
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We show it is enlightened by our model of pathogen predominant clonal evolution. We draw comparisons with other pathogens, in particular Leishmania. We underline the implications of the model for applied research.
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S0001-706X(15)00140-0 http://dx.doi.org/doi:10.1016/j.actatropica.2015.05.006 ACTROP 3618
To appear in:
Acta Tropica
Received date: Revised date: Accepted date:
9-3-2015 2-5-2015 6-5-2015
Please cite this article as: Tibayrenc, M., Ayala, F.J.,The population genetics of Trypanosoma cruzi revisited in the light of the predominant clonal evolution model, Acta Tropica (2015), http://dx.doi.org/10.1016/j.actatropica.2015.05.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1 1
The population genetics of Trypanosoma cruzi revisited in the light of the
2
predominant clonal evolution model.
3 4
Michel Tibayrenc1 and Francisco J. Ayala2
6
1
7
Montpellier Cedex 5, France
8
2
9
92697, USA
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5
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MIVEGEC (IRD 224-CNRS 5290-UM1-UM2), IRD Center, BP 64501, 34394
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Department Ecology and Evolutionary Biology, University of California, Irvine, CA
11
an
10
Corresponding author: Tibayrenc, M. ([email protected]).
M
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Comparing the population structure of Trypanosoma cruzi with that of other
14
pathogens, including parasitic protozoa, fungi, bacteria and viruses, shows that the
15
agent of Chagas disease shares typical traits with many other species, related to a
16
predominant
17
disequilibrium,
18
subdivisions somewhat blurred by occasional genetic exchange/hybridization) and
19
―Russian doll‖ patterns (PCE is observed, not only at the level of the whole species,
20
but also, within the near-clades). Moreover, T. cruzi population structure exhibits
21
linkage with the diversity of several strongly selected genes, with gene expression
22
profiles, and with some major phenotypic traits. We discuss the evolutionary
23
significance of these results, and their implications in terms of applied research
24
(molecular epidemiology/strain typing, analysis of genes of interest, vaccine and drug
evolution
(PCE)
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clonal
ed
13
multilocus
statistically significant
genotypes,
near-clades
linkage (genetic
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overrepresented
pattern:
Page 1 of 52
2 25
design, immunological diagnosis) and of experimental evolution. Lastly, we revisit the
26
long-term debate of describing new species within the T. cruzi taxon.
27 Key-words: predominant clonal evolution, near-clade, discrete typing unit,
29
recombinational load, drug resistance
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30 * see glossary of specialized terms.
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Page 2 of 52
3 33 34
Although much progress has been made in vector control, Chagas disease
35
remains a major health problem in Latin America, and is now threatening industrial
36
countries, in particular Spain and the USA. With the hope of developing radical control means for Chagas disease, extensive
38
studies have been conducted on Trypanosoma cruzi population genetics, and have
39
made this parasite probably one of the micropathogens whose intraspecific diversity
40
is best known.
us
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A comparative population genetic analysis of T. cruzi and of many other
42
pathogens, including parasitic protozoa, fungi, bacteria and viruses, has broaden our
43
view of the agent of Chagas disease’ population structure and has made it possible
44
to reveal the evolutionary strategies used by this pathogen. Moreover, such a
45
comparative population genetics has brought relevant considerations about the
46
possibility to describe new species within the T. cruzi taxon.
ed
M
an
41
47
50 51
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49
The predominant clonal evolution (PCE) model of pathogens
The model will be recalled only briefly here, since it has been exposed at length in several recents articles (Tibayrenc & Ayala, 2012, 2013, 2014 a and b).
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48
52
PCE is not defined by any precise mating system or cytological mechanism, but
53
only by strongly restrained genetic recombination* on an evolutionary scale. It
54
therefore deals with population structure of the whole species in the long run only.
55
This definition is accepted by most authors working on pathogen population genetics,
56
including parasitic protozoa, fungi and bacteria (Tibayrenc & Ayala, 2012) and is well
57
accepted in the T. cruzi literature (Barnabé et al., 2013; Flores-López & Machado,
Page 3 of 52
4 2011; Minning et al., 2011). According to our definition, PCE includes, not only mitotic
59
reproduction, but also selfing*/strong homogamy*, several cases of parthenogenesis*
60
(Tibayrenc & Ayala, 1991, 2002), and all cases of ―unisex‖ (Feretzaki & Heitman,
61
2013). PCE would group all means used by micropathogens to escape the
62
―recombinational load‖ (disrupting favorable multilocus genotypes by genetic
63
recombination) (Agrawal, 2006).
ip t
58
Equating selfing/strong homogamy and many cases of parthenogenesis to
65
clonality is not confined to microbiologists and extends to evolutionists working on
66
higher organisms too (Avise, 2008). Some authors, working on Leishmania
67
(Rougeron et al., 2009) or on T. cruzi (Llewellyn et al., 2009a) recommend to
68
distinguish selfing/homogamy from ―strict‖ clonality (= mitotic propagation). This is a
69
matter of definition. The problem deals with the means used for distinguishing
70
selfing/homogamy from ―strict‖ clonality.
M
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cr
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The main consequences of PCE are: (i) statistically significant linkage
72
disequilibrium, or nonrandom association of genotypes occuring at diffferent loci
73
(LD); (ii) overrepresented multilocus genotypes (MLGs), which can be lacking if the
74
mutation rate of the marker is high (all strains have different genotypes); (iii) the
75
presence of ―near-clades‖ (genetic subdivisions somewhat blurred by occasional
76
genetic exchange/hybridization); ―Russian doll‖ patterns (RDPs- PCE is observed,
77
not only at the level of the whole species, but also, within the near-clades) (Tibayrenc
78
& Ayala, 2013).
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71
79
We have coined the term ―near-clade‖ (Tibayrenc & Ayala, 2012) because it can
80
be suspected that virtually all micropathogen species undergo some bouts of
81
recombination, including among different species. This makes the demands of a strict
82
cladistic* analysis impossible. We have recommended to evidence the presence of
Page 4 of 52
5 near-clades with the tools of population genetics analysis (LD), then by a flexible
84
phylogenetic approach relying on the ―congruence principle‖ (Avise, 2004). The use
85
of more reliable information supports more and more the working hypothesis (here:
86
the presence of near-clades). For example, in the case of MLST*, the phylogenies of
87
individual genes may exhibit some discrepancies. However, the use of the whole set
88
of genes evidences a strong phylogenetic signal (Lauther et al., 2012). Or different
89
clustering/phylogenetic approaches, relying on different working hypotheses, give
90
convergent results, which confirm the robustness of the branchings (fig.1). Or the
91
phylogenies designed from different molecular markers corroborate each other
92
(Brisse, Barnabé & Tibayrenc, 2000; Tibayrenc et al., 1993). Identification of PCE
93
relies on the concept of a ―clonality threshold‖, beyond which the impact of clonal
94
evolution overcomes that of recombination and leads to the inescapable evolutionary
95
divergence of the near-clades. This concept of a ―PCE border‖ makes it possible to
96
replace vague and subjectives assertions (―substantial‖ recombination; de Paula
97
Baptista et al., 2014; "Gross incongruences", "multiple introgressions‖: Messenger et
98
al., 2012) with a clear-cut criterion. The alternative hypothesis to PCE is that
99
recombination is abundant enough to erase the effects of clonal evolution in the long
100
run. This corresponds to the ―epidemic clonality‖ model (Maynard Smith et al., 1993)
101
or ―semiclonal model‖ (Maiden, 2006)).
103 104
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Some important points of the model
105 106
- As recalled many times, the PCE model does not state that recombination is
107
absent, but rather, that it is not frequent enough to break the prevalent PCE pattern.
Page 5 of 52
6 108
The model considers that recombination/hybridization could play a major role in
109
pathogens’ evolution, but only on an evolutionary scale. It does not state by any
110
means that recombination is ―inconsequential‖ (Ramírez & Llewellyn, 2014) or ―of
111
little consequence‖ (Miles et al., 2009). - In a first step, PCE should be explored at the level of the entire species. The
113
species concept, as we will see further, is a matter of debate in the case of
114
micropathogens. However, the unit of analysis when PCE is explored should be the
115
presently described species. It has been argued that ―it makes little sense to address
116
each parasite species as a whole‖, because these ―genetic subdivisions (i.e.: the
117
near-clades) act as reproductive barriers‖ (Ramírez & Llewellyn, 2014). However,
118
only the PCE approach is able to evidence the existence of reproductive barriers
119
among the near-clades. Claiming that such reproductive barriers are self-evident
120
(Ramírez & Llewellyn, 2014) is quite untrue. Evidencing them is the very goal of the
121
PCE approach.
122
- To explore PCE, sampling should be twofold: (i) in a classical population genetics
123
strategy, it should be conducted in close sympatry* on very limited spans of time, in
124
order to ascertain that the organism under study has actual opportunity for mating
125
(de Meeûs et al., 2007). However, it is extremely difficult to ascertain a strict
126
sympatry when micropathogens are concerned. Even sampling at the level of
127
individual hosts is not a guarantee for it. If different genotypes of the species
128
concerned do not have the same tropism for different tissues of the host, they could
129
have little opportunity for mating. (ii) Since the PCE pattern is definitely a ―bird’s eye
130
view‖ of the overall genetic variability of the whole species in the long term, we
131
recommend a global sampling of the entire species on its complete ecogeographical
132
range, including all known hosts, and relying on retrospective studies to evaluate the
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Page 6 of 52
7 stability of the PCE pattern. Retrospective studies can concern the analysis of
134
ancient culture collections, or of ancient publications, or both. Such a sampling
135
strategy makes it possible to ascertain that ubiquitous MLGs, near-clades and RDPs
136
are stable in space and time, are specific properties of the species under study, and
137
cannot be explained by a Wahlund effect* (Rougeron et al., 2014). If inhibition of
138
recombination were due to trivial physical obstacles (Wahlund), overrepresented
139
MLGs, near-clades and RDPs would be strongly linked to space and/or time.
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- The PCE model predicts that different clonal genotypes, due to inhibition of
141
recombination among them, will tend to accumulate divergent mutations, which could
142
concern genes governing relevant phenotypes (virulence, resistance to drugs). This
143
should be especially true when the evolutionary divergence among clones is high.
144
However, the model does not predict that all phenotypes should be strictly linked to
145
the clonal population structure. This should be only a general tendency, and could be
146
not verified at microevolutionary levels, and when strongly selected phenotypes (drug
147
resistance) are concerned. Such selected phenotypes are bound to emerge
148
independently in distinct lineages. This is not an evidence for recombination in itself
149
(Rougeron, de Meeûs & Bañuls, 2015), although it can be consistent with
150
recombination events. Chloroquine resistance is observed in different Plasmodium
151
species. This is not the result of genetic exchange between these different species,
152
but only of a common selective pressure.
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153
- The clonet concept: Identical MLG is a relative notion, and is highly dependent
154
on the resolution power/mutation rate/molecular clock* of the marker employed. A set
155
of strains that appears as identical for a given marker could reveal genetic
156
heterogeneity when a more discriminative marker is used. If PCE obtains, this set
157
should be considered as a family of closely related clones, rather than a true clone.
Page 7 of 52
8 The term ―clonet‖ has been coined by us to refer to those sets of strains that appear
159
to be identical with a given set of genetic markers in a species that undergoes PCE
160
(Tibayrenc, Kjellberg & Ayala, 1991). A typical example of clonet is the zymodeme*
161
MON1 of Leishmania infantum. This MLEE* MLG is widespread in the ancient and
162
new world. The use of a more discriminating marker (microsatellites) shows that
163
MON1 is genetically heterogeneous (Amro et al., 2009; Chargui et al., 2009). One
164
important point with the clonet concept is that the population genetic tests and
165
phylogenetic analyses made on the basis of a given marker remain entirely valid
166
when more discriminating markers evidence additional genetic variability by
167
comparison with this marker. A given marker addresses a given level of resolution
168
and evolutionary divergence and is better adapted for given studies.
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170
PCE and Trypanosoma cruzi:
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169
T. cruzi is a paradigmatic case of PCE. It is actually the parasitic model used by
173
us for long to design the PCE approach (Tibayrenc et al., 1981, 1986), and to extend
174
it later to various other pathogens (Tibayrenc, Kjellberg & Ayala, 1990; Tibayrenc et
175
al., 1991, 2012).
ce pt
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Apart from our own studies, many data are available in the literature, back to the
177
70s (the pioneering studies by Miles et al., 1977, 1978, 1981), relying on various
178
markers, which makes comparative and retrospective studies easy (Zingales et al.,
179
2012). Retrospective studies (ancient strain collections and publications) are highly
180
relevant to evaluate the persistence of T. cruzi PCE features (widespread MLGs,
181
near-clades, RDPs). Many of the ancient studies dealing with the population genetics
182
and strain typing of T. cruzi and various other micropathogens rely on MLEE. It is
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Page 8 of 52
9 remarkable that MLEE results as a rule have been fully confirmed by modern DNA
184
markers. So ancient MLEE data, when properly interpreted, give a reliable basis for
185
retrospective studies. Lastly, the analysis of paleo DNA (Araujo, Reinhard & Ferreira,
186
2008) constitutes a promising approach to evaluate the antiquity of T. cruzi
187
population structure.
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188
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190
The PCE indices rely on many raw data sets that can be easily verified by anybody. They are as follows:
us
189
191
- LD: We do not consider LD and PCE as ―synonymous‖ (Rougeron, de Meeûs &
193
Bañuls, 2015). However, we and many authors working on micropathogen population
194
genetics (Tibayrenc & Ayala, 2012) consider LD as a valid circumstantial evidence
195
for clonal evolution, as long as the bias due to Wahlund effect is excluded. LD in T.
196
cruzi is observed: (i) among MLEE loci (Tibayrenc et al., 1981, 1986); (ii) among
197
different markers (―test g‖ of LD; Tibayrenc, Kjellberg & Ayala, 1990): between MLEE
198
and RAPD* (Tibayrenc et al., 1993; Brisse, Barnabé & Tibayrenc, 2000); between
199
microsatellites and ribosomal DNA restriction fragment length polymorphism (Oliveira
200
et al., 1998); between MLST*, MLEE and RAPD (Lauthier et al., 2012); between
201
microsatellites and the sequence of the Gpi gene (Lewis et al., 2011). LD in T. cruzi
202
is not limited to classical markers, and is observed between genetic markers and
203
copy number variants (Minning et al., 2011); microsatellites and DNA content (Lewis
204
et al., 2009); between MLEE and RAPD on one hand, 12 antigen genes (which are
205
under strong selection) on the other hand (Rozas et al., 2007); between classical
206
markers ad the cruzipain antigen gene (which is under strong selection too) (Lima et
207
al., 2012). Lastly, a statistical association is observed between the variability
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Page 9 of 52
10 208
revealed by molecular markers and the polymorphism of expressed genes (Telleria
209
et al., 2004, 2010, 2013).
210 - Overrepresented, widespread genotypes: we have called them: ―major clones‖
212
(Tibayrenc & Brenière, 1988). This is the case for example of the MLEE MLGs n° 19,
213
20, 32 and 39 in Tibayrenc et al. (1986), which have been observed in diverse
214
countries, years apart. For example; MLG 19 has been sampled in Brazil, Venezuela,
215
Colombia, Bolivia, in Chagasic patients, as well as in various mammalian hosts and
216
triatomine bug species, from 1977 to 1983. With more discriminating MLEE typing,
217
ubiquitous MLGs still are observed. One MLG repeated 31 times has been observed
218
in four different countries (Barnabé, Brisse & Tibayrenc, 2000). The remarks made
219
about the clonet concept (see above) should be recalled here. MLEE reveals
220
ubiquitous MLGs. However, when more discriminating markers (microsatellites) are
221
used, each strain may have a different genotype, due to the fast mutation rate of this
222
marker (Oliveira et al., 1998).
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223
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- Near-clades. The near-clade concept (Tibayrenc & Ayala, 2012) gives a clear
225
evolutionary definition to the descriptive term DTU* (discrete typing unit) coined by us
226
(Tibayrenc, 1998). On the basis of both MLEE and RAPD typing, it has been
227
proposed that T. cruzi is subdivided into 6 discrete genetic lineages, or DTUs
228
(Barnabé, Brisse and Tibayrenc, 2000; Brisse, Barnabé & Tibayrenc, 2000). T. cruzi
229
DTUs perfectly fit the definition of near-clades: well-individualized genetic lineages
230
that are somewhat blurred up by occasional genetic exchange and/or hybridization.
231
Hybridization events have been described since long in T. cruzi (Machado & Ayala,
232
2001; Brisse et al., 2003), although their precise evolutionary history still is a matter
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Page 10 of 52
11 of debate (de Freitas et al., 2006; Ferreira & Briones, 2012; Subileau et al., 2009;
234
Westenberger et al., 2005, 2006). The classification into 6 DTU/near-clades has
235
been recently validated by a panel of international experts (Zingales et al., 2012). To
236
a certain extent, T. cruzi near-clades correspond to the ―principal zymodemes‖ early
237
described by Miles et al. (1977, 1978). This illustrates their permanency in time. T.
238
cruzi near-clades have been corroborated by: (i) MLEE, RAPD (Brisse, Barnabé &
239
Tibayrenc, 2000); (ii) MLST (Lauthier et al., 2012; see fig. 1); (iii) fluorescent
240
fragment length barcoding (Hamilton et al., 2011); (iv) microsatellites, 3 mitochondrial
241
genes and the 24Sα rRNA gene (de Freitas et al., 2006); (v) the SSU rDNA,
242
Cytochrome B, Histone 2B genes, ITS1rDNA genotyping, RAPD (5 primers) and
243
PFGE* (Marcili et al., 2009); (vi) the sequences of 32 genes (Flores-López &
244
Machado, 2011). All T. cruzi near-clades are stable in time and widespread, although
245
their geographical distribution is not the same (Barnabé, Brisse & Tibayrenc, 2000;
246
Zingales et al., 2012). Although the precise history of hybridization still is questioned
247
(see above), it is considered that near-clades V and VI have a hybrid origin.
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- An additional near-clade, named ―TC-Bat‖ (for it has been isolated from bats only
249
until now) has been recently described. It has been recorded in Brazil and Panama
250
years apart, in different species of bats (Marcili et al. 2009; Pinto et al. 2012). This is
251
a striking illustration of the stability in space and time of the near-clades.
Ac
252
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248
253
- Russian doll patterns (RDPs). It is frequently inferred that genetic exchange is
254
inhibited among the genetic clusters (= near-clades) that subdivide a species, but not
255
within each of them (Campbell et al., 2005). When exploring this hypothesis, it is
256
important to be conscious that a lower evolutionary scale is under stidy. The
257
molecular markers used at the level of the whole species may lack resolution at such
Page 11 of 52
12 lesser levels. This, together with the fact that the sample size is generally much
259
lowered when considering the near-clades separately, increases by far the risk of
260
statistical type II error (impossibility to reject the null hypothesis of random genetic
261
exchange, not because it is verified, but because the test lacks power). This bias
262
being discarded, contrary to what has been recently stated (Ramírez & Llewellyn,
263
2014), RDP evidence is abundant in T. cruzi, as well as in other parasites
264
(Leishmania, Giardia). The near-clade ―TC I‖ (Zingalez et al., 2012) has never been
265
considered ―homogeneous‖ (Guhl & Ramírez, 2011). On the contrary, MLEE as well
266
as RAPD studies have always shown that it exhibits a high variability (Tibayrenc et
267
al., 1993; Barnabé, Brisse & Tibayrenc, 2000; Brisse, Barnabé & Tibayrenc, 2000).
268
However, until recently, it had been not possible to evidence LD and a clear
269
structuring within it (RDP). This is now done, thanks to the availability of more
270
discriminating markers and ampler samplings. Geographical distance has an obvious
271
role in the distribution of TCI genetic structuring (Llewellyn et al., 2009b). However, it
272
cannot account for the data cited thereafter. Within TCI selvatic strains, strong LD
273
evidences ―widespread clonality, infrequent recombination‖ (Llewellyn et al., 2011).
274
Contrary to what Ramírez et al. (2013b) state, within Colombian TCI strains, there is
275
LD too. As a matter of fact, the p values for the LD test and the Ia index of
276
association (another LD test) are 4 X 10
277
2013). As noted by Tomasini et al. (2014), this is evidence for LD, and not for the
278
opposite, as wrongly concluded by Ramírez et al. (2013). Moreover, absence of LD
279
would be inconsistent with the presence of 2 genetic subdivisions, clearly
280
corroborated by bootstrap analysis, within this sample. This last result is evidence for
281
RDP. As a matter of fact, not only TCI does exhibit LD, but it is also subdivided into
282
lesser near-clades that are widespread throughout different countries and exhibit
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-4
and 0.037, respectively (Ramírez et al.,
Page 12 of 52
13 host specificities (fig. 2; Guhl & Ramírez, 2011; Herrera et al., 2007). TCI lesser near-
284
clades have been corroborated by various markers, including the Cytochrome b gene
285
sequence (a mitochondrial gene) and the mini-exon gene (Ramírez et al., 2011),
286
PCR-RFLP of 5 coding regions (Ramírez et al., 2012), mini-exon and MLST
287
(Tomasini et al., 2011, 2014).
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Population genetics within other T. cruzi near-clades has been less explored.
289
However, in the near-clade TCIII, a strongly significant (p<0.001) LD (measured
290
using the Index of Association (IA)) was detected in all populations except one,
291
where only marginal significance was observed (p=0.032) (Llewellyn et al., 2009a).
292
This supports the hypothesis that TCIII exhibits also a RDP.
an
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- Indices of frequent recombination within near-clades?: A lack of congruence
294
between mitochondrial and nuclear phylogenies (Ramírez et al., 2012) indicates at
295
best limited introgression, as it can be observed even between different biological
296
species. The null hypothesis of the RDP model is not occasional bouts of genetic
297
exchange, which is consistent with PCE. It is random or quasi-random
298
recombination, as it can be observed in human populations for example. In humans,
299
the only obstacles to genetic exchange are geographical or cultural ones (quasi-
300
random recombination). TCI populations isolated from selvatic triatomine bugs in
301
Bolivia show data that are compatible with such frequent genetic exchange.
302
However, according to the authors themselves, these data are prone to type II error
303
and should be corroborated by stronger sampling (Barnabé et al., 2013). A survey in
304
Ecuador (Ocaña et al., 2010) is consistent with recombination in a small TCI
305
subpopulation. However, as we have shown (Tibayrenc & Ayala, 2013), (i) the
306
―domestic‖ subpopulation is poorly defined (33% of selvatic strains are included in it,
307
while the ―selvatic‖ clade comprises 12% of domestic strains); (ii) the risk of type II
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Page 13 of 52
14 error is high, due to very limited sampling (only 12 truly ―domestic‖ strains).
309
Additionally, the ―selvatic‖ subpopulation exhibits strong LD and clonality, which fits a
310
RDP. De Paula Baptista et al. (2014) have proposed that Brazilian strains of the TCII
311
near-clade undergo substantial recombination. However, this study is based also on
312
limited sampling and is subject to type II error. Moreover, there is a strong
313
contradiction between the hypothesis of substantial recombination (with lack of LD)
314
and the evidence for clear structuration of these populations, even in sympatry, as
315
shown by STRUCTURE analysis. In conclusion: it is quite possible that some
316
populations within T. cruzi near-clades undergo frequent recombination. However,
317
the limited evidence presently available obviously has to be completed by far more
318
extensive studies. On the contrary, the evidence above exposed for RDPs is robust.
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319
From the results above exposed, it is hardly questionable that T. cruzi at the level
321
of the whole species undergoes PCE. Moreover, when enough data are available, it
322
is apparent that PCE operates also within the near-clades (RDP). Genetic
323
recombination/hybridization in the agent of Chagas disease has probably an
324
important impact at an evolutionary scale. However, it is unable to counter the effects
325
of clonal evolution in the long run (―clonality threshold‖) and to prevent the
326
evolutionary divergence of the near-clades. When looking for recombination in a
327
given species amounts to ―searching for a needle in a haystack‖ (Ramírez &
328
Llewellyn, 2014), it does show that recombination is strongly restrained, in other
329
words, that the PCE model is corroborated. When it is not, like in the highly
330
recombining bacterium Helicobacter pylori, one finds easily clear indications for
331
recombination without searching a haystack.
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Page 14 of 52
15 In the framework of PCE, it is quite possible that local populations of genetically
333
related strains undergo more genetic exchange than clonal propagation (Barnabé et
334
al., 2013; Ocaña et al., 2010; de Paula Baptista et al., 2014). However, this has to be
335
better explored on more extensive samples. It is possible that in T. cruzi,
336
recombination is less restrained among identical genotypes (= selfing) or similar
337
genotypes (tending to selfing) than among more distantly related genotypes. This
338
happens in bacteria, where recombination is more frequent among ―sister cells‖ than
339
among cells that have radically different genotypes (Caugant & Maiden, 2009). This
340
leads to ―invisible sex‖ (Balloux, 2010) and is explained by the fact that the higher the
341
genetic divergence, the more the restriction systems are different (Caugant &
342
Maiden, 2009). Moreover, local introgressions of mitochondrial genomes are quite
343
possible in T. cruzi (Messenger et al., 2012; Ramírez et al., 2012). All this does not
344
challenge the PCE model. As exposed earlier, the alternative hypothesis to PCE is
345
that recombination is abundant enough to overcome the effects of clonal evolution in
346
the long run.
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347
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Lastly, restrained recombination in T. cruzi cannot be attributed to a lack of
349
opportunity for mating. As a matter of fact, multiclonal infections in the same host,
350
either vectors (Tibayrenc et al., 1985) or chagasic patients (Brenière et al., 1985) are
351
very frequent.
352
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348
353 354
Aneuploidy and the selfing/homogamy debate
355
Page 15 of 52
16 We recall here that selfing/homogamy is included in our PCE concept, a view
357
shared by many, if not most, specialists of pathogen population genetics (Tibayrenc
358
& Ayala, 2012) as well as by evolutionist working on metazoa undergoing uniparental
359
reproduction (Avise, 2008). However, other scientists recommend distinguishing
360
selfing/homogamy from ―strict‖ (= mitotic) clonality (Rougeron et al., 2009; Rougeron,
361
de Meeûs & Bañuls, 2015). When the macroevolution scale recommended by the
362
PCE approach is considered, the distinction in terms of population structure has
363
limited relevance, since both ―strict‖ clonality and selfing/homogamy lead to
364
restrained recombination and LD. Now selfing as a mode of restrained recombination
365
has the evolutionary advantage of permitting DNA repair, which is not the case of a
366
strictly mitotic mode of propagation. If one wants to distinguish selfing from ―strict‖
367
clonality, the problem lies on the way to do it. When bacteria are considered, since
368
they are haploid, selfing, which is certainly very frequent in these pathogens (Maiden
369
2008; Caugant & Maiden 2009), is impossible to distinguish from mitotic propagation
370
(―invisible sex‖; Balloux, 2010). When protozoa are concerned, the evidence
371
proposed for selfing relies on heterozygote deficit (Rougeron et al., 2009). However,
372
apart from selfing, many phenomena could cause heterozygote deficit, such as null
373
alleles, allelic drop-out, homoplasy (a strong concern in the case of microsatellites)
374
(Alam et al., 2009; Lehmann et al., 2004, Pearson et al., 2009), and genome-wide
375
mitotic gene conversion (Llewellyn et al., 2009a, 211). Experiments in the strictly
376
asexual species Daphnia pulex have shown that a loss of heterozygosity by mitotic
377
recombination is 1,000 times more frequent than an accumulation of divergent
378
mutations (Omilian et al. 2006). The probability of the possible causes of
379
heterozygote deficit should not be evaluated separately (Rougeron et al., 2009),
380
since they are not mutually exclusive. Moreover, heterozygote deficit inferred from
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Page 16 of 52
17 microsatellite analysis clashes with SNP* data for Leishmania mexicana and L.
382
braziliensis (Rogers et al., 2011) and T. cruzi (Yeo et al., 2011), where an excess of
383
heterozygotes is recorded. However, selfing should cause a heterozygote deficit for
384
all loci, including the ones involved in SNPs. The main problem with the way to
385
explore heterozygote deficit is that all tests proposed for it (de Meeûs et al., 2007)
386
are based on the hypothesis that the organism under survey is diploid*. Now strong
387
convergent results suggest that Leishmania undergoes widespread aneuploidy
388
(Downing et al. 2011; Lachaud et al. 2014; Mannaert et al. 2012; Rogers et al. 2011;
389
Sterkers et al. 2011, 2012, 2014), which renders the tests invalid, and at the same
390
time, is an explanation for apparent heterozygote deficit. As a matter of fact,
391
widespread aneuploidy should cause a rapid elimination of heterozygosity by
392
frequent passage through a haploid state (Sterkers et al. 2012). This should happen
393
even if aneuploidy is transitory in Leishmania, as proposed by Rougeron, de Meeûs
394
& Bañuls (2015). The evidence for aneuploidy in T. cruzi still is more limited than for
395
Leishmania, although progress is rapid (Minning et al. 2011; Souza et al. 2011).
396
When population genetic data are concerned, contrary to what has been claimed
397
(Ramírez et al., 2013a), in a survey dealing with more than 200 cloned stocks from
398
the same host, in mammals from Venezuela and Bolivia, ―multiple aneuploid clones
399
were isolated across those infrapopulations studied‖ (Llewellyn et al., 2011). It has
400
been proposed that microsatellite data do not suggest aneuploidy in Leishmania
401
(Rougeron, de Meeûs & Bañuls, 2015). However, Lachaud et al. (2014) disagree with
402
this statement. The conclusion of all this is that selfing in Leishmania and T. cruzi is a
403
reasonable hypothesis. However, the tests proposed to explore it may not be reliable.
404
As stated by Ramírez et al. (2012): ―In general, we urge caution in the interpretation
405
of heterozygosity statistics at STR loci in the context of parasite sexuality, especially
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Page 17 of 52
18 406
given that strong evidence for linkage disequilibrium often accompanies both
407
negative and positive values for FIS‖. Such difficulties in evidencing selfing in
408
parasitic protozoa gives additional ground to our proposal of including it in the PCE
409
model. It should be noted that LD tests do not suffer from this problem. As a matter of
411
fact, they can be practiced whatever be the ploidy of the organism under study, and
412
even, when this ploidy level is not known (Tibayrenc, Kjellberg & Ayala, 1990).
cr
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414
us
413
Evolutionary features that could mimic recombination
an
415
Recombination can be confounded with several evolutionary features. They
417
include: (i) homoplasy; (ii) lack of phylogenetic signal; (iii) natural selection; (iv)
418
different molecular clocks; (v) long branch attraction; (vi) different modes of
419
inheritance.
ed
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416
Homoplasy is a concern for many, if not most, molecular markers. Microsatellites
421
are considered as especially prone to it (Alam et al., 2009; Lehmann et al., 2004,
422
Pearson et al., 2009). This could lead to overestimate both recombination and
423
heterozygote deficit.
ce pt
420
Lack of phylogenetic signal: if the population is close to genetic monomorphism,
425
or if the molecular marker has too low a resolution power, it is impossible to evidence
426
LD and the presence of near-clades. This should not be taken as evidence for
427
recombination. A lack of phylogenetic signal could be due also to a saturation of the
428
molecular marker. As an example, microsatellites are not adapted to survey higher
429
levels of evolutionary divergence, since their molecular clock is too fast. MLSTs and
430
MLEE could reveal a strong phylogenetic signal where microsatellites cannot.
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424
Page 18 of 52
19 Natural selection is difficult to distinguish from recombination (Didelot & Maiden,
432
2010). Strongly selected characters, such as drug resistance, should be especially
433
prone to this bias. Resistance to the same antibiotics can be present in different
434
bacterial lines, and even in different species. This can be the result of genetic
435
exchange, but it is also easily explained by an identical selective pressure. Such
436
convergent selected characters should be therefore considered as evidence for
437
recombination only carefully (Rougeron, de Meeûs & Bañuls, 2015).
cr
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Differences in molecular clocks could lead to discrepancies between trees
439
designed from different genes. In Giardia duodenalis, Wielinga & Thompson (2007)
440
have attributed the discrepancies observed among trees built from different genes to
441
both homoplasy and molecular clock differences, not to recombination. Similarly, in
442
the case of the SARS coronavirus, phylogenetic incongruence among trees has been
443
attributed to different molecular clocks (Moya et al., 2004).
M
an
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438
Long branch attraction: analyzing the phylogeny of the SARS coronavirus, Yip et
445
al. (2009) have recommended to not confound recombination with both homoplasy
446
and long branch attraction.
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ed
444
Markers that have a different mode of inheritance will exhibit different evolutionary
448
patterns. For example, in humans, the population structures inferred from autosomal
449
genes, mitochondrial genes and Y chromosome genes, respectively, are quite
450
different (Zoossmann-Diskin, 2010). In T. cruzi, experimental mating has shown that
451
nuclear genes are inherited from both parental cells, while the kinetoplast is inherited
452
from only one parental cell (Gaunt et al., 2003). In the framework of the PCE model,
453
in case of occasional bouts of genetic exchange, this phenomenon is bound to
454
maximize the discrepancies between mitochondrial and nuclear phylogenies.
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447
Page 19 of 52
20 Lastly, these different phenomena are not exclusive of each other and could
456
combine their effects. In the case of T. cruzi, microsatellites are subject to
457
homoplasy. They have a fast molecular clock, they are explored at the level of the
458
nucleus, whose inheritance is biparental, and they are considered as selectively
459
neutral. Mitochondrial maxicircle genes correspond to coding sequences, which are
460
subject to natural selection. They have a slower molecular clock than microsatellites,
461
and their mode of inheritance is uniparental. So, although the discrepancies between
462
microsatellite and mitochondrial gene phylogenies are consistent with recombination
463
(Ramírez et al., 2012), alternative explanations are possible.
us
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455
an
464 465
A brief comparison with other micropathogens
468
We have performed a compared population genetic approach dealing with viruses
469
(22 species and categories), bacteria (43 species), parasitic protozoa (30 species)
470
and fungi (13 species) (Tibayrenc & Ayala, 2012 and in preparation). This shows that
471
T. cruzi population structure is not unique and, on the contrary, is very similar to that
472
of many major pathogen species. Each species exhibits specific traits. However, the
473
common denominator among many of them is strong, and suggests the existence of
474
common evolutionary strategies, which could be either ancestral or the result of
475
convergence.
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467
M
466
476
Well-established T. cruzi ―siblings‖ in terms of population structure are the species
477
for which the PCE features (LD, widespread MLGs, near-clades and RDPs) have
478
been recorded. It is assumed that these species have crossed the ―clonality
479
threshold‖ beyond which the impact of clonal evolution will overcome the effects of
Page 20 of 52
21 recombination. Such species include: (i) parasitic protozoa: the Leishmania infantum
481
complex (Tibayrenc & Ayala, 2013), Giardia duodenalis, (Tibayrenc & Ayala, 2014b),
482
Toxoplasma gondii (Tibayrenc & Ayala, 2014a); (ii) fungi: Candida albicans, the
483
Cryptococcus neoformans complex; (iii) bacteria: Escherichia coli, Mycobacterium
484
tuberculosis, Neisseria meningitidis, Staphylococcus aureus. Many other species
485
exhibit clonality traits, but the PCE pattern is not complete, either because these
486
species undergo more recombination, or because the studies are less advanced than
487
for the species above listed, or both. When parasitic protozoa are concerned, such
488
examples include: Leishmania braziliensis/peruviana, the Trypanosoma brucei
489
complex, T. congolense, Cryptosporidium spp.
an
us
cr
ip t
480
Plasmodium falciparum and P. vivax constitute distinctive cases. Since P.
491
falciparum undergoes meiosis in the vector, it has been long assumed that the agent
492
of the most malignant form of malaria was a panmictic* organism. Our cautious
493
proposal that ―uniparental and biparental lineages could coexist within this species‖,
494
based on the observation of departures from panmixia in some populations
495
(Tibayrenc, Kjellberg & Ayala, 1990), had been rejected but it was subsequently
496
confirmed (Anderson et al., 2000). Many P. falciparum and P. vivax populations
497
exhibit clonality traits. Selfing is probably at the origin of them. However, the
498
hypothesis that selfing is strictly correlated to the level of transmission is at odds with
499
many data (Tibayrenc & Ayala, 2014a). This is epidemiologically relevant, since LD
500
and the degree of clonality in P. falciparum cannot be taken as an indication for the
501
transmission rate (Tibayrenc & Ayala, 2014a), as it had been proposed (Volkman et
502
al., 2012). Now P. falciparum and P. vivax definitely do not fit the PCE model.
503
Clonality traits (LD, widespread MLGs and near-clades) that are observed in these
504
species are labile and change over time, due to frequent genetic recombination. Still
Ac
ce pt
ed
M
490
Page 21 of 52
22 505
the fact remains that clonal propagation introduces a major stratification effect in P.
506
falciparum and P. vivax natural populations, that should be taken into account in
507
studies dealing with malaria epidemiology, drug resistance and the analysis of genes
508
of interest. When considering the various species that fit the PCE model, two facts should be
510
underlined: (i) for several of them, PCE was not expected. Neisseria meningitidis was
511
considered a ―highly recombining‖ bacterium (Maiden, 2008). However, widespread
512
MLGs and near-clades in this species are extremely stable, as verified by
513
retrospective studies. Moreover, whole genome sequencing has revealed the
514
existence of deep phylogenies (Budroni et al., 2011), which is incompatible with the
515
―epidemic clonality model‖ (occasional bouts of clonal propagation in an otherwise
516
recombining species; Maynard Smith et al., 1993). Toxoplasma gondii constitutes
517
another unexpected case of PCE, since this parasite undergoes meiosis in the
518
mammalian host. We have proposed that T. gondii undergoes frequent, and possibly
519
predominant clonal evolution (Tibayrenc et al., 1991). This has been fully confirmed
520
by further studies (Sibley & Boothroyd, 1992). High resolution typing with many
521
different markers (Su et al., 2012) has shown the existence of deep phylogenies, with
522
6 major ―clades‖ (= near-clades), which definitely includes T. gondii in the PCE model
523
(Tibayrenc & Ayala, 2014a). (ii) the species listed pertain to phylogenetically highly
524
diverse categories (bacteria, fungi, and parasitic protozoa), and exhibit various host
525
ranges, modes of transmission (air-, vector-, food- and water-borne) and tissue
526
tropisms. This strongly suggests that PCE represents a common evolutionary
527
strategy of many pathogens, probably linked to parasitism.
Ac
ce pt
ed
M
an
us
cr
ip t
509
528 529
Experimental mating, meiosis genes and PCE
Page 22 of 52
23 530 The seminal work by Gaunt et al. (2003) has demonstrated that the potential for
532
recombination is present in T. cruzi. However, these experiments dealt only with
533
related strains pertaining to the near-clade TCI. It remains to be established whether
534
mating can also be obtained between strains pertaining to different near-clades.
535
More important: these experiments provide no indication about the frequency of
536
genetic exchange in natural populations, which matters when population structure is
537
concerned. Successful mating experiments cannot therefore be used to challenge
538
the PCE model.
us
cr
ip t
531
Genes homologous to meiosis genes are observed in many micropathogen
540
species, including T. cruzi (El-Sayed et al., 2005). However, this does not imply that
541
T. cruzi undergoes meiosis (Ramírez & Llewellyn, 2014), and if it did, the presence of
542
these genes does not give indications about the frequency of it. Moreover, genes
543
analogous to meiosis genes may have acquired totally different functions through
544
evolution. ―Evolution is constantly re-using old genes for new purposes‖ (Birky 2009).
545
We have suggested that, in micropathogens undergoing PCE, the meiosis kit could
546
actually
547
micropathogens to use either one or the other mode of propagation when facing
548
different evolutionary challenges (Tibayrenc & Ayala, 2012). In conclusion: like for
549
mating experiments, the presence of genes homologous to meiosis genes is
550
therefore not evidence against the PE model.
ce pt
ed
M
an
539
to
a
―sexuality/clonality
machinery
that
could
allow
Ac
correspond
551 552
The PCE model and the problem of pathogen species definition
553
Page 23 of 52
24 Defining species in micropathogens has been always a challenge, since the
555
mixiological species concept (Dobzhansky, 1937), which addresses only organisms
556
with obligatory mating, cannot be applied to them. Moreover, the phylogenetic
557
species concept (Cracraft, 1983) is difficult to apply, since it is most probable that
558
some genetic exchange always occurs in micropathogens natural populations, even
559
between different species. The near-clade concept, which relaxes the cladistic
560
demands, makes it possible to revisit the phylogenetic species concept for
561
micropathogens (Tibayrenc and Ayala, 2014b). Two situations occur. The first case is
562
when presently described species can be equated to near-clades in an evolutionary
563
point of view. Many, if not most, Leishmania species are in this case. As a matter of
564
fact, in general, they are clearly discriminated by molecular markers. However,
565
hybridizations are frequent among them. Actually, many Leishmania species are
566
perfectly equivalent to T. cruzi near-clades from an evolutionary point of view. The
567
second case is when presently observed near-clades have not been described as
568
Linnean species yet. This is the case for the Giardia duodenalis ―assemblages‖ (=
569
near-clades) (Tibayrenc & Ayala, 2014b). T. cruzi near-clades could constitute the
570
basis for splitting the agent of Chagas disease into several species. We do not
571
recommend it for several reasons. First, the inventory of T. cruzi genetic diversity
572
could still evidence new features and unknown near-clades. Second, the advent of
573
whole genome sequencing could lead to reconsider the presently accepted near-
574
clade classification (Zingales et al., 2012). Third, the epidemiological and
575
pathological specificity of T. cruzi near-clades is not clear-cut. The Chagas disease
576
scientific community will decide whether the description of these new species is
577
desirable. The near-clade concept may constitute a necessary, but not sufficient,
578
criterion for it. Since near-clading is widespread among micropathogens, it would be
Ac
ce pt
ed
M
an
us
cr
ip t
554
Page 24 of 52
25 579
misleading to equate all of them to Linnean species. This should be done only when
580
a given near-clade exhibits strong phenotypic specificities dealing with relevant
581
characters (host range, drug resistance, pathogenicity for example).
582
584
ip t
583 The PCE model and applied research.
cr
585
The PCE model provides applied research with relevant units of analysis that are
587
based on simple concepts, and are sharply defined. Their stability in space and time
588
has been fully ascertained by retrospective studies, as well as by population genetics
589
and phylogenetic analysis. Clonal MLGs (clonets) can be used for epidemiological
590
tracking. To characterize them, markers with different mutation rates and level of
591
resolution can be selected according to the study concerned: microsatellites for
592
microepidemiology, MLST and whole genome sequencing plus SNPs (when these
593
two last tools become more accessible) for epidemiological surveys at broader
594
scales. Near-clades and their subdivisions (RDPs) can, and should be used as units
595
of analysis for epidemiological tracking, drug design (Zingales et al., 2014), clinical
596
studies and experimental evolution. Taking the near-clades as units of analysis, we
597
have conducted long-term experimental evolution studies, which have shown that
598
many phenotypic characters (including drug sensitivity, virulence in mice and cell
599
infectivity), as well as gene expression patterns, were linked to the near-clades
600
(Revollo et al., 1998; Telleria et al., 2004, 2010, 2013).
Ac
ce pt
ed
M
an
us
586
601
A major application of the PCE model for strain typing/molecular epidemiology is
602
indirect typing, which is a remarkable property of LD. Since all genes are linked
603
together (―block of genes‖), the near-clades can be identified by only one or two
Page 25 of 52
26 markers (―tags‖; Tibayrenc, 1998). Several RAPD and isoenzyme tags have been
605
identified for the 6 near-clades, including near-clade TCI, (Brisse et al., 2000). By the
606
amplification of a unique gene (TcSC5D), Cosentino & Agüero (2012) were able to
607
identify the T. cruzi near-clades I, II, II, IV, V+VI, TC-Bat, and the subspecies T. cruzi
608
marinkellei. We recommend this ―phylogenetic character mapping‖ (PCM) approach
609
(Avise, 2004). A convenient population genetics framework is first designed through
610
the PCE approach. Once the relevant units of analysis (near-clades) are clearly
611
identified, phenotypes of interest (drug resistance, virulence) are mapped onto this
612
framework. This makes it possible to see the links between the overall evolution of
613
the organism under study and its phenotypes, and at the same time, to show the
614
specific phenotypic properties of the near-clades.
an
us
cr
ip t
604
616
M
615 Conclusion
ed
617
Due to the dynamism of many research groups throughout the world, with a major
619
contribution from Latin American teams, the intraspecific genetic variability of T. cruzi
620
is probably one of the best known among micropathogens. The agent of Chagas
621
disease could be considered a star model for evolution, like Drosophila
622
melanogaster, Mus musculus, Caenorabditis elegans and Escherichia coli.
Ac
623
ce pt
618
The PCE model, which has been
elucidated and enriched by wide surveys
624
dealing with many pathogens (Tibayrenc & Ayala, 2012), sets in perspective the
625
particular case of T. cruzi population genetics. It shows that T. cruzi shares many
626
features with other major pathogens, including parasitic protozoa, fungi and bacteria.
627
Promising avenues of evolutionary research are represented by experimental
628
mating (less advanced for T. cruzi than for T. brucei), within near-clade population
Page 26 of 52
27 genetics, experimental evolution, and the development of more powerful tools of
630
analysis (whole genome sequencing, SNP analysis). These tools constitute a
631
challenge for analysis, since the computing power required for treating these data is
632
huge. However, the use of these tools becomes routine for viruses and bacteria, and
633
should be more and more accessible when parasitic protozoa are concerned, making
634
it possible to develop a population genomics of these pathogens.
Ac
ce pt
ed
M
an
us
cr
635
ip t
629
Page 27 of 52
28 636 637
Legends of the figures
638 Figure 1: The six Trypanosoma cruzi near-clades evidenced by MLST analysis (after
640
Lauthier et al., 2012). Left: neighbor-joining tree; right: split decomposition analysis.
641
The two modes of analysis, which rely on different computing methods and different
642
working hypotheses, show the same near-clading pattern. This is an indication that
643
the clustering of the near-clades is robust, according to the congruence principle
644
(Avise, 2004).
us
cr
ip t
639
an
645
Figure 2: Geographic distribution of the five lesser near-clades evidenced within the
647
Trypanosoma cruzi near-clade TCI (see fig. 1) (after Gull & Ramírez, 2011). The
648
lesser near-clades are widespread and occur sympatrically, which is an evidence of a
649
―Russian doll Pattern‖ (Tibayrenc & Ayala, 2013).
ed
M
646
Ac
ce pt
650
Page 28 of 52
29 651 652
Glossary of specialized terms:
653 Allopatry: a situation where different populations occur at different locations; cf
655
sympatry*
ip t
654
656
Aneuploidy: a situation in which the chromosome number of an individual is not an
658
exact multiple of the haploid set of the species. Copy numbers may vary among
659
chromosomes.
us
cr
657
an
660
Clade: evolutionary line defined by cladistic analysis. A clade is monophyletic* and is
662
genetically differentiated (i.e., evolves independently) from other clades.
M
661
663
Cladistic analysis: phylogenetic analysis based on the distribution of characters that
665
are
666
characteristics. Only apomorphic characters shared by all members of a given clade*
667
(synapomorphic character) convey relevant phylogenetic information.
670
ancestral
(plesiomorphic)
and
derived
(apomorphic)
ce pt
669
between
Diploid: possessing 2 copies of each chromosome.
Ac
668
shared
ed
664
671
DTU = Discrete Typing Unit: sets of strains that are genetically closer to each other
672
than to any other stock and are identifiable by common genetic, molecular or
673
immunological markers called tags (Tibayrenc, 1998).
674 675
Haploid: possessing only one copy of each chromosome.
Page 29 of 52
30 676 677
Homogamy: preferential mating between individuals that are genetically identical or
678
extremely similar to each other.
679 Homoplasy: distinct evolutionary units possess similar characters that do not
681
originate from a common ancestor.
ip t
680
cr
682
MLEE: = Multilocus Enzyme Electrophoresis. Analysis by electrophoresis of protein
684
extracts from biological samples. MLEE banding reflects the aminoacid sequence of
685
the proteins, coded by the DNA sequences. It is an efficient genetic typing method.
686
However, identical MLEE bands may correspond to different DNA sequences
687
(homoplasy*).
M
an
us
683
688
MLST = Multilocus sequence typing : a highly standardized typing technique
690
based on the sequencing of 450-bp segments of a set of housekeeping genes
691
(usually seven). It has been widely used for a high number of bacterial species and
692
some eukaryotic pathogens as well.
693
Molecular clock : in its strict, original sense (more correctly named: ―DNA clock
694
hypothesis‖), the concept that the rate of nucleotide substitutions in DNA remains
695
constant. In a broader sense, simply the rate of evolution of the genomic part that
696
codes for the variability of a given marker. This fastness is driven by the rate of
697
substitution/mutation. It may be regular or irregular.
Ac
ce pt
ed
689
698 699
Monophyletic: the case where a given phylogenetic lineage has only one ancestor
700
(see clade*).
Page 30 of 52
31 701 702
Panmixia, panmictic: a situation where genetic exchange occurs at random.
703 Parthenogenesis: Reproduction by the development of a single gamete without
705
fertilization by a gamete of the opposite sex.
ip t
704
706
PFGE = Pulse Field Gel Electrophoresis: separation of large DNA fragments by a
708
particular electrophoresis technique using alternately pulsed, perpendicularly
709
oriented electrical fields. Strains that share the same PFGE are referred to as pulse
710
types. In the case of bacteria, the large DNA fragments result from the action of a
711
―low-frequency cutter‖ (a bacterial endonuclease which restriction action has a low
712
frequency) on the bacterial chromosome. . In the case of parasitic protozoa
713
(Trypanosoma and Leishmania) and of yeasts, the large DNA fragments correspond
714
to entire chromosomes (―molecular karyotype‖).
ed
M
an
us
cr
707
715
RAPD = Random Amplified Polymorphic DNA: In the classical PCR method, the
717
primers used are known DNA sequences, whereas the RAPD technique relies on
718
primers whose sequence is arbitrarily determined.
719
Ac
ce pt
716
720
Recombination, recombining : Reassortment of genotypes occurring at different
721
loci.
722 723
Selfing : self-fertilization of an organism. If the organism is haploid (bacteria), or if all
724
loci are homozygous, selfing is undistiguishable from mitotic clonality. In diploid
Page 31 of 52
32 725
organisms, selfing leads to a deficit of heterozygotes, and to a lack of genetic
726
recombination
727 SNP = Single Nucleotide Polymorphism : a DNA sequence variation occurring
729
commonly within a population, in which a single nucleotide — A, T, C or G differs
730
between individuals.
ip t
728
cr
731
Sympatry: a situation where different populations are present in the same place; cf
733
allopatry*.
us
732
an
734 735
Wahlund
736
subpopulations (isolated in time or space or both) that have different genotype
737
compositions, population genetic tests performed at the level of the whole population
738
show departures from panmictic expectations, even if each subpopulation is
739
panmictic*.
742
sampled
population is composed
of
several
ed
M
the
ce pt
741
when
Zymodeme : MLEE* MLG.
Ac
740
effect:
Page 32 of 52
33 743 744
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We expose our view on Trypanosoma cruzi population genetics
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We show it is enlightened by our model of pathogen predominant clonal evolution. We draw comparisons with other pathogens, in particular Leishmania. We underline the implications of the model for applied research.
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