An organism’s genetic ‘blueprint’ is encoded in DNA packaged within structures called chromosomes. Most organisms have two copies of each chromosome and, through sexual reproduction, the DNA within a pair of chromosomes can recombine randomly in a process that could be likened to shuffling a deck of cards. This process generates genetic diversity and means that any undesirable combinations of genes or mutations can be eliminated from the population by natural selection. While these activities are thought to promote the long-term survival of a species, some organisms appear not to have sex at all.

Evolutionary theory predicts that ‘asexual’ organisms should face extinction in the long-term and that a lack of sexual recombination should leave a characteristic genetic ‘signature’ in their DNA. The theory also predicts that pairs of chromosomes will evolve independently, a phenomenon that is termed the ‘Meselson effect’. However, while it was first predicted almost twenty years ago, evidence for this signature has been elusive.

Now, Weir et al. have used the asexual parasite (Trypanosoma brucei gambiense), which causes African sleeping sickness in humans, to search for signs of the Meselson effect. Sequencing the whole genome of a large number of parasites revealed that the population of this parasite arose from a single individual within the last 10,000 years. Over this time, mutations have built up and the lack of sexual recombination means that the two chromosomes in each pair have evolved independently of the other. These results provide the first demonstration of the Meselson effect at a genome-wide level in any organism.

Weir et al. also uncovered evidence that this parasite uses a mechanism called “gene conversion” to compensate for its lack of sex. This mechanism essentially repairs the inferior, or mutated, copy of a gene on a chromosome by ‘copying and pasting’ the superior copy from the chromosome’s partner. The findings also suggest that gene conversion can only go some way to compensating for a lack of sex. A future challenge will be to investigate how effective this mechanism can be in the long term and to predict whether the parasite will ultimately become extinct.

Obligate asexual reproduction has been argued to carry considerable negative evolutionary consequences (Maynard Smith, 1986) hence most clonal species undergo some degree of recombination, albeit infrequent, which generates novel genotypes (Heitman, 2006). Here we investigate the reproductive strategy of a putatively asexual yet successful human pathogen, Trypanosoma brucei gambiense Group 1, the main causative agent of human African trypanosomiasis, which contrasts with closely-related, sexually reproducing sub-species. T.b. gambiense has been divided into two groups. T.b. gambiense Group 1, found in West and Central Africa, is the main human-infective sub-species, causing >97% of all human cases of trypanosomiasis (Simarro et al., 2010). T.b. gambiense Group 2 was detected in the 1980/90s in Côte d’Ivoire and Burkina Faso but may now be extinct (Capewell et al., 2013). A third human infective sub-species, T.b. rhodesiense, is found in East Africa and causes <3% of human cases (Simarro et al., 2010). Each of these human infective sub-species appears to have arisen independently from the non-human infective T. brucei and possesses a different mechanism of human infectivity (Capewell et al., 2013; Capewell et al., 2011; Uzureau et al., 2013; Van Xong et al., 1998). All sub-species of T. brucei, with the important exception of T.b. gambiense Group 1, show evidence for mating in natural populations (Capewell et al., 2013; Duffy et al., 2013; Gibson and Stevens, 1999) and have the ability to undergo sexual reproduction with Mendelian allelic segregation and independent assortment of unlinked markers (Cooper et al., 2008; MacLeod et al., 2005). In addition, haploid gametes have been observed in T.b. brucei (Peacock et al., 2014) and although meiosis genes appear to be expressed in T.b. gambiense Group 1 (Peacock et al., 2014), no haploid gametes have ever been observed in these parasites (Peacock et al., 2014). This is consistent with clonality in all Group 1 populations analysed (Koffi et al., 2009; Morrison et al., 2008; Tibayrenc and Ayala, 2012), however, these studies were based on limited sets of genetic markers, which lack the necessary discriminatory power to distinguish between predominantly clonal evolution, with occasional bouts of genetic exchange, and strictly asexual propagation. Genomic-level analyses of T. brucei diversity to date have concentrated on T.b. brucei and T.b. rhodesiense and for T.b. gambiense Group 1, include only the genome reference strain (DAL972) (Goodhead et al., 2013) or two (Sistrom et al., 2014) field isolates. We hereby present a population-level genomic analysis as a means to determine whether this species is truly asexual, when the switch to asexuality arose and to provide insights into the genomic consequences of asexual evolution, including possible compensating strategies for eliminating deleterious mutations.

The genomes of 75 isolates of T.b. gambiense Group 1 (Supplementary file 1) were sequenced, including multiple samples from geographically separated disease foci within Guinea (n=37), Côte d’Ivoire (n=36) and Cameroon (n=2) collected over fifty years (1952–2004). For comparative purposes, isolates of T.b. rhodesiense (n=4), T.b. gambiense Group 2 (n=4) and T.b. brucei (n=2) were also sequenced. A total of 230,891 single nucleotide polymorphisms (SNPs) were identified compared to the haploid consensus assembly of the T.b. brucei reference genome (Berriman et al., 2005). These were evenly distributed over the eleven major chromosomes, covering 85% of the genome (Figure 1—figure supplement 1). T.b. gambiense Group 1 showed a 5–10 fold lower number of SNPs (11,398) and SNP density compared to the other groups (Figure 1—source data 1), despite an over-representation in terms of the number of samples. Phylogenetic network analysis revealed that T.b. gambiense Group 1 genotypes showed an extremely low level of intra-group diversity (e.g. the two most distantly related isolates differed only at 435 SNP loci) and formed a monophyletic group (Figure 1A). The network features reticulation among non-Group 1 parasites indicating the presence of recombinant genotypes; this stands in contrast to Group 1 parasites and is consistent with an absence, or rarity, of recombination in this group. Network analysis of T.b. gambiense Group 1 revealed the population is geographically sub-structured (Figure 1B). A significant deviation from Hardy-Weinberg Equilibrium (HWE) was observed at 97.4% of SNP loci (P<10^-17 at each locus) and this was found to be associated with every sampled genotype being heterozygous at these loci (Figure 1—source data 2). To control for geographical and temporal population sub-structure, isolates from three sub-populations were analysed and HWE deviation and heterozygote excess was confirmed (Figure 1—source data 2). F_IS was calculated for each SNP locus, giving a uni-modal distribution with a median of -1 (Figure 1—figure supplement 2 and Figure 1—source data 3), as would be predicted for a strictly asexual population. Using a genome-wide panel of SNP loci, strong evidence of linkage disequilibrium (LD) was obtained for each chromosome and the whole genome formed a single genetic linkage group (Figure 1—figure supplement 3).

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Figure 1—source data 1

Number of SNP loci with respect to different sub-species.

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Figure 1—source data 2

Testing Hardy-Weinberg Equilibrium (HWE) across the T.b. gambiense Group 1 genome.

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Figure 1—source data 3

F_IS by sub-population.

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Figure 1—source data 4

Number and type of T.b. gambiense Group 1 SNPs.

https://doi.org/10.7554/eLife.11473.007

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Inspection of the SNP distribution across the genome of Group 1 isolates identified multiple long tracts of homozygosity, termed Loss of Heterozygosity (LOH) (Figure 2—figure supplement 1). Examination of read depth variation confirmed that this is not due to the loss of part of a homologue and is consistent with mitotic gene conversion; similarly there is no evidence of aneuploidy. In T.b. gambiense Group 1, LOH has occurred on every chromosome, with many isolates showing the same LOH patterns (Figure 2—figure supplement 1), strongly suggesting that many of these regions arose early in Group 1 evolution. Chromosome 10 displayed the greatest variation in LOH sites, with a total of eighteen different patterns of variation among Group 1 genomes (Figure 2). This varied from 2% to 82% of the chromosome length, with LOH occurring predominantly towards one telomere. Distinct LOH patterns were associated with particular phylogenetic lineages, indicating LOH has occurred at various points in Group 1 evolution (Figure 2—figure supplement 2).

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Taken together, these analyses clearly demonstrate that Group 1 parasites are evolving asexually. A key predicted feature of asexual diploid species is the independent evolution and divergence of alleles on chromosome homologues (Birky, 1996), often referred to as the ‘Meselson effect’ (Birky, 1996; Butlin, 2002; Mark Welch and Meselson, 2000). To test whether this phenomenon occurred in T.b. gambiense Group 1, three regions of the genome were chosen where it was possible to manually phase the genomic data, using LOH events to guide the phasing in non-LOH closely related isolates (Materials and methods). For all three regions, a clear sequence of the accumulating mutations could be inferred (Figure 3), with each haplotype evolving independently, thus illustrating the Meselson effect (Judson and Normark, 1996).

Figure 3

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Estimated time since the most recent common ancestor.

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To test whether haplotypes have evolved independently at the whole-chromosomal level, the sequence dataset for all sub-species was computationally phased and haplotypes inferred for each isolate. Excluding LOH regions, phylogenetic trees for each chromosome were generated, revealing that A and B haplotypes separate into distinctive clusters (example in Figure 4A) and for every chromosome this pattern was maintained (Figure 4—figure supplement 1). Co-phylogenetic analysis revealed congruence between the A and B haplotype trees across different chromosomes (Figure 4B; Figure 4—figure supplements 2 and 3) illustrating that these haplotypes are evolving in parallel.

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Co-phylogenetic analysis.

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The time of emergence of T.b. gambiense Group 1 was determined. We estimated the genome-wide mutation rate using the number of accumulated mutations (both genome-wide and on Chromosome 9) together with the year of isolation for each isolate, using root-to-tip linear regression (Drummond et al., 2003) (Figure 3—source data 1). A rate of 1.82 x 10^–8 substitutions per site per year was estimated, similar to that of the COII-NDI ‘clock’ locus in T. cruzi (Lewis et al., 2011). Given the observed number of accumulated mutations per isolate, the genome size, and our calculated mutation rate, the time since the existence of the most recent common ancestor (MRCA) of the Group 1 isolates analysed in the study is estimated to be in excess of one thousand years before present (Figure 3—source data 1). Similarly, using the mutation rates for two different T. cruzi clock genes (COII-NDI and GPI) (Lewis et al., 2011), the date of emergence was estimated to be between 750 and 9,500 years before present (Figure 3—source data 1).

The theory of clonality in parasitic protozoan populations has been proposed and debated over the last quarter of a century (Tibayrenc and Ayala, 2002; Tibayrenc et al., 1990). To advance our understanding of clonal evolutional, we have undertaken a whole-genome population-level analysis of T.b. gambiense Group 1 focussing on a large number of isolates sampled from two countries, together with a small out-group from a more distant West African country. We provide robust evidence that this important human parasite reproduces exclusively asexually, demonstrating complete genetic linkage across the genome and the absence of allelic segregation. Despite meiosis-specific genes being intact and expressed (Peacock et al., 2014), the population genetic data is incompatible with sexual reproduction, self-fertilisation, aneuploidy (Schurko et al., 2009), a parasexual cycle (Ramírez and Llewellyn, 2014; Forche et al., 2008) or the atypical meiosis observed in Rotifers (Signorovitch et al., 2015). The barrier to sexual reproduction in T.b. gambiense Group 1 remains unclear. The lack of decay of ‘meiosis-associated’ genes may be explained by a number of non-mutually exclusive hypotheses including the relatively recent emergence of this asexual lineage, such that insufficient time has elapsed to allow decay. Alternatively, these genes may perform additional roles in processes such as DNA repair or VSG-related recombination.

Our data indicates the parasite population comprises just two independently evolving haplotypes; this remarkable observation confirms the Meselson effect at a whole-genome level for the first time. Despite being predicted for almost twenty years, empirical evidence for this phenomenon has been elusive. The original report of the Meselson effect focused on Bdelliod rotifers (Welch, 2000), however this was later shown to instead be due to the entirely different phenomenon of cryptic tetraploidy (Mark Welch et al., 2008). Subsequent attempts to demonstrate the Meselson effect have been thwarted, such as in the case of the obligately apomictic crustacean, Daphnia (Omilian et al., 2006). The relatively high rate of mitotic recombination in comparison to the mutation rate results in novel heterozygous sites being eliminated by gene conversion (LOH) events much faster than they are generated and consequently allelic divergence is not observed (Omilian et al., 2006). More recently, in the genome of Timema stick insects, nuclear alleles have been shown to display a higher level of divergence in asexual rather than sexual species (Schwander et al., 2011). In that system, as in T.b. gambiense Group 1, the mitotic recombination rate is sufficiently low so as not to obscure the pattern of accumulating mutations, and this has underpinned our ability to detect and confirm the Meselson effect.

The similarity of the genomes studied from different geographical locations, together with a lack of recombination in the evolution of T.b. gambiense Group 1, suggests this sub-species emerged from a single progenitor. Each clade represents a separate country and these are partitioned by the earliest branches of the phylogenetic tree, implying early radiation and colonisation. The emergence of T.b. gambiense Group 1 within the last 10,000 years coincided with an important period in human history when civilisation and livestock farming were developing in West Africa (Oliver, 1966), but whether asexual reproduction was a prerequisite for human infection or occurred subsequently is uncertain. This successful asexual human pathogen contrasts markedly with the virtually extinct sexual T.b. gambiense Group 2 parasite that occupied similar biological and geographical niches and this may be an example of the dominance of an asexual lineage over its sexual counterpart (Charlesworth, 1980). Another remarkable feature of the T.b. gambiense Group 1 genome is the extensive loss of heterozygosity across large regions of each chromosome, which likely arose from gene conversion/mitotic recombination. Gene conversion provides a mechanism for removing a proportion of deleterious mutations in asexual eukaryotes, as hypothesised in other systems (Tucker et al., 2013). A fitter allele on one haplotype would be positively selected, resulting in long-range tracts of LOH that encompass the loci under selection together with extensive flanking regions. However, it has been predicted that following an LOH event, individuals will experience a slight decline in long-term fitness due to newfound homozygosity featuring pre-existing sub-optimal alleles, leaving a signature equivalent to inbreeding (Tucker et al., 2013) in the progeny. This process has been described as a more powerful evolutionary force than the accumulation of point mutations (Omilian et al., 2006) and thus may drive Muller’s Ratchet more quickly than de novo mutations. This suggests that although LOH may effectively counteract the accumulating mutational load in the short-term, it is unclear whether it can prevent this uniquely well-adapted pathogen from ultimately entering an extinction vortex.

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Author details

1. William Weir

   Wellcome Centre for Molecular Parasitology, College of Medical, Veterinary and Life Sciences, Institute of Biodiversity Animal Health and Comparative Medicine, University of Glasgow, Glasgow, United Kingdom

   Contribution

   WW, performed the genomic and genetic analysis and wrote the paper

   Competing interests

   The authors declare that no competing interests exist.

2. Paul Capewell

   Wellcome Centre for Molecular Parasitology, College of Medical, Veterinary and Life Sciences, Institute of Biodiversity Animal Health and Comparative Medicine, University of Glasgow, Glasgow, United Kingdom

   Contribution

   PC, performed the computational phasing

   Competing interests

   The authors declare that no competing interests exist.

3. Bernardo Foth

   Sanger Institute, Wellcome Trust Genome Campus, Cambridge, United Kingdom

   Contribution

   BF, performed the SNP calling

   Competing interests

   The authors declare that no competing interests exist.

4. Caroline Clucas

   Wellcome Centre for Molecular Parasitology, College of Medical, Veterinary and Life Sciences, Institute of Biodiversity Animal Health and Comparative Medicine, University of Glasgow, Glasgow, United Kingdom

   Contribution

   CC, prepared parasite material and purified DNA

   Competing interests

   The authors declare that no competing interests exist.

5. Andrew Pountain

   Wellcome Centre for Molecular Parasitology, College of Medical, Veterinary and Life Sciences, Institute of Biodiversity Animal Health and Comparative Medicine, University of Glasgow, Glasgow, United Kingdom

   Contribution

   AP, was involved in LOH analysis

   Competing interests

   The authors declare that no competing interests exist.

6. Pieter Steketee

   Wellcome Centre for Molecular Parasitology, College of Medical, Veterinary and Life Sciences, Institute of Biodiversity Animal Health and Comparative Medicine, University of Glasgow, Glasgow, United Kingdom

   Contribution

   PS, was involved in LOH analysis

   Competing interests

   The authors declare that no competing interests exist.

7. Nicola Veitch

   West Medical Building, Office 350, University of Glasgow, Glasgow, Scotland

   Contribution

   NV, prepared parasite material and purified DNA

   Competing interests

   The authors declare that no competing interests exist.

8. Mathurin Koffi

   UFR Environnement, Laboratoire des Interactions Hôte-Microorganismes-Environnement et Evolution (LIHME), Université Jean Lorougnon GUEDE, Daloa, Côte d'Ivoire

   Contribution

   MK, Co-ordinated and performed field sampling and prepared parasite DNA

   Competing interests

   The authors declare that no competing interests exist.

   Additional information

   Member of TrypanoGEN, part of the H3Africa consortium

9. Thierry De Meeûs

   1. Institut de Recherche pour le Développement, Campus International de Baillarguet, Montpellier, France
   2. Centre International de Recherche-Développement de l’Elevage en zone Subhumide, Bobo-Dioulasso, Burkina Faso

   Contribution

   TDM, provided expert support in analysing an interpreting the data

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0001-8807-241X

10. Jacques Kaboré

    1. Centre International de Recherche-Développement de l’Elevage en zone Subhumide, Bobo-Dioulasso, Burkina Faso
    2. Université Polytechnique de Bobo-Dioulasso, UFR Sciences et Techniques, Bobo-Dioulasso, Burkina Faso

    Contribution

    JK, Co-ordinated and performed field sampling and prepared parasite DNA

    Competing interests

    The authors declare that no competing interests exist.

    Additional information

    Member of TrypanoGEN, part of the H3Africa consortium

11. Mamadou Camara

    Programme National de Lutte contre la Trypanosomiase Humaine Africaine, Conakry, Guinea

    Contribution

    MC, Co-ordinated and performed field sampling and prepared parasite DNA

    Competing interests

    The authors declare that no competing interests exist.

    Additional information

    Member of TrypanoGEN, part of the H3Africa consortium

12. Anneli Cooper

    Wellcome Centre for Molecular Parasitology, College of Medical, Veterinary and Life Sciences, Institute of Biodiversity Animal Health and Comparative Medicine, University of Glasgow, Glasgow, United Kingdom

    Contribution

    AC, was involved in LOH analysis

    Competing interests

    The authors declare that no competing interests exist.

13. Andy Tait

    Wellcome Centre for Molecular Parasitology, College of Medical, Veterinary and Life Sciences, Institute of Biodiversity Animal Health and Comparative Medicine, University of Glasgow, Glasgow, United Kingdom

    Contribution

    AT, wrote the paper

    Competing interests

    The authors declare that no competing interests exist.

14. Vincent Jamonneau

    1. Institut de Recherche pour le Développement, Campus International de Baillarguet, Montpellier, France
    2. Centre International de Recherche-Développement de l’Elevage en zone Subhumide, Bobo-Dioulasso, Burkina Faso

    Contribution

    VJ, Co-ordinated and performed field sampling and prepared parasite DNA

    Competing interests

    The authors declare that no competing interests exist.

    Additional information

    Member of TrypanoGEN, part of the H3Africa consortium

15. Bruno Bucheton

    1. Institut de Recherche pour le Développement, Campus International de Baillarguet, Montpellier, France
    2. Programme National de Lutte contre la Trypanosomiase Humaine Africaine, Conakry, Guinea

    Contribution

    BB, Co-ordinated and performed field sampling and prepared parasite DNA

    Competing interests

    The authors declare that no competing interests exist.

    Additional information

    Member of TrypanoGEN, part of the H3Africa consortium

16. Matt Berriman

    Sanger Institute, Wellcome Trust Genome Campus, Cambridge, United Kingdom

    Contribution

    MB, supervised the sequencing

    Competing interests

    The authors declare that no competing interests exist.

    "This ORCID iD identifies the author of this article:" 0000-0002-9581-0377

17. Annette MacLeod

    Wellcome Centre for Molecular Parasitology, College of Medical, Veterinary and Life Sciences, Institute of Biodiversity Animal Health and Comparative Medicine, University of Glasgow, Glasgow, United Kingdom

    Contribution

    AMACL, Conceived the study and wrote the paper

    For correspondence

    annette.macleod@glasgow.ac.uk

    Competing interests

    The authors declare that no competing interests exist.

    Additional information

    Member of TrypanoGEN, part of the H3Africa consortium

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