Admixture and recombination among Toxoplasma gondii
lineages explain global genome diversity
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Citation
Minot, S. et al. “Admixture and Recombination Among
Toxoplasma Gondii Lineages Explain Global Genome Diversity.”
Proceedings of the National Academy of Sciences 109.33
(2012): 13458–13463. CrossRef. Web.
As Published
http://dx.doi.org/10.1073/pnas.1117047109
Publisher
National Academy of Sciences (U.S.)
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Final published version
Accessed
Wed May 25 18:52:58 EDT 2016
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http://hdl.handle.net/1721.1/77588
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Detailed Terms
Admixture and recombination among Toxoplasma
gondii lineages explain global genome diversity
Samuel Minota,b,1, Mariane B. Meloa,1, Fugen Lia, Diana Lua, Wendy Niedelmana, Stuart S. Levinea,
and Jeroen P. J. Saeija,2
a
Biology Department, Massachusetts Institute of Technology, Cambridge, MA 02139; and bDepartment of Microbiology, University of Pennsylvania Perelman
School of Medicine, Philadelphia, PA 19104
Edited by Louis H. Miller, National Institutes of Health, Rockville, MD, and approved July 10, 2012 (received for review October 19, 2011)
Toxoplasma gondii is a highly successful protozoan parasite that
infects all warm-blooded animals and causes severe disease in
immunocompromised and immune-naïve humans. It has an unusual global population structure: In North America and Europe,
isolated strains fall predominantly into four largely clonal lineages,
but in South America there is great genetic diversity and the North
American clonal lineages are rarely found. Genetic variation between Toxoplasma strains determines differences in virulence,
modulation of host-signaling pathways, growth, dissemination,
and disease severity in mice and likely in humans. Most studies
on Toxoplasma genetic variation have focused on either a few loci
in many strains or low-resolution genome analysis of three clonal
lineages. We use whole-genome sequencing to identify a large
number of SNPs between 10 Toxoplasma strains from Europe
and North and South America. These were used to identify haplotype blocks (genomic regions) shared between strains and construct a Toxoplasma haplotype map. Additional SNP analysis of
RNA-sequencing data of 26 Toxoplasma strains, representing
global diversity, allowed us to construct a comprehensive genealogy
for Toxoplasma gondii that incorporates sexual recombination. These
data show that most current isolates are recent recombinants and
cannot be easily grouped into a limited number of haplogroups. A
complex picture emerges in which some genomic regions have not
been recently exchanged between any strains, and others recently
spread from one strain to many others.
evolution
| hapmap | pathogen | selection
T
he protozoan, Toxoplasma gondii, a highly prevalent parasite
of warm-blooded animals including humans, is an excellent
model to study sexual recombination and its effects on the
population structure of eukaryotic unicellular pathogens. It has
two distinct reproductive mechanisms: (i) asexual reproduction in
intermediate hosts, in which tissue cysts containing haploid parasites infect other hosts through carnivorism and omnivorism;
and (ii) sexual recombination in felines, the definitive host. The
latter is more productive—an infected feline sheds millions of
extremely stable, infectious oocysts (1). Because a single parasite
can produce both micro- and macrogametes and self-mate, novel
recombined genotypes only form in the rare event that a feline is
infected simultaneously or in rapid succession with multiple strains
(2). When coinfection does occur, myriad new, genetically distinct
organisms are generated. The new genotypes with the greatest
fitness can then be rapidly expanded by self-mating in felines (3).
Initial analyses showed that the majority of strains isolated in
Europe (E) and North America (NA) belong to three distinct
clonal haplotypes, types I, II, and III (hereafter called the clonal
lineages) (4). Genetic variation between these lineages ranges
from 1 to 3%, and variation within a lineage is very low (<0.01%),
suggesting recent expansion (5). A fourth clonal lineage prevalent in NA wild animals, haplogroup (HG) 12, was recently described (6). Strains with atypical or novel allele combinations
have also been isolated from patients with unusual clinical presentations (7) or on other continents, particularly South America
(SA) (8). The causes of these geographically confined population
13458–13463 | PNAS | August 14, 2012 | vol. 109 | no. 33
structures are unknown. Because some (usually SA) strains can
cause severe disease even in immunocompetent humans (7),
identifying the factors affecting the movement and spread of
certain strains is of the utmost importance.
Evidence supports a model of infrequent sexual recombination
followed by strong selective sweeps. Genome-wide SNP comparison of clonal-lineage strains determined that the ancestor of
type II (II*) crossed with ancestral strains α and β ∼10,000 y ago
to generate types I and III, respectively (9–11). Types I and III
are frequently isolated in E and NA, suggesting that they have
spread rapidly since their recent creation. It is unclear why types
I and III and no other progeny were so prolific, but a likely explanation is that recombination brought together a specific combination of alleles that provided them with a selective advantage
(12). The clonal lineages share a common chromosome Ia (chrIa)
(9, 13) and a small segment of the distal part of chrXI (9), suggesting these regions might be especially advantageous. Although
the genetic determinants remain elusive, we know the clonal lineages are extremely successful, albeit limited to E and NA. Surprisingly, many SA isolates carry a chrIa nearly identical to that of
the clonal lineages (8, 11).
More recently, SNP analysis of eight introns in five genes from
many—including atypical and SA—isolates sorted all strains into
14 major HGs, with the clonal lineages designated haplotypes I,
II, and III and atypical strains distributed among HGs 4 through
14 (11). Further analyses indicated that all 14 HGs may have
derived from admixture of 6 ancestral stains (11). These 14 HGs
have distinct geographical distributions: I–III, 7, 11, and 12 are
mainly confined to E and NA; 4, 5, 8, 9, and 10 to SA; and 13 to
Asia (14). HG6 is found worldwide (11).
A drawback of the previous analyses that led to the current
evolutionary model is its reliance on sequence information of
only five loci. Moreover, the sequences were concatenated and
analyzed as if they share a single evolutionary history. However,
Toxoplasma undergoes sexual recombination, so different loci may
have completely different evolutionary histories (15). We argue
that evolutionary analysis of Toxoplasma (or any sexual organism)
can only be achieved by sequencing a large number of loci, determining which loci have a shared recombinational history, then
estimating the evolutionary history of each of those haplotype
blocks independently. Analysis of haplotype blocks is greatly aided
by the fact that Toxoplasma only replicates as a haploid.
Author contributions: S.M. and J.P.J.S. designed research; S.M., M.B.M., D.L., and W.N.
performed research; S.M. and F.L. contributed new reagents/analytic tools; S.M., M.B.M.,
F.L., S.S.L., and J.P.J.S. analyzed data; and S.M., M.B.M., D.L., and J.P.J.S. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS direct submission.
Data deposition: The sequence reported in this paper has been deposited in Short Read
Archive, http://www.ncbi.nlm.nih.gov/sra (accession nos. SRA047110 and SRA050293).
1
S.M. and M.B.M. contributed equally to this work.
2
To whom correspondence should be addressed. E-mail: jsaeij@mit.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1117047109/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1117047109
Results
Pairwise Strain Comparisons of SNP Frequencies Identify Haplotype
Blocks. Recombination between any two strains results in progeny
whose genomes are a mosaic of the parental genomes. We
obtained the genome sequences of seven Toxoplasma strains
representing HGs 4 through 11, except 7, and public genome
data for the clonal lineages [Toxodb.org (16)]. To identify genomic regions with RCA, we made genomewide relative SNP
rate plots for the 45 pairwise comparisons between the 10 strains.
We will focus our initial analysis on the comparisons between
strains that have previously been suggested to be related to each
other based on the haplogroup mixture model (8) (Fig. 1). These
SNP rate plots show distinct SNP patterning on each chromosome that demarcates regions (Dataset S1 A–D) shared between
certain strains (Fig. S1 A–D). For example, it has been suggested
that a cross between type II* and β (HG9-like) created the type
III lineage (Fig. 1C) (9, 11). If this were the case, one would
expect large regions in the type III genome to be very similar to
either type II or HG9. From the pairwise SNP rates between
ME49 (type II), P89 (HG9), and VEG (type III) on chrVIII, for
example, we see it is clearly divided into three distinct regions
(Fig. 2). For region 1, types II and III are almost identical. For
region 3, types III and 9 are almost identical. Region 2 has a high
number of SNPs between types II, III, and 9. Although regions 1
and 3 are consistent with the model of mating shown in Fig. 1C,
region 2 is not. The SNP rate between types II and 9 is similar for
all three regions of chrVIII. Across all 14 chromosomes (Fig. S1C),
42% of type III was highly similar to type II and 29% to HG9.
Thus, at a low resolution it might indeed appear that type III arose
from a cross between II* and β (9). However, our high-resolution
whole-genome analysis shows that 29% of the type III genome is
neither similar to II* nor β, establishing that the parents of type III
were significantly dissimilar to the modern strains II and 9.
We similarly analyzed other strain “families” as defined by the
haplotype mixture model (8): crosses A (9 × 4), B (II x 6), and D
(9 × 6) (Fig. 1). Although the types I and III and HG8 genomes
are largely derived from strains that are closely related to the
A
P89 (9) x MAS (4)
0.23 0.37
TgCatBr5 (8)
B
ME49 (II) x BOF (6)
0.11 0.49
GT1 (I)
C
ME49 (II) x P89 (9)
0.42 0.29
VEG (III)
D
P89 (9) x BOF (6)
0.0 0.0
GUY-KOE (5)
Fig. 1. Schematic of the four Toxoplasma crosses investigated in this study.
Shown are four crosses (A–D) between strains (ME49, MAS, BOF, and P89)
from four ancestral HGs (HGII, HG4, HG6, and HG9) that might have led to
the creation of strains (TgCatBr5, GT1, VEG, and GUY-KOE) from HG8
(cross A), HGI (cross B), HGIII (cross C), and HG5 (cross D), respectively (9,
13). The fraction of the genome of TgCatBr5, GT1, VEG, and GUY-KOE that
was almost identical to the putative parental strains is indicated below
those strains.
Minot et al.
strains in Fig. 1, 30–40% of their genomes resemble neither
parental strain. This could indicate that they are not the true
ancestral parents and/or that types I and III and HG8 were
generated by more than one recent cross. Of note, GUY-KOE
(HG5) did not have a single region in its genome that was very
similar to either HG6 or HG9 (Fig. S1D), indicating that it was
not derived from admixture between ancestral lineages similar to
these HGs, as previously proposed (8).
From the other pairwise comparisons, we identified many genomic regions that were nearly identical between strains, suggesting they share an RCA. We thus divided the Toxoplasma
genome into 131 haplotype blocks containing evidence of RCA
between at least two strains based on boundaries identified from
all 45 possible pairwise SNP plot analyses (Dataset S2A). For
some haploblocks, no strain was similar (similar defined as SNP
rate < 2.5 × 10−4) to any other strain, indicating that all 10 strains
have distinct ancestries (“unexchanged” in Dataset S2A). Our
analysis focuses on haploblocks with evidence of extremely recent ancestry: pairwise SNP rates < 2.5 × 10−4, corresponding
to ≥10-fold decrease in SNP rate compared with other genomic
regions. Thus, our results, based on a genome-wide SNP dataset,
establish that greater than six distinct ancestries are necessary to
explain the relationship between these 10 HGs. Furthermore, the
almost complete absence of SNPs in haploblocks shared among
strains suggests that recent recombination events created most of
these strains. Only the CASTELLS, GUY-KOE, and COUGAR
genomes did not show evidence of RCA with any other strains.
Construction of a Toxoplasma Haplotype Map. The 10 Toxoplasma
genomes provided a high-resolution SNP map that precisely
defined haploblocks, but more strains were needed to draw firm
conclusions about their ancestry. We therefore also identified
SNPs in genome-wide RNA-sequencing data of 26 Toxoplasma
strains representing 12 of the 14 currently defined HGs. We
identified 606,742 SNPs; 132,744 of which contained biallelic
sequence information for all 26 strains. These SNPs were used to
identify RCA (shared haploblocks) among strains as described
above. Adding the RNA-seq data increased the number of haploblocks from 131 to 157 (Dataset S2B). For example, CAST
(HG7) has genomic regions showing RCA with strains from
types I (RH/GT1) and HG6 (BOF/FOU/GPHT) (e.g., chrVIIb,
Dataset S2B and Fig. S2); TgCatBr44 (HG4) has genomic
regions showing RCA with TgCatBr5 (HG8), MAS (HG4), and
type II (ME49/Pru/DEG) (e.g., chrVIII, Dataset S2B and Fig.
S2). B73 seems to be an F1 progeny from a cross between types
II and III; chrV, VI, and VIIa are derived from type III, and the
rest of its genome is from type II (Dataset S2B and Fig. S2).
Similarly, B41 (HG12) seems to be an F1 progeny from a type II
and RAY/WTD3 (HG12) cross, as it is comprised of haploblocks
almost identical to either type II or RAY/WTD3 (Dataset S2B
and Fig. S2). It also becomes clear that many strains previously
defined as being in the same HG are actually highly divergent.
The best examples are HG5 (GUY-KOE/GUY-MAT/RUB) and
HG10 (VAND/GUY-DOS), which do not share RCA for any
haploblock (note the high SNP rate in Fig. S2 and see Dataset
S4A). Strains from HG4 (MAS/CASTELLS/TgCatBr44) also
have distinct ancestries for many haploblocks. On the other
hand, types I (GT1/RH), II (ME49/Pru/DEG), and III (VEG/
CEP), HG6 (BOF/FOU/GPHT), and HG12 (RAY/WTD3 but
not B41) show recent common ancestry for all 157 haploblocks,
suggesting they recently decended from a common progenitor.
Many strains had very recent common ancestry for chrIa, the
right end of chrXI, and parts of chrXII. In particular, chrIa is
highly similar between many strains, as previously observed (8, 11).
If these haploblocks were transferred from a single source to
many different lineages, it suggests a selective sweep driven by
genetic determinants at those loci. Therefore, it is important to
identify their exact genealogy, whether they have strong fitness
PNAS | August 14, 2012 | vol. 109 | no. 33 | 13459
POPULATION
BIOLOGY
We used whole-genome SNP analysis of 26 Toxoplasma strains,
representing global diversity, to construct a haplotype map and
determine phylogeny for all haplotype blocks independently. Our
results show that the observed extent of Toxoplasma genetic diversity cannot be explained by the mating of four to six ancestral
strains, nor do most strains fit into the 14 proposed haplogroups.
Instead, most strains appear to have formed through recent recombination events. Some of these recombinations likely led to
particularly fit genotypes that geographically spread or swept.
Particularly, three haploblocks, on chrIa, XI, and XII, have recent common ancestry (RCA) across multiple diverse lineages,
perhaps suggesting that these regions may contain fitness loci.
Our approach improves upon current methods because it analyzes recombination-defined haploblocks, building a more precise and comprehensive genealogy of Toxoplasma.
SNPs/50,000
0.004 0.008
ME49 (II) vs P89 (9)
ME49 (II) vs VEG (III)
P89 (9) vs VEG (III)
2
3
0
1
0
2
4
Position on chr VIII
6
Fig. 2. Genomewide strain comparisons of SNPs identify regions with recent common ancestry. Nonexon SNPs between two distinct strains were
identified for 50-kb bins along T. gondii chromosome VIII and the relative
SNP rate was plotted (5-kb step size). The chromosome position in Mb (x axis)
and SNPs/50 kb (y axis) are shown. Numbered arrows indicate regions with
distinct pairwise SNP rates.
effects, and if so, the genes that are responsible. One of the primary difficulties in analyzing recombination among environmental
isolates is that parentage is ambiguous. Thus, we must limit the
conclusions drawn from such a dataset. We anchor our analysis in
the genomic regions that are dissimilar between all sequenced
strains. Their low similarity suggests they are derived from strains
existing independently before the recent recombinations under
investigation. We therefore refer to them as “unexchanged.” Using the phylogenetic topology derived from these regions as
a basal genealogy, we compare the topology of the trees derived
from haploblocks containing RCA to infer the ancestry of intermediate strains. For simplicity, we refer to the “directionality
of transfer” of a given genomic region, disregarding true parentage
and identity. Our approach is as follows: suppose 18 hypothetical
strains (A–R) fall into two groups, NA-like and SA-like (Fig. 3A).
For one region of the genome (tree 2), strains F and J have very
low SNP rates and the rest of the genomes (tree 1) are dissimilar.
To distinguish the directionality of transfer for that locus, we
construct a phylogenetic tree for these two haploblocks. If the
exchanged locus were derived from a J-like ancestor, we expect F
to group with J (the red branch). Alternatively, if the locus were
derived from an F-like ancestor, we expect J to group with F (the
green branch). For each haploblock, SNP frequencies were used
to calculate the pairwise distance between strains and construct
neighbor-joining phylogenetic trees (Fig. S2). For most trees there
are two major clades, one containing the NA strains (type II,
HG12, and COUGAR) and the other containing all other
strains, many of which are from SA. Thus, COUGAR and HG12
are sister taxa of type II (Fig. S2) and an early separation and
diversification occurred between NA and SA ancestral strains.
Our data recapitulate the striking biallelism for most Toxoplasma genes observed in multiple studies (9, 12).
Our basal topology approach allows us to infer directionality
of transfer by comparing different haploblocks with multiple
outgroups. For example, chrIb_7 (Dataset S2B) falls into the
unexchanged group, and chrXI_1 represents a haploblock shared
between types II and III. The strains with topological differences
between these two haploblocks are underlined (Fig. 3B). One can
infer that a type II–like strain was the source of region chrXI_1 for
CEP and VEG (type III) because COUGAR and HG12 cluster
basally to II–III. A tree consisting of merely type II, CEP, and
VEG would be ambiguous with regards to directionality, but the
inclusion of multiple outgroups gives us enough information to
make inferences about the directionality of genetic transfer between strains. Although types I and III are NA strains, their
genomes are of mixed ancestry. Portions of their genomes derive
from an NA type II–like strain while other parts derive from SA
strains (e.g., P89-like and BOF-like, Fig. 1). ChrIa_1 (Fig. 3C),
chrIa_4 (Fig. S2), and chrXI_7 (Fig. 3D) that have RCA among
many strains have HG12 and COUGAR as an outgroup and
13460 | www.pnas.org/cgi/doi/10.1073/pnas.1117047109
cluster most closely with type II. The fact that the unexchanged
topology of type II, COUGAR, and HG12 is preserved suggests
that these conserved haploblocks were most likely derived from
a type II–like ancestral strain. The alternate explanation, that the
unexchanged topology of type II, COUGAR, and HG12 was
recreated through some other set of matings is extremely unlikely. In contrast, the chrXII haploblocks with RCA among
many strains have SA strains as an outgroup (e.g., Fig. 3E,
chrXII_4) and were therefore most likely derived from an SA
ancestral strain. In other words, the SA strain topology was preserved following the exchange of this region, suggesting that an SA
strain was the source. However, the lack of a clear outgroup for
most of the SA strains precludes identification of a single ancestral strain. Similarly, chrIa_2 (Fig. 3C) and chrIa_3 (Fig. S2)
have no outgroup, so no definitive ancestry can be concluded.
However, because chrIa_1 and chrIa_4 are derived from a type
II–like strain it is reasonable to propose that these were also
derived from the same type II–like strain. We performed similar
analyses to determine the ancestry of most haploblocks from
the Toxoplasma haplotype map (Dataset S2B).
We further determined the recombination-independent relationship between strains by assembling the 35kb genome of the
Toxoplasma apicoplast (a maternally inherited organelle), identifying polymorphisms between strains, and constructing a phylogenetic tree (Fig. 3F). These data show that VEG, GT1, and
TgCatBr5 inherited their apicoplast from P89-like, ME49-like,
and MAS-like strains (Fig. 1). The fact that the MAS/TgCatBr5
strains clustered close to type II, with COUGAR as an outgroup,
provides some evidence that MAS not only inherited chrIa from
a type II–like ancestor, but also its apicoplast, which it passed
to TgCatBr5.
Crosses That Created Most Current Strains Happened Around the
Same Time. The SNP rate in haploblocks shared between two
strains is related to the time of the most recent common ancestor
(TMRCA). However, without knowing the Toxoplasma mutation
rate, it is difficult to translate SNP rate into years. We restricted
analysis to SNPs in noncoding sequences from our DNA shotgun
sequencing data, which are less likely to be subject to positive or
diversifying selection and therefore evolve in a more clocklike
manner. The average SNP rate for haploblocks that have RCA
between different strains is low (<1 × 10−4) but variable (∼15-fold)
(Table S1), which suggests that the crosses that produced them
happened relatively recently but at different times. For comparison with published TMRCA, using an intronic SNP rate of
1.94 × 10−8 (10) and the SNP rate from our data (multiplied with
a conversion factor of 2.55, which accounts for the fact that our
SNP rate is lower than the real SNP rate due to incomplete
genome coverage) the 6.9 × 10−5 SNP rate for haploblocks with
RCA between types I and II corresponds to a TMRCA of ∼9 ×
103 y, in concurrence with other studies (10, 11). The different
SNP rates of the haploblocks shared between types II and III
suggest they were transferred from a type II–like ancestor to type
III two separate times (Table S1). Pairwise SNP rates for unexchanged haploblocks (Dataset S2A and Table S2) are higher
than those for exchanged regions by ∼60-fold. Therefore, whatever the absolute timescale, the evolutionary histories of different haploblocks clearly vary by one to two orders of magnitude.
Identification of Highly Polymorphic Genes and Genes Undergoing
Positive Selection. Many strains had very recent common ancestry
for haploblocks chrIa_1_2_3_4, chrXI_7, and chrXII_2_4 (Fig.
S2 and Dataset S2B), suggesting genes in these regions might
confer a selective advantage. We focused on regions chrIa_2,
chrXI_7, and chrXII_2_4, as they introgressed into the most
strains. Using the annotated ME49 genome (www.toxodb.org),
we identified for each strain (for which we had whole-genome
sequence data) which SNPs were in coding regions and if they
Minot et al.
0 . 00 0 8
0 . 00 0 4
100
98
100
2
91
76
97
P 89
FOU
B OF
G PH T
TgCATBr44
TgCATBr5
GUYMAT
VAND
G U YDO S
GUYKOE
RUB
C AST
C A S TE L L S
MA S
GT1
RHdelta
RAY
W TD3
M E 4 9 .1
B 41
ME 4 9
CEPdelta
B7 3
VEG
D EG
PRUdelta
C E P d e lt a
VEG
CO U G A R
GUYKOE
P8 9
VAND
G U Y DO S
TgCATBr5
GUYMAT
RUB
SA origin
RAY
W TD 3
VE G
ME 49. 1
M E 49
C E Pd e l t a
B 41
B7 3
F OU
RHdelta
GT1
BO F
GP H T
D EG
PRUdelta
GU YD OS
RUB
C AS T
GUYMAT
GUYKOE
VAND
C A S T E L LS
chrXI_7
M AS
TgCATBr44
P8 9
TgCATBr5
C A ST E L LS
COUGAR
GUYMAT
RUB
VAND
G UY D OS
GUYKOE
F
NA origin
SNP rate
4e-4
6e-4
2e-4 4e-4 6e-4 8e-4
0
RAY
W TD 3
D EG
M E49. 1
VE G
TgCATBr5
TgCATBr44
R H d e lta
PRUdelta
P8 9
M E4 9
M AS
GT1
GPHT
FO U
C E Pd e l t a
C A ST
BO F
B4 1
B73
CA ST EL L S
VAND
G PHT
B OF
F OU
RAY
W T D3
PRUdelta
ME49.1
B41
ME49
B73
C E P d e lta
VE G
P89
M AS
TgCATBr44
TgCATBr5
D EG
R H d e lta
CA ST
GT1
0.0012
NA origin
chrXII_4
D
chrIa_2
SA origin
0
2e-4
C O U G AR
SA origin
C A ST EL L S
MA S
TgCATBr5
R H d e lta
C A ST
G T1
G P HT
BO F
FOU
P8 9
TgCATBr44
CEPdelta
VE G
GUYKOE
GUYMAT
VAND
GUYDOS
RUB
C O U G AR
RAY
WT D 3
DEG
PRUdelta
M E 4 9 .1
ME49
B4 1
B 73
0
2e-4
JF
chrIa_1
SA origin
C AS TE L L S
MA S
TgCATBr44
C A ST
I
0
E
6e-4
4e-4
C D EF
NA origin
KLM
NA origin
NA II-like to III
chrXI_1
TgCATBr5 to TgCATBr44
G PH T
B OF
F OU
G T1
R H d e l ta
0
C DEF
N OPQR
H
KLM
I J
NA F-like to J B
?
SA J-like to F
SA origin
chrIb_7
Unexchanged
0
H
SNP rate
2e-4 4e-4 6e-4 8e-4
C OUGA R
G
N OPQR A
NA origin
DEG
PRUdelta
M E 4 9 .1
B73
B 41
ME 4 9
RAY
W TD 3
G
A
B
SA origin
2
B
C
NA origin
C O U G AR
SA origin
1
2e-4
SNP rate
4e-4 6e-4
NA origin
GUYMAT
RUB
G U YD O S
GUYKOE
A
MAS (4)
TgCatbr5 (8)
ME49 (II)
GT1 (I)
COUGAR (11)
CASTELLS (4)
GUY-KOE (5)
BOF (6)
VEG (III)
100 P89 (9)
Fig. 3. Phylogenetic analysis of haplotype blocks. (A) Two phylogenetic trees (1, 2) showing the relatedness for a group of 18 hypothetical strains (A–R) for
distinct genomic regions. For most of the genome (tree 1), strains F and J are highly divergent, but for a particular haploblock (tree 2), they have very low rates
of SNP diversity. To distinguish the directionality of transfer for that haploblock, a phylogenetic tree showing relatedness among strains is constructed. If the
exchanged locus was derived from a J-like ancestor, we would expect the red tree, where both strains have the position of strain J in tree 2. Alternatively, if
the locus was from an F-like ancestor, we would expect the green tree, where both strains are found in the F position. For each of the 157 haplotype blocks
identified (Dataset S2B), phylogenetic trees were constructed based on the pairwise distance between strains (calculated from SNP frequencies) (Fig. S2). The y
axis indicates relative SNP rate. (B) Pairwise distance tree for haploblock chrIb_7 and haploblock chrXI_1. Because of the low similarity among strains [except
among strains from types I (RH, GT1), II (DEG, PRU, ME49), III (CEP, VEG), and HG12 (RAY, WTD3) lineages], we conclude that haploblock chrIb_7 is derived
from different strains that existed before the recent recombinations here investigated. For haploblock chrXI_1, the position of the type III strains (CEP and
VEG) and of TgCatBr44 has shifted, and for this haploblock, the SNP rate between types II and III strains and between TgCatBr44 and is very low. Colors match
haploblocks as indicated in Dataset S2B. ME49.1 represents the ME49 genome data and ME49 represents the RNA-seq data. (C–E) Pairwise distance trees were
constructed as described for the indicated regions. (F) Phylogenetic tree of the apicoplast sequence constructed using neighbor joining. Numbers at branches
are bootstrap values (1,000 replicates). Branch lengths indicate number of nucleotide differences.
Minot et al.
proteins with a signal peptide (P = 0.0005 and P = 4.8 × 10−7,
respectively, hypergeometric distribution). A list of genes that
are most polymorphic and divergent (and have ≥4 SNPs), have
a signal peptide and are highly expressed (>90th percentile,
toxodb.org) in at least one life stage, is shown in Table S4. This
table contains four rhoptry protein kinases, two of which, ROP16
and ROP18, are known to be involved in mediating strain-specific differences in virulence (20, 21). ROP17 and ROP39 have
all residues important for kinase activity (22) but their function is
unknown. In addition, Table S4 contains three genes: Tgd057,
GRA6, and GRA4, which encode proteins with H-2K(b) (Tgd057)
and H-2L(d) (GRA4 and GRA6) T-cell epitopes (23, 24). The
other genes in this list should also encode proteins that are
good candidates for either direct host-cell modulation or
strong antigenicity.
Discussion
Here we present a comprehensive representation of global Toxoplasma diversity based on whole genome analysis. Our genomewide SNP data were used to make a haplotype map from which
we inferred the ancestry of each haploblock. It is interesting to
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BIOLOGY
were synonymous (S) or nonsynonymous (NS) (Dataset S3) and
selected the top five most polymorphic genes as candidates for
conferring selective advantage (Table S3). For region
chrXII_2_4, a promising candidate is the ROP5 cluster of
pseudokinases recently shown to determine strain differences in
virulence (17, 18). This cluster is present on chrXII_4, a region
that is highly similar between types I, III, HG6, CAST, P89,
TgCatBr44, and TgCatBr5 (Fig. 3E). Further, GT1 and VEG
contain the virulent ROP5 alleles (17, 18). The chrIa_2 region,
which is highly similar between 19 of the 26 strains (Fig. 3C),
only contains 17 predicted genes, most of unknown function. For
chrXI_7, a potential candidate is Tgd057, recently shown to contain an H-2K(b) CD8+ T-cell epitope (19).
We also identified polymorphic genes under positive selection
for the whole genome. From the 45 pairwise SNP comparisons,
we focused on genes with the highest average SNPs/kb and the
highest NS/S rate in the greatest number of pairwise comparisons. We excluded duplicated genes, which have a high rate of
false positive SNP calls. Polymorphic genes (≥10 times ranked in
top 200 polymorphic genes for all 45 pairwise strain comparisons)
and divergent genes (NS/S > 2) were enriched for genes encoding
note that haploblocks recently derived from a type II–like strain
are present in 18 of the 25 other strains for which we have
genomewide SNP data. The only strains not containing type II–
like haploblocks with low SNP rates were CASTELLS and the
strains isolated in a Guianese rainforest GUY-KOE/GUYMAT/GUY-DOS/VAND and RUB (7). In such a remote area,
one can speculate that these Guianese strains have not had recent contact with NA type II–like strains. Previous analysis of five
concatenated loci indicated that the COUGAR strain was particularly divergent compared with all others and it was estimated
that the TMRCA with the other strains was ∼10 Mya (8). Our
sequencing results, however, indicate that COUGAR clustered
most closely with the type II strains and never clustered basally to
all other strains. In fact, COUGAR and ME49 are as similar as
MAS is to CASTELLS, two strains that are currently placed in
the same HG. Our data do not support the model that most
current strains are admixtures of four to six ancestral strains, as
large regions of the genomes of all supposedly admixed strains
are very different from those of the ancestral strains (Fig. 3B and
Dataset S2B). This illustrates the importance of whole-genome
analysis on studying evolution in sexual organisms, as the genomic
location of strain-typing markers greatly affects the interpretation
of evolutionary relationships.
We identified ≥106 SNPs which may determine strain-specific
phenotypes. Indeed, some of the most polymorphic genes we
identified were recently described to be important for determining
strain-specific differences in virulence (ROP18 and ROP16)
(25, 26), modulation of host cell signaling (ROP16) (20), and
interaction with the host adaptive immune response (GRA4,
GRA6, and Tgd057) (23, 24). We expect that future wholegenome sequencing will enable association studies between HGs
(or even specific SNPs) and phenotypes. With the exceptional tools
available for gene manipulation in Toxoplasma, high-throughput
testing of candidate genes (Tables S3 and S4) responsible for these
phenotypes may soon uncover an unprecedented wealth of information about Toxoplasma proliferation and fitness.
We next propose a model to explain Toxoplasma’s sexual history
and current geodiversity. Toxoplasma has an unusual global population distribution. Type II, followed by HG12, III, and I strains,
are the dominant clonotypes in NA and E, whereas clonality is
largely absent in SA. We address this transcontinental disparity
by using our genomewide pairwise SNP rates and haplotype map.
It was proposed that Toxoplasma originated in NA and was
concurrently introduced to SA with Felidae members following
reconnection of the Panamanian land bridge (∼2.5 Mya), after
which NA and SA strains diverged (8). Our data corroborate this
model: NA type II, HG12 (RAY/WTD3), and HG11 (COUGAR)
mostly form a separate cluster from SA strains, except for the
haploblocks that were originally derived from a type II–like
strain and ended up in many SA strains (such as chrIa).
It has been suggested that the greater diversity of SA Toxoplasma strains might be related to the greater diversity of SA
Felidae species (8). However, until the late Pleistocene extinction (∼11,500 y ago), a variety of Felidae species existed in NA as
well. We instead propose that this massive extinction event,
which eliminated ∼80% of large vertebrates in NA, including the
NA cheetah, the American lion, and the saber-toothed tigers
(Smilodon and Homotherium) (27), could have extirpated most
Felidae members in NA, causing a severe bottleneck and the
demise of most NA Toxoplasma strains except type II, HG11,
and HG12. This might explain the limited diversity that is currently present in NA. Furthermore, the limited availability of its
definitive host in NA might have led to adaptations in NA strains,
such as their reduced virulence (8), enhanced cyst formation, and
greater oral infectivity (28), to enhance propagation between
intermediate hosts. Recent NA repopulation with SA Felidae
migrants could have reintroduced some SA Toxoplasma strains
to NA. Molecular genetic analysis of mountain lion (Puma
13462 | www.pnas.org/cgi/doi/10.1073/pnas.1117047109
concolor) DNA indicates that the current NA population is most
likely derived from a population that migrated into SA ∼2.5 Mya
and subsequently returned northward more recently, ∼10,000 y
ago (29). Coincident with this remigration of SA Felidae to NA,
a sudden burst of outcrossings could have occurred in which
a NA type II–like strain and SA strains similar to HG9 and HG6
crossed to create types I and III, and HG6. The type I, type III,
and HG6 lineages inherited the region containing the virulent
ROP5 gene cluster (chrXII_4) from the SA strain, and chrIa_2
and chrXI_7 from the type II–like strain, which together might
have conferred a strong selective advantage. Recent global trade
and the spread of the domestic cat probably contributed to further
Toxoplasma dissemination and could account for the rare SA
isolates in other parts of the world, e.g., MAS, isolated in France.
Part of a type II–like chrIa is present in many SA strains,
suggesting that it confers a selective advantage. Hybridization of
genomic DNA of multiple strains to a microarray that contained
probes to distinguish types I, II, and III was recently used to
determine relative similarity of multiple strains to type I, II, or
III (11). Confirming our analyses, it was reported that there are
regions where P89 and VEG are highly similar and regions where
GT1 and FOU (HG6) are highly similar. However, the absence
of type II–like regions in SA strains was used to argue that it was
unlikely that chrIa was derived from a type II–like NA strain,
which is supported by our data. However, the hybridization approach has several limitations when used to identify haploblocks:
(i) 53% of the genome of type II is almost identical to type III or
type I (Dataset S2A) and these regions can therefore not be
investigated by this approach. (ii) The similarity of nontype I, II,
and III strains to each other cannot be investigated. (iii) The
method has lower resolution, as there are only 1,500 usable
probe sets for the whole genome. Our high-resolution genomewide SNP comparisons clearly show that some of these strains do
contain type II–like regions: haploblocks VIIa_1_2 and XI_6_7
(Fig. 3D) from the HG6 strains and haploblock VIIb_10_11 and
VIII_1_2_3_4 from TgCatBr44 have a type II–like ancestry (Fig.
S2). The fact that part of the HG6 genome is derived from a type
II–like strain provides an important clue to a potential route of
transfer of an NA type II–like chrIa into many SA strains. HG6 is
one of the few HGs that is found worldwide and ∼7% of its genome has RCA with the HG9 strain P89, which has been isolated
from both NA and SA. Many other strains contain haploblocks
with RCA with HG6 and HG9 (Fig. S2 and Dataset S2B) including type I, type III, TgCatBr44, CAST, and TgCatBr5. It is
therefore possible that a relatively recent cross introduced chrIa
from a type II–like strain to an ancestral HG6-like strain, which
might have crossed with other ancestral HG6 strains diluting the
type II–like genomic portion, and then recently crossed with an
ancestral HG9 strain and so on. With each new cross, the type II
portion of the genome would be diminished and only the parts
with a selective advantage retained. What trait chrIa confers is
currently unknown but previous studies indicate it is not enhanced
virulence or oral transmission (8). The phenotype may only be
detected in the definitive feline host, such as enhanced fecundity
or oocyst viability. Our genome analysis refined the region of
chrIa that is conserved among most strains to a region of only 17
predicted genes and feline studies may identify the fitness advantage it confers.
The model we describe combines the striking evidence of
ancestry and recombination that we uncovered using wholegenome sequencing with the history of mammalian migration
and extinction discerned from the fossil record. It is a model of
Toxoplasma evolution that will be continually tested as more
complete genomes are sequenced and we see the full impact of
its sexual history.
Minot et al.
Materials and Methods
Genomic Sequencing. T. gondii was cultured on human foreskin fibroblasts as
described previously (30); all strains in this study have been described and
some are available at BRC Toxoplasma (http://www.toxocrb.com) (8, 30).
DNA was isolated from lysed parasites using DNAzol (Invitrogen) and prepared for high-throughput sequencing as per the Illumina single-end genomic
DNA kit protocol (COUGAR, CASTELLS, and MAS). Thirty-six nucleotides of
each library was sequenced on an Illumina GAII and processed using the
standard Illumina pipeline. Paired-end sequencing libraries were constructed
for P89, GUY-KOE, TgCatBr5, and BOF using the Nextera Illumina-compatible DNA sample prep kit (Epicentre) and amplified with the modified PCR
protocols (31). Libraries were barcoded (four strains per lane) and pairedend sequenced on Illumina HiSeq2000 (40+40 nucleotides). All libraries were
spiked with trace amounts of the phiX174 bacteriophage DNA for quality
control. Sequence reads were aligned to the ME49 genome using the Maq
software package (32). Reference genomes from ME49, GT1, and VEG were
obtained from toxodb.org (release 6.3). Genome coverage is shown in Table
S5. All data presented are from reads aligned to the ME49 genome. There
was no particular bias of read density or coverage for any individual
chromosome or region. Data are accessible at the Short Read Archive
under accession no. SRA047110.
barcoded and paired-ended sequenced (SRA050293). Number of reads that
mapped to the ME49 genome per strain is shown in Table S6.
T. gondii Interstrain SNP Mapping. The complete GT1 and VEG genome
sequences were aligned to the ME49 genome using the mummer software
package and SNPs were identified using nucmer with standard settings (33).
Reads for all other genomes and RNA-seq data were aligned to the ME49
genome and high-confidence SNPs were called using the easyrun option of
the Maq software package (32). To construct the apicoplast genome for the
sequenced strains, we aligned reads to the published (GenBank: U87145.2)
complete RH (type I) apicoplast genome. The GT1, VEG, and ME49 apicoplast
genomes were constructed by aligning reads from the trace archive to
the RH apicoplast genome using blast (34) and assembling them using
Sequencher software (Gene Codes Corporation). The location of each
polymorphism was assigned based on the physical map of the ME49
genome or the RH apicoplast genome. More details are in SI Materials
and Methods.
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High-Throughput RNA Sequencing. Murine bone marrow–derived macrophages (30) were infected with different T. gondii strains. After 20 h, total
RNA was extracted (Qiagen RNeasy Plus kit) and integrity, size, and concentration of RNA were checked (Agilent 2100 Bioanalyser). The mRNA was
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ACKNOWLEDGMENTS. We thank C. Su and M. Darde for contributing
T. gondii strains; Asis Khan, David Sibley, and John Boothroyd for useful
discussions; the Massachusetts Institute of Technology biomicrocenter for
excellent technical assistance; The Institute for Genomics Research and the
Wellcome Trust Sanger Centre, Cambridge, United Kingdom for the GT1,
ME49, and VEG genome sequences; and the Toxoplasma gondii database
for use of T. gondii genome data. J.P.J.S. was supported by a Massachusetts
Life Sciences Center New Investigator Award, National Institutes of Health
R01-AI080621, and the Pew Scholars Program in the Biomedical Sciences;
M.B.M. was supported by the Knights Templar Eye Foundation; W.N. and
D.L. were supported by Pre-Doctoral Grant in the Biological Sciences 5-T32GM007287-33; and S.M. was supported by training Grant T32AI060516.
Minot et al.
PNAS | August 14, 2012 | vol. 109 | no. 33 | 13463