Phylogeny of the malarial genus Plasmodium, derived from rRNA gene sequences.

Abstract

Malaria is among mankind's worst scourges, affecting many millions of people, particularly in the tropics. Human malaria is caused by several species of Plasmodium, a parasitic protozoan. We analyze the small subunit rRNA gene sequences of 11 Plasmodium species, including three parasitic to humans, to infer their evolutionary relationships. Plasmodium falciparum, the most virulent of the human species, is closely related to Plasmodium reichenowi, which is parasitic to chimpanzee. The estimated time of divergence of these two Plasmodium species is consistent with the time of divergence (6-10 million years ago) between the human and chimpanzee lineages. The falciparum-reichenowi clade is only remotely related to two other human parasites, Plasmodium malariae and Plasmodium vivax, which are also only remotely related to each other. Thus, the parasitic associations of the Plasmodium species with their human hosts are phylogenetically independent. The remote phylogenetic relationship between the two bird parasites, Plasmodium gallinaceum and Plasmodium lophurae, and any of the human parasites provides no support for the hypothesis that infection by Plasmodium falciparum is a recent acquisition of humans, possibly coincident with the onset of agriculture.

Full text

Proc.
Natl.
Acad.
Sci.
USA
Vol.
91,
pp.
11373-11377,
November
1994
Evolution
Phylogeny
of
the
malarial
genus
Plasmodium,
derived
from
rRNA
gene
sequences
(Plasmodium
falciparum/host
switch/small subunit
rRNA/human
malaria)
ANANIAS
A.
ESCALANTE
AND
FRANCISCO
J.
AYALA*
Department
of
Ecology
and
Evolutionary
Biology,
University
of
California,
Irvine,
CA
92717
Contributed
by
Francisco
J.
Ayala,
August
5,
1994
ABSTRACT
Malaria
is
among
mankind's
worst
scourges,
affecting
many
millions
of
people,
particularly
in
the
tropics.
Human
malaria
is
caused
by
several
species
of
Plasmodium,
a
parasitic
protozoan.
We
analyze
the
small
subunit
rRNA
gene
sequences
of
11
Plasmodium
species,
including
three
parasitic
to
humans,
to
infer
their
evolutionary
relationships.
Plasmo-
dium
falciparum,
the
most
virulent
of the
human
species,
is
closely
related
to
Plasmodium
reiehenowi,
which
is
parasitic
to
chimpanzee.
The
estimated
time
of
divergence
of
these
two
Plasmodium
species
is
consistent
with
the
time
of
divergence
(6-10
million
years
ago)
between
the
human
and
chimpanzee
lineages.
The
falkiparun-reichenowi
lade
is
only
remotely
related
to
two
other
human
parasites,
Plasmodium
malariae
and
Plasmodium
vivax,
which
are
also
only
remotely
related
to
each
other.
Thus,
the
parasitic
associations
of
the
Plasmodium
species
with
their
human
hosts
are
phylogenetically
indepen-
dent.
The
remote
phylogenetic
relationship
between
the
two
bird
parasites,
Plasmodium
gallinaceum
and
Plasmodium
lo-
phurae,
and
any
of
the
human
parasites
provides
no
support
for
the
hypothesis
that
infection
by
Plasmodium
falcipaum
is
a
recent
acquisition
of
humans,
possibly
coincident
with
the
onset
of
agriculture.
Malaria
counts
among
the
worst
scourges
of
mankind.
The
global
incidence
of
malaria
is
estimated
to
be
110
million
persons
per
year,
some
270
million
carry
the
parasite,
and
the
number
of
people
at
risk
is
2100
million
(ref.
1,
p.
30).
The
number
of
human
deaths
in
subSaharan
Africa
alone
is
estimated
at
1.5-3
million
annually
(2).
Malaria
is
a
parasitic
disease
caused
by
protozoa
of
the
genus
Plasmodium
(phylum
Apicomplexa
Levine
1970)
that
parasitize
reptiles,
birds,
or
mammals.
Four
species
are
parasitic
to
humans:
Plasmodium
falciparum,
Plasmodium
malariae,
Plasmodium
ovale,
and
Plasmodium
vivax.
Most
virulent
is
P.
falciparum,
the
agent
of
the
lethal
tertian
malaria.
It
has
been
suggested
(ref
3,
cited
in
ref.
4)
that
the
virulence
of
P.
falciparum
is
derived
from
it
recently
becom-
ing
a
human
parasite
by
way
of
a
host
switch
from
a
nonhuman
parasite.
Support
from
this
hypothesis
has
been
provided
by
Waters
et
al.
(4-6)
who
concluded
that
P.
falciparum
is
phylogenetically
more
closely
related
to
those
Plasmodium
species
parasitic
to
birds
than
to
the
species
that
parasitize
humans
or
primates.
To
reconstruct
the
phylogeny
of
the
genus
and,
particu-
larly,
to
test
the
hypothesis
that
P.
falciparum
is
monophyl-
etic
with
the
bird
parasites,
we
have
analyzed
small
subunit
(SSU)
rRNA
genes
of
11
Plasmodium
species,
including
Plasmodium
reichenowi,
a
chimpanzee
parasite,
which
was
not
considered
in
refs.
4-6.
Our
analysis
shows
that
the
closest
relative
of
P.
falciparum
among
the
taxa
examined
is
P.
reichenowi
and
that
the
clade
formed
by
these
two
species
is
only
remotely
related
to
other
Plasmodium
species,
in-
cluding
those
parasitic
to
birds
and
other
human
parasites,
such
as
P.
vivax
and
P.
malariae.
MATERIALS
AND
METHODS
We
have
investigated
the
18S
SSU
rRNA
sequences
of
the
11
Plasmodium
species
listed
in
Table
1.
This
table
also
gives
the
known
host
and
geographical
distribution.
The
sequences
are
for
type
A
genes,
which
are
expressed
during
the
asexual
stage
of
the
parasite
in
the
vertebrate
host,
whereas
the
SSU
rRNA
type
B
genes
are
expressed
during
the
sexual
stage
in
the
vector
(12).
Four
or
five
copies
of
type
A
genes
have
been
found
in
P.
cynomolgi
that
are
all
evolving
in
a
concerted
fashion
(13).
The
11
Plasmodium
sequences
were
aligned
by
means
of
the
CLUSTAL-V
program
(14).
Segments
for
which
a
reliable
alignment
could
not
be
inferred
were
eliminated.
Moreover,
the
available
sequence
of
P.
mexicanum
is
not
complete.
Thus,
only
a
1620-bp
segment
was
considered
in
our
analysis.
The
alignment
is
available
upon
request
from
F.J.A.
Phylogenetic
relationships
are
inferred
by
two
methods:
(i)
Neighbor
joining
(NJ)
(15)
with
Tamura's
three-parameter
distance
(16)
takes
into
account
the
possibility
of
high
bias
in
the
transition/transversion
ratio
and
in
G+C
content,
as
is
the
case
in
these
Plasmodium
genes.
The
estimate
for
G+C
content
used
is
the
average
for
all
the
sequences
(39%;
range,
37-41%).
Distances
are
calculated
using
all
aligned
nucleo-
tides
and
eliminating
gaps.
The
reliability
of
the
trees
is
assessed
by
the
bootstrap
method
(17)
with
1000
pseudorep-
lications.
All
NJ
analyses
are
performed
using
the
program
MEGA
Version
1.0
(18).
(ii)
Maximum
likelihood
(ML)
(19)
assumes
specific
tran-
sition/transversion
ratios.
Given
that
a
strong
bias
in
G+C
content
occurs
in
these
sequences,
we
used
several
transi-
tion/transversion
ratios
(2,
4,
6, 8,
10,
and
15),
all
of
which
yielded
very
similar
results.
Alternative tree
topologies
are
compared
using
the
Kishino
and
Hasegawa
test
(20).
These
analyses
are
performed
by
means
of
the
algorithm
DNAML
of
the
PHYLIP
package,
Version
3.5c
(ref.
21;
program
available
from
J.
Felsenstein,
Department
of
Genetics,
University
of
Washington,
Seattle).
The
tree
root
is
estimated
by
the
algorithm
DNAMLK
of
the
PHYLIP
program,
which
assumes
a
molecular
clock.
To
verify
this
assumption,
trees
obtained
by
the
DNAML
and
the
DNAMLK
algorithms
are
compared
by
the
test
in
ref.
20.
If
the
trees
are
not
statistically
different,
one
can,
at
least
provi-
sionally,
assume
that
the
gene
is
evolving
as
a
molecular
clock
and,
therefore,
accept
the
tree
root
position
estimated
by
DNAMLK.
We
also
estimate
the
tree
root
by
a
second
method.
Three
independent
alignments
of
the
11
Plasmodium
species
are
Abbreviations:
ML,
maximum
likelihood;
My,
million
years;
NJ,
neighbor
joining;
SSU
rRNA,
small
subunit
rRNA.
*To
whom
reprint
requests
should
be
addressed.
11373
The
publication
costs
of
this
article
were
defrayed
in
part
by
page
charge
payment.
This
article
must
therefore
be
hereby
marked
"advertisement"
in
accordance
with
18
U.S.C.
§1734
solely
to
indicate
this
fact.

11374
Evolution:
Escalante
and
Ayala
Table
1.
Plasmodium
species
and
their
hosts
and
accession
numbers
for
the
SSU
rRNA
sequences
Geographic
Accession
Code
Species
Host
distribution
number
Ref.
Pfa
P.
falciparum
Human
Tropics
worldwide
M19172
7
Pma
P.
malariae
Human
Worldwide
M54897
8
Pvi
P.
vivax
Human
Tropics
worldwide
X13926
9
Pre
P.
reichenowi
Chimpanzee
African
tropics
Z25819
*
Pfr
P.
fragile
Monkey
(Macaca
sinica)
Asian
tropics
M61722
4
Pkn
P.
knowlesi
Monkey
(Macaca
fascicularis)
Asian
tropics
L07560
6
Pcy
P.
cynomolgi
Monkey
(M.
fascicularis
&
Presbytis
spp.)
Asian
tropics
L07559
6
Pbe
P.
berghei
Rodent
African
tropics
M14599
10
Pga
P. gallinaceum
Bird
(Gallus
gallus)
Asia
M61723
4
Plo
P.
lophurae
Bird
Old
World
X13706
11
Pme
P.
mexicanum
Lizard
(Sceloporus
spp.)
North
America
L11716
t
*A.
W.
Thomas,
M.
Dubbled,
B.
Mons,
D.
Narum,
and
A.
P.
Waters
(1993)
GenBank,
unpublished
sequence.
tJ.
B.
Dame,
C.
A.
Yowell,
S.
R.
Telfold,
Jr.,
D.
Higgins,
and
T.
F.
McCutchan
(1993)
GenBank,
unpublished
sequence.
made
using
three
different
outgroup
species,
Babesia
bovis,
Theileria
parva,
and
Sarcocystis
fusiformis
(Table
2),
and
a
fourth
alignment
incorporating
jointly
all
three
outgroup
taxa
and
the
11
Plasmodium
species.
These
alignments
are
avail-
able
upon
request
from
A.A.E.
The
three
outgroup
genera
belong
to
the
Apicomplexa
phylum;
Babesia
and
Theileria
are
in
the
class
Hematozoa
to
which
Plasmodium
also
belongs,
whereas
Sarcocystis
belongs
to
the
class
Coccidia.
Toxoplasma
gondii
(which
belongs
to
the
class
Coccidia
and
the
family
Sarcocystidae,
the
same
as
S.
fusiformis)
was
also
used
as
an
outgroup,
but
the
root
of
the
Plasmodium
tree
could
not
be
unambiguously
determined,
which
suggests
that
for
the
present
purposes
Toxoplasma
may
be
genetically
far
too
removed
from
Plasmodium.
Phylogenetic
relationships
between
the
outgroup
taxa
and
the
Plasmodium
species
were
inferred
by
the
two
methods
mentioned
above.
RESULTS
Table
3
shows
the
genetic
distances
among
the
11
Plasmo-
dium
species.
Fig.
1
shows
the
NJ
tree
derived
from
this
distance
matrix.
The
numbers
on
the
branches
are
the
bootstrap
values
that
indicate
the
percent
number
of
times
out
of
1000
replications
that
the
species
in
the
cluster
to
the
right
appear
as
a
clade.
The
phylogeny
obtained
by
ML
(DNAML
or
DNAMLK)
has
precisely
the
same
topology
as
the
one
shown
in
Fig.
1.
Alternative
topologies
give
low
boot-
strap
values
and
are
significantly
worse
(by
the
method
of
ref.
20)
than
the
tree
shown
in
Fig.
1.
P.
falciparum
clusters
with
P.
reichenowi,
the
chimpanzee
parasite,
with
bootstrap
reliability
of
100%.
P.
vivax
clusters
unambiguously
(100%)
with
the
three
monkey
parasites
(P.
fragile,
P.
knowlesi,
and
P.
cynomolgi).
The
two
bird
para-
sites
(P.
gallinaceum
and
P.
lophurae)
cluster
with
P.
mex-
icanum,
the
reptile
parasite.
P.
malariae,
a
human
parasite,
and
P.
berghei,
a
rodent
parasite,
do
not
closely
cluster
with
any
of
the
other
clades
nor
with
each
other.
The
root
of
the
tree
in
Fig.
1
defines
two
major
clusters,
one
with
the
rodent
and
monkey
parasites
and
the
human
P.
vivax
and
the
second
cluster
with
all
other
species,
which
is
consistent
with
ref.
6.
The
root
is
determined
by
using
the
DNAMLK
model
that
assumes
that
all
lineages
are
evolving
at
the
same
rate.
The
test
of
ref.
20
shows
that
the
assumption
of
a
molecular
clock
is
not
warranted
for
the
whole
data
set.
However,
if
P.
mexicanum
is
removed,
the
data
set
is
consistent
with
the
molecular
clock
assumption,
and
the
root
appears
in
the
same
location
as
in
Fig.
1.
Table
4
gives
the
pairwise
genetic
distances
for
the
11
Plasmodium
and
the
three
outgroup
species.
Fig.
2
depicts
the
NJ
tree
derived
from
this
distance
matrix.
As
in
Fig.
1,
falciparum
clusters
with
reichenowi,
vivax
clusters
with
the
monkey
species,
and
also,
the
two
bird
species
cluster
with
P.
mexicanum,
the
reptile
parasite.
Moreover,
P.
malariae
andP.
berghei
cluster
with
the
vivax-monkey
clade
with
high
(95%)
reliability.
The
pair
falciparum-reichenowi
appears
as
a
sister
group
to
all
other
Plasmodium
species,
but
only
in
64%
of
the
bootstrap
replications
(70%o
or
higher
is
the
value
required
for
statistical
reliability-ref.
24).
We
have
also
obtained
three
separate
Plasmodium
phy-
logenies
by
using
in
turn
each
of
the
three
outgroup
species.
As
in
Figs.
1
and
2,
P.
falciparum
and
P.
reichenowi
appear
as
sister
taxa
in
all
three
trees,
with
very
high
reliability
(91-100%6).
P.
vivax
and
the
three
monkey
species
also
form
a
reliable
cluster
(99-100%o).
The
two
bird
Plasmodium
species
cluster
with
the
reptile
P.
mexicanum
when
Babesia
or
Theileria
are
used
as
outgroups
(95-98%
reliability);
but
not
when
Sarcocystis
is
the
outgroup.
Trees
with
Babesia
and
Theileria
as
outgroups
show
the
couplet
falciparum-
Table
2.
Three
outgroup
species
and
accession
number
for
the
SSU
rRNA
sequences
Accession
Code
Species
number
Ref.
Bbo
Babesia
bovis
L19077
22
Tpa
Theileria
parva
L02366
23
Sfu
Sarcocystis
fusiformis
U03071
*
*0.
J.
Holmdahl,
J.
G.
Mattsson,
A.
Uggla,
and
K.
Johansson
(1993)
GenBank,
unpublished
sequence.
I
0
.01
FIG.
1.
Phylogenetic
relationships
among
11
Plasmodium
species
inferred
from
the
SSU
rRNA
gene
sequences
(see
Table
1
for
abbreviations
of
species
names).
The
tree
is
derived
by
the
NJ
method
(15)
from
the
matrix
of
genetic
distances
given
in
Table
3.
The
scale
is
in
units
of
substitutions
per
site.
The
numbers
on
the
branches
are
bootstrap
values
based
on
1000
replications
and
indi-
cate
the
percent
of
cases
in
which
all
the
species
to
the
right
appear
as
a
cluster.
The
root
(arrowhead
on
left)
is
located
by
a
ML
method,
implemented
in
the
DNAMLK
program
of
PHYLIP,
which
assumes
constant
evolutionary
rate.
Proc.
Nad.
Acad.
Sci.
USA
91
(1994)

Proc.
Nadl.
Acad.
Sci.
USA
91
(1994)
11375
Table
3.
Genetic
distances
between
the
SSU
rRNA
sequences
of
11
Plasmodium
species
Nucleotide
substitutions,
no.
per
site
Pfa
Pma
Pvi
Pre
Pfr
Pkn
Pcy
Pbe
Pga
Plo
Pme
Pfa
0.051
0.058 0.009
0.051
0.049
0.052
0.061
0.043
0.045
0.069
Pma
0.006
0.058 0.054
0.053
0.053
0.054 0.066
0.047
0.053
0.081
Pvi
0.006
0.006
0.060
0.023
0.018
0.009
0.063
0.056
0.061
0.091
Pre
0.002
0.006
0.007
0.053
0.052
0.053
0.065
0.045
0.047
0.072
Pfr
0.006
0.006
0.004
0.006
0.012 0.018
0.059
0.056
0.062
0.085
Pkn
0.006
0.006
0.003
0.006
0.003
0.016
0.058 0.051
0.056
0.081
Pcy
0.006
0.006
0.002
0.006
0.003
0.003
0.058
0.052
0.057
0.085
Pbe
0.007
0.007
0.007
0.007
0.006
0.006 0.006
0.068
0.074
0.095
Pga
0.005
0.006
0.006
0.006
0.006
0.006
0.006
0.007
0.021
0.060
Plo
0.006
0.006
0.007
0.006
0.007
0.006
0.006
0.007
0.004
0.061
Pme
0.007
0.008
0.008
0.007
0.008
0.008
0.008
0.008
0.007
0.007
Data
are
expressed
as
nucleotide
substitutions
per
site
(mean)
and
based
on
1620
nucleotides
sequenced
in
all
species
(above
diagonal).
Below
the
diagonal
are
the
standard
errors.
The
distances
are
calculated
with
Tamura's
three-parameter
method
(16)
using
transitions
and
transversions.
Abbreviations
are
in
Table
1.
reichenowi
as
a
sister
clade
to
all
other
species,
whereas
this
couplet
is
the
sister
clade
to
the
bird
parasites,
although
without
dependable
bootstrap
reliability,
when
Sarcocystis
is
the
outgroup.
In
these
trees,
P.
malariae
and
P.
berghei
appear
phylogenetically
associated
with
the
vivax-monkey
cluster,
but
without
consistent
reliability.
Two
ML
phylogenies
derived
from
comparisons
with
the
three
outgroup
species
are
shown
in
Fig.
3.
These
two
trees
are
not
statistically
different
from
each
other
by
the
test
of
ref.
20.
The
one
topological
difference
between
them
is
that
in
the
left-side
tree
the
bird-plus-reptile
species
are
a
sister
clade
to
the
rest
of
Plasmodium
species
other
than
falci-
parum
and
reichenowi,
which
is
the
configuration
found
in
Fig.
2;
whereas
in
the
right-side
tree,
the
bird-plus-reptile
species
are
the
sister
clade
of
falciparum-reichenowi,
as
in
Fig.
1.
DISCUSSION
We
have
tested
the
hypothesis
that
"P.
falciparum
is
mono-
phyletic
with
the
avian
subgroup
[of
Plasmodium
species],
indicating
that
P.
falciparum
and
avian
parasites
share
a
relatively
recent
avian
progenitor"
(ref.
4,
p.
3140;
see
refs.
5
and
6),
which
is
"consistent
with
the
commonly
held
notion
that
infection
by
P.
falciparum
is
a
recent
acquisition
of
humans
and
possibly
coincident
with
the
onset
of
an
agri-
culture-based
life
style"
(ref.
4,
p.
3141).
Our
results
do
not
support
these
statements;
rather,
P.
falciparum
is
monophyl-
etic
with
P.
reichenowi,
a
chimpanzee
parasite,
but
shares
only
a
distant
common
ancestor
with
the
avian
parasites.
The
results
are
ambiguous
concerning
whether
the
falciparum-
reichenowi
clade
is
more
closely
related
to
the
avian
parasites
than
to
other
clades.
Our
analysis
confirms
that
P.
falciparum
is
only
remotely
related
to
the
other
human
Plasmodium
parasites,
P.
vivax
and
P.
malariae
(and
these
two
to
each
other).
The
evolu-
tionary
divergence
of
these
three
human
parasites
greatly
predates
the
origin
of
the
hominids.
Thus,
the
parasitic
associations
of
these
Plasmodium
species
with
humans
are
phylogenetically
independent.
This
result
is
consistent
with
the
diversity
of
physiological
and
epidemiological
character-
istics
of
these
three
Plasmodium
species
(25,
26).
Various
estimates
exist
of
the
rate
of
nucleotide
substitu-
tion
in
SSU
rRNA
genes.
Based
on
the
study
of
prokaryotic
endosymbionts
of
aphids,
Moran
et
al.
(27)
have
estimated
a
rate
of
1-2%
per
50
million
years
(My;
rounded
from
actual
estimates
ranging
from
0.0076
to
0.0232).
This
is
consistent
with
earlier
estimates
of
1%
per
50
My
(28,
29).
Differences
between
estimates
may
be
due
to
statistical
variance
and
to
heterogeneous
evolutionary
rates
in
different
lineages
or
at
different
times.
Variation
in
estimates
of
rRNA
evolutionary
rates
may
also
arise
because
the
set
of
nucleotides
that
are
compared
differ
from
one
to
another
case.
Generally,
as
the
set
of
lineages
compared
becomes
more
ancient,
and
hence
more
diverse,
it
becomes
increasingly
difficult
to
align
the
more
variable
Table
4.
Genetics
distances
between
the
SSU
rRNA
sequences
of
11
species
of
Plasmodium
and
three
other
Apicomplexa
species
Nucleotide
substitutions,
no.
per
site
Pfa
Pma
Pvi
Pre
Pfr
Pkn
Pcy
Pbe
Plo
Pga
Pme
Bbo
Tpa
Sfu
0.019
0.024 0.004
0.004
0.018
0.024
0.004
0.004 0.026
0.002
0.005
0.005
0.004
0.004
0.002
0.005
0.004
0.004
0.002
0.004
0.004
0.004
0.002
0.004
0.004
0.004 0.004
0.005
0.003
0.004
0.004
0.004
0.003
0.004
0.004
0.004
0.005
0.005 0.005
0.005
0.016
0.016
0.016
0.016
0.013
0.014
0.013
0.013
0.014
0.014
0.014
0.014
0.020
0.017
0.007
0.024
0.001
0.002
0.003
0.004
0.004
0.005
0.016
0.014
0.014
0.020
0.015
0.004
0.023
0.002
0.000
0.003
0.004
0.004
0.005
0.016
0.014
0.014
0.020
0.015
0.003
0.023
0.003
0.000
0.003
0.004
0.004
0.005
0.016
0.014
0.014
0.022
0.016
0.017
0.025
0.014
0.013
0.013
0.004
0.004
0.005
0.016
0.014
0.014
0.013
0.020
0.021
0.015
0.022
0.019
0.019
0.024
0.002
0.004
0.016
0.014
0.014
0.014
0.018
0.019
0.017
0.020
0.018
0.018
0.022
0.005
0.004
0.016
0.013
0.014
0.026
0.030
0.035
0.030
0.033
0.031
0.031
0.035
0.024
0.020
0.017
0.014
0.014
0.225
0.168
0.243
0.178
0.235 0.173
0.225
0.170
0.239
0.176
0.239
0.176
0.239
0.176
0.242
0.176
0.237
0.178
0.237
0.175
0.246
0.182
0.110
0.010
0.012
0.008
Pfa
Pma
PNi
Pre
Pfr
Pkn
Pcy
Pbe
Plo
Pga
Pme
Bbo
Tpa
Sfu
0.185
0.191
0.188
0.186
0.191
0.191
0.191
0.192
0.191
0.189
0.195
0.144
0.079
Data
are
expressed
as
nucleotide
substitutions
per
site
(mean)
and
based
on
1340
nucleotides
aligned
for
all
species
(above
diagonal).
Below
the
diagonal
are
the
standard
errors.
The
distances
are
calculated
with
Tamura's
three-parameter
method
(16)
using
transitions
and
transversions.
Abbreviations
are
in
Tables
1
and
2.
Evolution:
Escalante
and
Ayala

11376
Evolution:
Escalante
and
Ayala
95
Pfa
Pro
100
Pga
7
Plo
Pme
Pnma
5
Pvi
gc
g
Pcy
97
Pkn
f
Pfr
Pbe
I
Otti
Tpa
0
.01
FIG.
2.
Phylogenetic
tree
of
11
Plasmodium
species
and
three
outgroup
species
(see
Tables
1
and
2
for
abbreviations),
derived
by
the
NJ
method
from
the
genetic
distance
matrix
given
in
Table
4.
Other
information
is
as
in
Fig.
1.
regions
of
the
homologous
genes.
Consequently,
only
the
more
conserved
regions
of
the
molecule
are
compared,
and
the
estimated
rate
of
evolution
becomes
smaller.
This
effect
is
clearly
apparent
by
comparing
Table
3
(where
1620
bp
are
taken
into
account)
with
Table
4
(where
only
1340
bp
could
be
reliably
aligned):
the
substitution
rates
in
Table
4
are
about
half
those
in
Table
3.
Table
5
gives
divergence-time
estimates
for
various
lin-
eages;
the
rate
of
1%
for
50
My
is
used
in
column
a
for
the
data
in
Table
4,
rates
of
2%
(column
b)
and
1%
(column
c)
per
50
My
are
used
for
the
data
in
Table
3.
From
Table
5,
columns
a
and
b,
the
divergence
time
between
P.
falciparum
and
P.
reichenowi
is
estimated
as
10.5
±
5.0
My
and
11.2
±
2.5
My,
respectively.
These
estimates
are
consistent
with
the
time
of
divergence
between
the
lineages
of
humans
and
chimpanzees
(6-10
My;
see,
e.g.,
ref.
30),
their
corresponding
hosts.
The
rate
of
1%
per
50
My
would
seem
appropriate
for
the
more
conserved
fraction
of
the
molecule
examined
in
Table
4
but
not
for
the
complete
molecule
(Table
3),
since
it
yields
a
divergence
estimate
of
22.5
My,
which
is
much
too
ancient
for
the
divergence
between
humans
and
chimpanzee.
(Of
course,
the
theoretical
possibility
exists
that
the
two
para-
sites,falciparum
and
reichenowi,
diverged
much
before
their
host
species.)
For
the
data
in
Table
3,
the
rate
of
2%
per
50
My
yields
a
95%
confidence
interval
of
6.2-16.2
My,
which
is
consistent
with
the
divergence
of
the
hosts.
Table
5,
columns
a
and
b,
yields
estimates
of
11.7
±
5.0
My
and
20.9
±
3.8
My
for
the
average
divergence
between
the
human
P.
vivax
and
the
monkey
parasites,
estimates
that
are
low
for
the
divergence
between
the
Old
World
monkeys
and
the
hominoids.
Two
possible
explanations
for
this
discrep-
ancy
are
(i)
the
rRNA
genes
in
these
Plasmodium
species
are
evolving
at
a
slower
rate
than
in
falciparum
and
reichenowi
FIG.
3.
Two
ML
phylogenetic
trees
obtained
by
grouping
11
Plasmodium
species
as
indicated
(the
six
unlisted
species
are
grouped
as
Plasmodium
spp.).
Statistically,
the
two
topologies
are
not
significantly
different.
or
(ii)
the
phylogeny
of
these
parasites
does
not
parallel
the
phylogeny
of
the
hosts,
so
that
parasite
or
host
polymorphism
may
exist
(now
or
in
the
past),
or
lateral
transfers
may
have
occurred.
(We
mean
by
parasite
polymorphism
that
a
given
host
species
is
parasitized
by
more
than
one
of
these
Plas-
modium
species;
by
host
polymorphism,
we
mean
that
a
given
Plasmodium
species
parasitizes
more
than
one
of
these
host
species;
by
lateral
transfer,
we
mean
that
a
parasite
of
one
species
may
be
acquired
by
another
species.)
A
third
possibility
is
that
Tamura's
method
for
estimating
distances
is
not
properly
correcting
for
the
actual
number
of
substitu-
tions
that
have
occurred.
Consistent
with
hypothesis
ii
is
that
P.
cynomolgi
clusters
with
human
P.
vivax,
rather
than
with
the
two
other
macaque
parasites.
Moreover,
P.
cynomolgi
parasitizes
Macaca
fascicularis
and
other
species,
whereas
M.
fascicularis
is
also
parasitized
by
P.
knowlesi
(25,
31).
The
rate
of
1%
per
50
My
applied
to
the
data
in
Table
3
yields
a
divergence
time of
152.5
±
17.5
My
between
the
reptile
and
bird
Plasmodium
(Table
5,
column
c),
which
is
consistent
with
the
divergence
time of
the
birds
from
their
closest
reptile
lineages.
This
may
imply
that
the
other
rates
used
in
Table
5,
columns
a
and
b,
are
much
too
fast
in
this
case.
But
it
may
also
be
that
the
patterns
mentioned
in
the
previous
paragraph
may
be
operating.
A
discrepancy
in
evolutionary
rates
was
noted
earlier:
the
reptile
parasite
P.
mexicanum
appears
to
evolve
at
a
faster
rate
than
other
Plasmodium
lineages.
Pairwise
comparisons
among
the
four
main
Plasmodium
lineages
yield
an
estimate
of
151.5
±
17.5
My
(Table
5,
column
c)
for
the
origin
of
these
lineages.
This
estimate
would
imply
that
the
genus
Plasmodium
originated
about
the
time
of
divergence
of
birds
from
reptiles,
a
conjecture
consistent
Table
5.
Divergence
time
between
Plasmodium
lineages
Time
since
divergence,
My
Paleontological
host
data
Lineage
comparison
a
b
c
Event
Time,
My
1.
falciparum
vs.
reichenowi
10.0
±
5.0 11.2
±
2.5
22.5
±
5.0
Human-chimp
8
2.
vivax
vs.
monkey
11.7
±
5.0
20.9
±
3.8
41.7
±
7.5
Origin
of
hominoids
30
3.
mexicanum
vs.
bird
55.0
±
10.0
76.2
±
8.8
152.5
±
17.5
Origin
of
birds
150
4.
falciparum
vs.
vivax
vs.
mexicanum
vs.
malariae
vs.
berghei
52.4
±
10.0
75.7
±
8.8
151.5
±
17.5
Origin
of
genus
Plasmodium
?
5.
Plasmodium
vs.
outgroup
502.5
±
37.50
-
Origin
of
Apicomplexa
phylum
?
Estimates
are
obtained
as
follows:
Columns:
a,
rate
1%
per
50
My,
distances
from
Table
4;
b,
rate
2%
per
50
My,
distances
from
Table
3;
c,
rate
1%
per
50
My,
distances
from
Table
3.
Standard
errors
are
calculated
from
distance
errors.
Lineage
comparisons:
2,
average
between
vivax
and
each
of
the
three
monkey
parasites;
3,
average
between
mexicanum
and
each
of
the
two
bird
parasites;
4,
average
of
all
comparisons
between
species
of
the
five
Plasmodium
lineages;
5,
average
of
Plasmodium
species
each
separately
compared
with
each
of
the
three
outgroup
species.
Proc.
Natl.
Acad.
Sci.
USA
91
(1994)

Proc.
Natl.
Acad.
Sci.
USA
91
(1994)
11377
with
other
considerations
(32).
Some
authors
(for
review,
see
ref.
33)
have
questioned
whether
the
reptile
parasites
should
be
included
within
the
genus
Plasmodium.
Our
analysis
favors
the
affirmative,
since
P.
mexicanum
generally
clusters
within
the
set
of
all
other
Plasmodium
species.
(The
only
exception
is
the
tree
obtained
with
Sarcocystis
as
outgroup,
in
which
P.
mexicanum
is
the
sister
group
to
the
other
Plasmodium
species,
but
with
a
bootstrap
value
of
only
61%.)
Two
of
the
three
outgroup
species
used,
Babesia
bovis
and
Theileria
parva,
belong
to
the
class
Hematozoa,
the
same
as
Plasmodium,
although
they
belong
to
different
families.
The
third
outgroup
species,
S.
fusiformis,
belongs
to
the
Coc-
cidia,
which
is
thought
to
be
the
closest
class
to
the
Hema-
tozoa,
and
perhaps
ancestral
to
it.
Table
5
gives
an
estimate
of
502.5
±
37.5
My
as
the
time
of
divergence
between
Plasmodium
and
the
three
outgroups.
This
is
almost
certainly
an
underestimate
of
the
time
of
origin
of
the
Apicomplexa
phylum
for
two
reasons.
(i)
These
outgroup
species
are
presumed
to
be
less
distantly
related
to
Plasmodium
than
taxa
from
other
Apicomplexa
clades.
(ii)
The
rate
of
nucle-
otide
substitution
used,
1%
per
My,
is
probably
too
high
for
the
set
of
nucleotides
compared
between
the
Plasmodium
and
the
outgroup
species.
-This
rate
is
also
too
high
for
comparisons
between
distant
Plasmodium
species
(Table
5,
lineage
comparisons
3
and
4;
compare
column
a
with
column
b
or
c).
We
are
grateful
to
Eladio
Barrio,
Walter
Fitch,
Richard
Hudson,
Anthony
James,
and
Andrds
Moya
for
comments
that
have
contrib-
uted
to
improving
this
manuscript.
A.A.E.
has
been
supported
by
fellowships
from
the
Fundaci6n
Gran
Mariscal
de
Ayacucho-Latin
American
Program
of
American
Universities
and
the
New
Technol-
ogies
Program
from
Banco
Interamericano
de
Desarrollo-Consejo
Nacional
de
Ciencia
y
Tecnologfa,
Venezuela.
F.J.A.
acknowledges
the
support
of
National
Institutes
of
Health
Grant
GM42397.
1.
World
Health
Organization
(1991)
Tropical
Diseases:
Progress
in
Research
1989-1990
(W.H.O.,
Geneva).
2.
Selormey,
J.
A.
(1994)
Afr.
Technol.
Forum
7,
27-28.
3.
Boyd,
M.
F.
(1949)
in
Malariology,
ed.
Boyd,
M.
F.
(Saunders,
Philadelphia),
Vol.
1,
pp.
3-25.
4.
Waters,
A.
P.,
Higgins,
D.
G.
&
McCutchan,
T.
F.
(1991)
Proc.
Nati.
Acad.
Sci.
USA
88,
3140-3144.
5.
Waters,
A.
P.,
Higgins,
D.
G.
&
McCutchan,
T.
F.
(1993)
Parasitol.
Today
9,
246-250.
6.
Waters,
A.
P.,
Higgins,
D.
G.
&
McCutchan,
T.
F.
(1993)
Mol.
Biol.
Evol.
10,
914-923.
7.
McCutchan,
T.
F.,
de
la
Cruz,
V.
F.,
Lal,
A.
A.,
Gunderson,
J.
H.,
Elwood,
H.
J.
&
Sogin,
M.
L.
(1988)
Mol.
Biochem.
Parasitol.
28,
63-68.
8.
Goman,
M.,
Mons,
B.
&
Scaife,
J.
G.
(1991)
Parasitology
45,
281-288.
9.
Waters,
A.
P.
&
McCutchan,
T.
F.
(1989)
Nucleic
Acids
Res.
17,
2135.
10.
Gunderson,
J.
H.,
McCutchan,
T.
F.
&
Sogin,
M.
L.
(1986)
J.
Protozool.
33,
525-529.
11.
Waters,
A.
P.,
Unnasch,
T.,
Wirth,
A.
P.
&
McCutchan,
T.
F.
(1989)
Nucleic
Acids
Res.
17,
1763.
12.
Waters,
A.
P.,
Syn,
C.
&
McCutchan,
T.
F.
(1989)
Nature
(London)
342,
438-441.
13.
Corredor,
V.
&
Enea,
V.
(1994)
Genetics
136,
857-865.
14.
Higgins,
D.
G.,
Bleasby,
A.
J.
&
Fuchs,
R.
(1992)
Comput.
Appl.
Biosci.
8,
189-191.
15.
Saitou,
N.
&
Nei,
M.
(1987)
Mol.
Biol.
Evol.
4,
406-425.
16.
Tamura,
K.
(1992)
Mol.
Biol.
Evol.
9,
678-687.
17.
Felsenstein,
J.
(1985)
Evolution
39,
783-791.
18.
Kumar,
S.,
Tamura,
K.
&
Nei,
M.
(1993)
MEGA,
Molecular
Evolutionary
Genetics
Analysis
(Pennsyl.
State
Univ.,
Univer-
sity
Park),
Version
1.0.
19.
Felsenstein,
J.
(1981)
J.
Mol.
Evol.
17,
368-376.
20.
Kishino,
H.
&
Hasegawa,
M.
(1989)
J.
Mol.
Evol.
29,170-179.
21.
Felsenstein,
J.
(1989)
Cladistics
5,
164-166.
22.
Allsopp,
B.
A.,
Baylis,
H.
A.,
Allsopp,
M.
T.,
Cavalier-Smith,
T.,
Bishop,
R.
P.,
Carrington,
D.
M.,
Sohanpal,
B.
&
Spooner,
P.
(1993)
Parasitology
107,
157-165.
23.
Allsopp,
M.
T.,
Cavalier-Smith,
T.,
de
Waal,
T.
&
Allsopp,
B.
A.
(1994)
Parasitology
lOS,
147-152.
24.
Hillis,
D.
M.
&
Bull,
J. J.
(1993)
Syst.
Biol.
42,
182-192.
25.
Coatney,
R.
G.,
Collins,
W.
E.,
Warren,
M.
&
Contacos,
P.
G.
(1971)
The
Primate
Malarias
(Gov.
Print.
Off.,
Washington,
DC).
26.
LUpez-Antufano,
F.
&
Schumunis,
G.
A.
(1993)
in
Parasitic
Protozoa,
ed.
Kreier,
J.
P.
(Academic,
New
York),
2nd
Ed.,
Vol.
5,
pp.
135-265.
27.
Moran,
N.
A.,
Munson,
M.
A.,
Baumann,
P.
&
Ishikawa,
H.
(1993)
Proc.
R.
Soc.
London
B
253,
167-171.
28.
Ochman,
H.
&
Wilson,
A.
C.
(1987)
J.
Mol.
Evol.
26,
74-86.
29.
Wilson,
A.
C.,
Ochman,
H.
&
Prager,
E.
M.
(1987)
Trends
Genet.
3,
241-247.
30.
Holmes,
E.
C.,
Pesole,
G.
&
Saccone,
C.
(1989)
J.
Hum.
Evol.
18,
775-794.
31.
Collins,
W.
E.
&
Aikawa,
M.
(1993)
in
Parasitic
Protozoa,
ed.
Kreier,
J.
P.
(Academic,
New
York),
2nd
Ed.,
Vol.
5,
pp.
105-133.
32.
Manwell,
R.
D.
(1955)
Indian
J.
Malariol.
9,
247-253.
33.
Telford,
S.
R.,
Jr.
(1994)
in
Parasitic
Protozoa,
ed.
Kreier,
J.
P.
(Academic,
New
York),
2nd
Ed.,
Vol.
7,
pp.
1-71.
Evolution:
Escalante
and
Ayala