, 86 (2008); 322 Science
et al. Stephen A. Smith,
History in Flowering Plants
Rates of Molecular Evolution Are Linked to Life
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Rates of Molecular Evolution Are Linked
to Life History in Flowering Plants
Stephen A. Smith* and Michael J. Donoghue
Variable rates of molecular evolution have been documented across the tree of life, but the
cause of this observed variation within and among clades remains uncertain. In plants, it has been
suggested that life history traits are correlated with the rate of molecular evolution, but
previous studies have yielded conflicting results. Exceptionally large phylogenies of five major
angiosperm clades demonstrate that rates of molecular evolution are consistently low in trees
and shrubs, with relatively long generation times, as compared with related herbaceous plants,
which generally have shorter generation times. Herbs show much higher rates of molecular change
but also much higher variance in rates. Correlates of life history attributes have long been of
interest to biologists, and our results demonstrate how changes in the rate of molecular evolution
that are linked to life history traits can affect measurements of the tempo of evolution as
well as our ability to identify and conserve biodiversity.
metabolic rate, DNA repair, and generation time
(e.g., 1–4). In plants, differences in rates of mo-
lecular evolution have been noted between an-
nuals and perennials (5) and between woody and
been presumed to reflect differences in genera-
tion time (the time from seed germination to the
relationship between life history and the average
through replication or repair [nucleotide genera-
tion time (1)] is complicated by the fact that so-
matic mutations can accumulate during growth
and can be transmitted through gametes (8, 9).
Variation in breeding systemand/or seed-banking
by annual plants (9) may also affect the ability to
detect a correlation between molecular rate and
Previous studies have been inconclusive with
respect to the extent and the correlates of rate
heterogeneity in plants (5, 7, 9, 10). Studies fo-
cused on individual smaller clades, or on single
gene regions, have yielded results of uncertain
have suffered from limited taxon sampling and,
hence, comparisons among very distant relatives
(11). Some tests have failed to account for phy-
logenetic relatedness (9).
We assembled molecular sequence data for
five major branches within the flowering plants:
three clades of asterids (Apiales, Dipsacales, and
used group-to-group profile alignments (12) that
take advantage of previously recognized clades
within the groups analyzed (13) and yield denser
data matrices (containing less missing data) than
has been attributed to a number of fac-
tors, including differences in body size,
those produced using other strategies (14). Spe-
cifically, we identified alignable clusters of homol-
data, only phylogenetically informative clusters
(with at least four taxa) were used. The gene re-
gions varied among the five matrices but in each
case included markers from the chloroplast, nu-
clear, and mitochondrial genomes (figs. S1 and
S2 and table S2). The average gene region in our
analyses contained 305 species; the smallest con-
tained 10 species. This process resulted in an
Apiales matrix of 1593 species by 9522 sites
(>15 megabases); for Dipsacales, it was 366 by
11374 (>4 megabases); for Primulales, 529 by
457 by 7820 (>3.5 megabases); and for Comme-
linidae, 4657 by 22391 sites (>104 megabases).
Phylogenetic trees (Fig. 1) were inferred
under maximum-likelihood (ML) with RAxML
(vers.7.0.0) (15), with gene regions treated as
separate partitions (13). We conducted 100 rapid
bootstrap analyses, using every 10th bootstrap
tree as a starting tree for a full ML search, and
chose the tree with the highest likelihood score;
owing to the size of the Commelinidae matrix,
only a single ML search was conducted. For all
clades but Commelinidae, we used nonparame-
tric rate smoothing (16) to set branch lengths
proportional to time; we used the PATHd8 meth-
od (17) for the exceptionally large commelinid
analysis. Published studies were used to cali-
brate each phylogeny, using multiple calibra-
tion points to limit the impact of clade-specific
rate heterogeneity(13,18–21).For Apiales and
Primulales, we separately calibrated the major
subclades identified in previous analyses, which
also accommodated the fact that our analyses
included some taxa not represented in previous
Ancestral states of the life history trait “trees/
shrubs” versus “herbs” (a proxy for generation
time) (6, 7, 22) were inferred with ML methods
(Fig. 1) (13); palms (Arecaceae, Commelinidae),
were scored as trees/shrubs. For each branch on
branch lengths estimated from the dated molecu-
lar trees. Branch calculations were binned on the
basis of inferred life history to produce box plots
as artifacts of divergence-time estimation (e.g.,
consistently evolving more slowly than related
herbaceous plants. Median rates of nucleotide
divergence were 2.7 to 10 times as high in herbs
as in trees/shrubs; herbs also showed higher
ranges and variances (Fig. 1). None of the tree/
shrub lineages examined here showed high rates
of molecular evolution, but some herbaceous
lineages were inferred to have low evolutionary
rates, in the range characteristic of trees/shrubs.
that, although most trees/shrubs are not able to
reproduce within the first few years (23, 24), as
most herbs can, some herbs take as long as trees
to flower. Consistent with the view that genera-
tion time influences the rate of molecular
evolution within the Commelinidae (Fig. 1), the
longer-lived bromeliads [which take up to 18
years to reproduce (25)] have remarkably short
branches, with even fewer substitutions per site
per million years than palms (0.00059 and
0.0014, respectively). Other factors, such as
population size, breeding system, and seed-
banking, may also relate to the observed
asymmetry; for example, the rate of fixation of
mutations by selection increases in large popula-
in explaining the observed variance, they are less
clearly correlated with the life history distinction
than is generation time [e.g., (26)].
To explore whether the difference in rates of
molecular evolution has remained constant over
time, we compared substitutions per site per mil-
lion years through 10-million-year segments for
with some noteworthy exceptions in the earliest
estimated to have a high rate of evolution before
the herbaceous habit is inferred to have evolved
Dipsacales were herbaceous), by faster evolution
of woody lineages during earlier times (e.g., due
to warmer climate in the early Tertiary), or by the
extinction of early woody lineages.
Because these comparisons do not directly
take into account phylogenetic relationships or
examine the effects of evolutionary change from
one life history state to the other, we calculated
branch length contrasts (27) around each inferred
evolutionary shift in life history (Fig. 3) (13).
Specifically, we calculated the average accumu-
lation of molecular changes from each branch tip
to the shared ancestor of a tree/shrub clade and
Department of Ecology and Evolutionary Biology, 21 Sachem
*To whom correspondence should be addressed. E-mail:
3 OCTOBER 2008 VOL 322
on October 2, 2008
compared this to the average accumulation in its
nested clades and worked toward the root, ex-
cising any nested contrasts from the more in-
clusive calculations to avoid measuring any node
more than once. We omitted contrasts containing
only one tree/shrub or one herb branch to less-
en the impact of incorrectly estimating singleton
with two or more species).
Of the 13 contrasts identified using these crite-
ria (Table 1 and Fig. 3), 12 showed a slower rate
(sign test, P= 0.00342). On average, herbs evolve
2.5 times as fast as trees/shrubs. A maximum
rate difference of 4.75 times was found between
The single exception occurred within Sambucus
a slightly higher rate than the herbs (0.0075 and
Fig. 1. Phylogeniesoffive
angiosperm clades with
ferred life history states
(brown for trees/shrubs;
per million years for the
inferred life history cate-
the median, hinges mark
the first and third quar-
tiles, whiskers extend to
have values >1.5 times
beyond the first or third
Fig. 2. Dated phylog-
enies for Apiales and
Dipsacales with substi-
tutions per site per mil-
lion years plotted for
through the life of the
resent inferred life his-
tory states (brown for
trees/shrubs; green for
herbs). Box plots as in
Fig. 1. PM, Pittospora-
ceae and Myodocarpa-
ceae; Dips, Dipsacaceae;
M, Morinaceae; L, Linna-
0 102030 40 506070
VOL 3223 OCTOBER 2008
on October 2, 2008
numbers of species (three shrubby species versus
three herbs) and also presented the greatest diffi-
ture rapidly). As such uncertainties are inherent
er alternative phylogenetic hypotheses (13) af-
fected the results for the smallest clade examined
here, the Dipsacales, as well as the effect of scor-
ing all Sambucus species as trees/shrubs. These
alternatives (Table 1 and Fig. 3) yielded a simi-
larly strong historical correlation (P = 0.00049),
as did the exclusion of these contrasts altogether
(P = 0.00195).
On the basis of our trees and broader phylo-
see also (29)], the likely direction of evolution of
condition in Apiales, Dipsacales, and Primulales,
and with less certainly in Moraceae/Urticaceae.
The palms (Arecaceae) within the Commelinidae
present the one clear instance in our sample of the
evolution of trees/shrubs from herbaceous ances-
tors (30). From our comparisons and a broader
analysis of monocotyledons (11), the shift to the
tree/shrub habit in palms was associated with a
marked decrease in the rate of molecular evo-
lution (palms evolve 2.7 times as slow as their
sister commelinids), as predicted by the hypoth-
esis that generation time drives the rate of mo-
Differences in rates of evolution associated
ly in synonymous substitutions within coding se-
sequences, pruning species lacking an rbcL se-
quence in GenBank from our Commelinidae phy-
logeny and using RAxML to estimate branch
lengths for several partitions of the data (Table 2)
(13). As expected, estimated amino acid branch
lengths showed the least difference in rate be-
tween life history classes (2.1 times as fast as in
herbs), with first and second nucleotide positions
being next smallest (3.2 times as fast). The rate
difference in the full Commelinidae data set (all
species, all genes) fell between these two values
(2.7 times as fast in herbs). The third positions
showed the greatest difference in rate (4.98 times
as fast in herbs). These findings are similar to
those based on a much smaller sample of rbcL
sequences from grasses and palms (11).
Our findings highlight the need for the meth-
ods used to date phylogenies to address the form
A rate of nucleotide substitution obtained from
clade of trees/shrubs, or vice versa, without con-
founding age estimates. Likewise, relaxed clock
ly evolving groups are younger, and that rapidly
evolving groups are older, than their true ages. It
differentlifehistoriesin designing datingstudies.
Otherwise, as we have attempted here, the use of
multiple calibration points spanning clades that
differ in life history may help alleviate this prob-
lem. Also, as shown here for Commelinidae, the
use of amino acid sequences (or the removal of
third sites) may be useful. Bayesian models that
evolution [e.g., (33)] are promising, but current
We hope that our results will also focus new
attention on the extent to which molecular and
Are rates of morphological evolution also slower
in trees/shrubs than in herbs [e.g., (36)]? Until
this question is addressed, we urge caution in as-
suming that morphological change scales with mo-
lecular change and in using molecular branch
lengths alone to assess “feature diversity” and de-
sign conservation strategies [e.g., (37)]. A related
difference herb vs trees/shrubs
Fig. 3. Branch-lengthcontrastsfortrees/shrubsversusherbs.(A)Linesaredrawnbetweentheaccumulated
average molecular branch lengths for each tree/shrub clade and its sister herbaceous clade (numbers
correspond tothose inTable1).Allevolutionary shifts were inferred tobefrom trees/shrubstoherbsexcept
for the evolution of palmswithin monocotyledons (arrowhead in contrast 4).Contrasts 1 to 13were used in
an initial sign test (P = 0.00342). Alternative contrasts within the Dipsacales (14 and 15) are marked by
third test (P = 0.00195). (B) Magnitude of change between each tree/shrub clade and its herbaceous sister
clades; values above 1 show higher rates of molecular evolution in herbs than in trees/shrubs.
Table 1. Branchlengthcontrasts1to13derivefromthetreesinFig.1[see(13) formoreexactlocations
of the nodes in question]. Plants in the first taxon in each pair of representative taxa are trees/shrubs;
14 and 15 for 11 to 13 in one test and omitting contrasts 11 to 15 in another.
Major cladeRepresentative taxaTrees/shrubs HerbsDifference
3 OCTOBER 2008 VOL 322
on October 2, 2008
for identifying plant species from short DNA se-
quences [reviewed in (33)]. We predict that the
chloroplast genes proposed as universal barcode
loci will be most successful in resolving herba-
ly distinguishing closely related woody species.
Finally, our studies underscore the need for
better and more accessible information on the
underlying drivers of rates of molecular evolu-
tion. In addition to data on generation times, we
need better knowledge of effective population
sizes. Past analyses (e.g., in mammals) have as-
sumed that larger, longer-lived organisms have
large population sizes (31). Our analyses imply
that somatic mutation has not counteracted the
influence of generation time on rates of evolu-
of such mutations (8). In any event, our analyses
demonstrate a general pattern that must now be
taken into account in evolutionary studies and
whose existence demands the elaboration of a
cohesive causal explanation.
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Supporting Online Material
Materials and Methods
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14 July 2008; accepted 5 September 2008
Chemokine Signaling Controls
Endodermal Migration During
Sreelaja Nair and Thomas F. Schilling*
Directed cell movements during gastrulation establish the germ layers of the vertebrate embryo
and coordinate their contributions to different tissues and organs. Anterior migration of the
mesoderm and endoderm has largely been interpreted to result from epiboly and convergent-
extension movements that drive body elongation. We show that the chemokine Cxcl12b and its
receptor Cxcr4a restrict anterior migration of the endoderm during zebrafish gastrulation, thereby
coordinating its movements with those of the mesoderm. Depletion of either gene product causes
disruption of integrin-dependent cell adhesion, resulting in separation of the endoderm from the
mesoderm; the endoderm then migrates farther anteriorly than it normally would, resulting in
bilateral duplication of endodermal organs. This process may have relevance to human
gastrointestinal bifurcations and other organ defects.
and ectoderm) during gastrulation (1). Interac-
tions between the endoderm and mesoderm
specify organ locations and symmetries (2).
Defects in the endoderm alter the morphogene-
sis of mesodermal organs (e.g., heart, kidneys,
crucial feature of vertebrate embryo-
genesis is the coordinated morphogene-
sis of germ layers (endoderm, mesoderm,
and blood), whereas mesodermal defects disrupt
the locations of the liver and pancreas (2–5).
Morphogenesis is regulated by Wnt (6) and
Nodal signaling (7) when cells are intermingled
in a bipotential “mesendoderm” (8). However,
relatively little is known about germ layer–
specific pathways that establish organ rudiments.
In zebrafish, mesendodermal organ progenitors
involute at the gastrula margin (blastopore) and
move anteriorly toward the animal pole (future
head) while converging toward the midline (con-
The chemokine receptor CXCR4 controls
directional migration in many contexts and is
expressed in the endoderm. It is up-regulated by
the endodermal determinants Mixer and Sox17b
(9–13) and is required for gastrointestinal
vascularization (14). Of the two closely related
zebrafish Cxcr4s, Cxcr4b regulates the migration
of many cell types (12, 15–18), but no roles have
been reported for Cxcr4a during embryogenesis.
Zebrafish embryos deficient in Cxcr4a or
Cxcl12b, generated by injection with antisense
morpholino oligonucleotides (MO), appeared
morphologically normal (Fig. 1, A to C, and
fig. S1). However, analysis of Tg(gutGFP)s854
transgenic embryos in which the entire gut
fluoresces [(19); GFP, green fluorescent protein]
revealed duplications of endodermal organs at
56 hours post-fertilization (hpf) (Fig. 1, D to F,
and fig. S2), including the pancreas (normally
on the right; fig. S3, A to F) and liver (normally
Table 2. BranchlengthcontrastestimatesfordifferentpartitionsofrbcLsequencesfromCommelinidae.
Estimate data PalmsRest of CommelinidaeDifference
First, second, and third sites
First and second sites
Department of Developmental and Cell Biology, University
of California, Irvine, CA 92697, USA.
*To whom correspondence should be addressed. E-mail:
VOL 3223 OCTOBER 2008
on October 2, 2008