Journal of Experimental Botany, Vol. 57, No. 13, pp. 3471–3503, 2006
Major Themes in Flowering Research Special Issue
doi:10.1093/jxb/erl128
Morphological and molecular phylogenetic context of the
angiosperms: contrasting the ‘top-down’ and ‘bottom-up’
approaches used to infer the likely characteristics of the
first flowers
Richard M. Bateman1,*, Jason Hilton2 and Paula J. Rudall1
1
Jodrell Laboratory, Royal Botanic Gardens Kew, Richmond, Surrey TW9 3AB, UK
School of Geography, Earth and Environmental Sciences, University of Birmingham, Edgbaston,
Birmingham B15 2TT, UK
2
Received 4 May 2006; Accepted 13 July 2006
Abstract
Recent attempts to address the long-debated ‘origin’ of
the angiosperms depend on a phylogenetic framework
derived from a matrix of taxa versus characters; most
assume that empirical rigour is proportional to the size
of the matrix. Sequence-based genotypic approaches
increase the number of characters (nucleotides and
indels) in the matrix but are confined to the highly
restricted spectrum of extant species, whereas morphology-based approaches increase the number of
phylogenetically informative taxa (including fossils) at
the expense of accessing only a restricted spectrum of
phenotypic characters. The two approaches are currently delivering strongly contrasting hypotheses of
relationship. Most molecular studies indicate that all
extant gymnosperms form a natural group, suggesting
surprisingly early divergence of the lineage that led to
angiosperms, whereas morphology-only phylogenies
indicate that a succession of (mostly extinct) gymnosperms preceded a later angiosperm origin. Causes of
this conflict include: (i) the vast phenotypic and
genotypic lacuna, largely reflecting pre-Cenozoic extinctions, that separates early-divergent living angiosperms from their closest relatives among the living
gymnosperms; (ii) profound uncertainty regarding
which (a) extant and (b) extinct angiosperms are
most closely related to gymnosperms; and (iii) profound uncertainty regarding which (a) extant and (b)
extinct gymnosperms are most closely related to
angiosperms, and thus best serve as ‘outgroups’
dictating the perceived evolutionary polarity of character transitions among the early-divergent angiosperms.
These factors still permit a remarkable range of contrasting, yet credible, hypotheses regarding the order
of acquisition of the many phenotypic characters,
reproductive and vegetative, that distinguish ‘classic’
angiospermy from ‘classic’ gymnospermy. The flower
remains ill-defined and its mode (or modes) of origin
remains hotly disputed; some definitions and hypotheses of evolutionary relationships preclude a role for
the flower in delimiting the angiosperms. We advocate
maintenance of parallel, reciprocally illuminating programmes of morphological and molecular phylogeny
reconstruction, respectively supported by homology
testing through additional taxa (especially fossils)
and evolutionary-developmental genetic studies that
explore genes potentially responsible for major phenotypic transitions.
Key words: Angiosperm, character optimization, congruence,
development, evolutionary-developmental genetics, flower,
fossil, gene duplication, gymnosperm, morphology, ontogeny,
outgroup, phylogeny, pteridophyte, taxon sampling, tree rooting.
Introduction
‘An early hope was that the relationships indicated
among species by DNA were more likely to be correct
than those based on morphology; this now seems naı̈ve.’
(Judd et al., 1999, p. 99)
* To whom correspondence should be addressed. E-mail: [email protected]
ª The Author [2006]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
For Permissions, please e-mail: [email protected]
3472 Bateman et al.
‘The primary motivating force for preparing this book
was the dramatic change in our understanding of
angiosperm phylogeny during the past 10 years. Many
long-standing [morphological] views of deep-level relationships were altered suddenly and substantively as a direct
result of molecular analyses.’ (Soltis et al., 2005, p. ix)
‘Occam’s razor? But that’s for circumcision, surely?’
(Tom Sharpe, 1995; Grantchester Grind, London:
Macmillan, p. 82)
In this review we address one of the most popular
discussion topics in evolutionary biology: the origin of the
flower, and, by implication, the origin of the flowering
plants (note that we pay much less attention to the better
documented subsequent, family-level radiation of the
angiosperms; cf. Friis et al., 2006). We do not attempt to
offer definitive answers on the fundamental nature of the
flower but, instead, aim to establish a more rigorous context
for future research. Our main objective is to review and,
where possible, clarify several of the key issues in this
broad field.
We willingly follow the modern convention of placing
our discussion in the context of an explicit phylogenetic
framework, but within this framework we have chosen to
exercise certain prejudices. Unlike most recent contributions to this debate, we do not focus on a single phylogenetic hypothesis, preferring instead to consider the
implications of a variety of matrix-based phylogenies
derived from highly contrasting kinds of data. In assessing
the relative merits of these hypotheses, we emphasize
conceptual rigour over statistical robustness. We are
especially concerned with identifying the optimal roles
for different categories of data that pertain either directly or
indirectly to phylogeny reconstruction. We therefore discuss both molecular and morphological data sources,
including fossils, are discussed. This issue is explored in
the context of two contrasting perspectives on land-plant
phylogeny: the top-down perspective looks backward
through evolutionary time from the present, whereas the
bottom-up perspective looks forward through evolutionary
time from the deep past.
What is a flower?
Given the vast tracts of published text devoted to the origin
of the flower (and of the flowering plants), this is one
question that could reasonably be assumed to have been
unequivocally answered. Indeed, most glossaries included
in textbooks and reviews omit the term ‘flower’, presumably on the assumption that the term is universally
understood and the underlying concept is familiar to even
the most lackadaisical student of botany. However, comparison of the definitions assembled in Table 1 reveals little
unanimity. Four ostensibly distinct elements can be ext-
Table 1. Representative spectrum of definitions of a flower
‘. . . a growth comprising the reproductive organs of seed plants’
(Chambers Dictionary, 1998, p. 617)
‘. . . an axis on which the rest of the floral organs are borne’ (Fahn,
1974, p. 425)
‘. . . [a] specialized reproductive shoot, consisting of an axis
(receptacle) on which are inserted four different sorts of organs
[sepals, petals, stamens, carpels]’ (Penguin Dictionary of Biology,
1971, p. 102)
‘. . . an axis (or receptacle) bearing, in its complete form, four zones of
appendages that are considered the homologs of leaves’ (Bierhorst,
1971, p. 511)
‘. . . a highly modified shoot bearing specialized appendages (modified
leaves)’ (Judd et al., 1999, p. 53)
‘. . . an amphisporangiate strobilus of determinate growth and with an
involucrum of modified bracts’ (Arber and Parkin, 1907)
‘. . . the reproductive structure of Anthophyta, derived evolutionarily
from a leafy shoot in which leaves have become modified into petals,
sepals and into the carpels and stamens in which the gametes are
formed’ (Dictionary of Biological Terms, 1995; Harlow: Longman,
p. 206)
‘. . . a shoot beset with sporophylls: . . . leaves bearing sporangia’
(Goebel, 1905, p. 469)
‘. . . a section of a shoot, or branch resembling a short shoot, which
bears leaf organs which serve for sexual reproduction and which are
transformed accordingly’ (Weberling, 1989, p. 2)
‘. . . a particular type of determinate sporogenous shoot . . . one of its
most definitive organs is the carpel; . . . a megasporophyll [that is]
morphologically distinctive because the ovules (i.e. megasporangia)
are usually enclosed within a hollow basal portion designated as the
ovary’ (Foster and Gifford, 1971, pp. 593–594)
‘. . . a compressed, bisexual reproductive axis composed of stamens,
carpels and a sterile perianth’ (Baum and Hileman, 2006)
‘. . . a bisexual structure with more or less recognizable carpels; that
is, laminar structures largely enclosing the ovules on their upper
(adaxial) surface, and with more or less recognizable stamens’
(Frohlich, 2006)
‘. . . a determinate axis terminating in megasporangia that are
surrounded by microsporangia and are collectively subtended by at
least one sterile laminar organ’ (present study)
racted from this aggregate of definitions: form, function,
homology, and taxonomy.
The majority of definitions identify (sexual) reproduction
as the primary function of the flower; an uncontentious
statement, but one that does not in itself separate a flower
from the reproductive structure(s) of any other land plant.
Two definitions include explicit references to taxonomically (and phylogenetically) delimited groups of organisms: the clade of groups that bear flower-like structures
(Anthophytes) and, more controversially, the more inclusive clade of groups that bear seeds (Spermatophytes).
The definition from Chambers Dictionary encompasses
gymnosperms as well as angiosperms. It therefore begs
immediate rejection by all knowledgeable botanists, who
routinely equate the structure ‘flower’ with the (monophyletic) group ‘angiosperms’, and who consequently
attempt to define gymnosperms in part by their absence
of flowers.
With regard to form, several definitions describe the
flower as a composite structure, referring explicitly to the
presence of four differentiable categories of (usually
physically discrete) organs that are organized in a specific
Phylogenetic context of the flower
and reliable linear sequence along the axis towards its
apex: sepals, petals, stamens, and carpels. A few definitions
emphasized the function of stamens and carpels for
generating gametes (male and female, respectively), and
Foster and Gifford (1971) and Frohlich (2006) further
identified the carpel as being the most diagnostic feature of
a flower. Foster and Gifford also noted that definitions of
a flower omitting reference to these structures, such as that
of Goebel (1905), implicitly encompass not only all
angiosperms but also all gymnosperms and even some of
the more derived, reproductively complex, pteridophyte
groups (thereby rendering less bizarre the aforementioned
Chambers Dictionary definition: Table 1).
Crucial to the majority of definitions is an almost
universally accepted interpretation of the flower as a determinate reproductive shoot with a defined number of
floral organs, typically (but not universally) arranged in at
least four distinct whorls: sepals, petals, stamens, and
carpels. Specifically, the flower is homologized with an
axis bearing sporophylls (i.e. evolutionarily modified
sporangium-bearing leaves or, perhaps more conservatively, leaf-like structures). Implicit in these definitions is
the dynamic concept of a transition from an earlier,
profoundly different (and thus recognizable) ancestral
form of reproductive organ. The identity of the ancestral
group is almost universally accepted, namely the gymnosperms; an origin among the (by definition) seed-less
pteridophytes requires a set of morphological transitions
that seemingly is too radical for any modern botanist to
seriously contemplate.
Thus, we can draw on several categories of information
in order to define a flower. And, having defined a flower,
surely we have by default defined a flowering plant—an
angiosperm. Why then have the more informed among the
land-plant morphologists appended firm cautionary notes to
their preferred definitions? For example, according to
Foster and Gifford (1971, p. 593), ‘As angiosperms are
commonly designated the flowering plants, it might be
assumed that there is rather general agreement about the
scientific concept of a flower. Unfortunately, this is not the
case, and the literature on floral organography, ontogeny,
and structure displays widely divergent viewpoints of the
fundamental nature of the flower as well as on the
interpretation of its component organs (sepals, petals,
stamens, and carpels). One of the basic difficulties lies in
our complete [sic] ignorance of the evolutionary history of
the flower . . .; it becomes largely a matter of conjecture
whether it is justifiable to draw comparisons between
modern angiospermous flowers and the spore-producing
structures of other tracheophytes [vascular land-plants]. If
such comparisons are attempted, it is quite possible to reach
either a very broad or a very restricted concept or definition
of a flower.’
Bierhorst (1971, p. 511) preceded his definition by
stating that a flower is ‘a structure that, because of its
3473
various degrees of completeness, cannot adequately be
defined in words’. This, and many other similar statements
in the literature, refer primarily to the considerable floral
variation revealed by surveys of extant angiosperms. Some
species lack sepals, petals, or both. For example, in one
clade (Saururaceae plus Piperaceae) of the magnoliid order
Piperales, flowers are entirely perianth-less, a feature that is
closely correlated (at least, in this group) with possession
of indeterminate inflorescences (Remizowa et al., 2005).
Many other species show morphological gradation between
sepals, petals, and/or stamens. Yet others contravene the
requirement for bisexuality by bearing functional stamens
and carpels on separate flowers, either on the same
individual (monoecy) or on separate individuals (dioecy);
organs of the opposite gender have either been rendered
sterile or completely suppressed. For example, the muchdiscussed ‘primitive’ extant angiosperm Amborella (Fig.
1A, B) is commonly (though not reliably) dioecious,
bearing either exclusively male flowers (lacking even
sterile carpels) or female flowers that occasionally bear
staminodes, sometimes with a developmental transition
between stamens and carpels (Buzgo et al., 2004). Many
other early-divergent extant angiosperms even lack carpel
closure by tissue fusion (Endress and Igersheim, 2000a, b;
Endress, 2001a) or double fertilization (Williams and
Friedman, 2002). The columellar infratectum in the microspore wall of angiosperms, functionally linked to chemical
recognition systems on the stigmatic surface, is also unreliably present in basally divergent extant angiosperms
(Sampson, 2000; Doyle, 2006; Frohlich, 2006).
Thus, several supposedly definitive features of the flower
are frequently absent from angiosperms, especially earlydivergent angiosperms: these include hermaphroditism,
fully closed carpels, and a distinctly whorled arrangement.
Admittedly, the presence on the ovule of a second (outer)
integument, and the remarkably conservative structure of
the stamen (two thecae joined by a connective, each consisting of two embedded microsporangia), are more consistent features of the angiosperm flower, though they too
have potential homologues in some gymnosperm groups
(Doyle, 2006).
Furthermore, the flower-subtending bract, normally
regarded as extra-floral, can strongly influence, or even
be considered part of, the flower (e.g. Remizowa et al.,
2005; Buzgo et al., 2006). Bringing a palaeobotanical
perspective to bear, Stewart and Rothwell (1993, p. 440)
coined the phrase ‘accessory reproductive structures’ to
encompass sterile organs associated with reproductive
functions—not only perianth members (tepals, or petals
plus sepals) but also another (less well-researched) leaf-like
organ, the flower-subtending bract. Bracts (and bracteoles)
share with perianth members the characteristics of exhibiting many leaf-like features but being positionally fixed with
respect to either a flower or an inflorescence. In some
angiosperms, the bract has become intimately integrated
3474 Bateman et al.
Fig. 1. (A) Female and (B) male flowers of the putative basally divergent extant angiosperm Amborella trichopoda. (C) Male (above) and female
(below) reproductive structures of the much-discussed Jurassic fossil gymnosperm Caytonia (Caytoniales) and (D) hermaphrodite reproductive structure
of the Jurassic fossil gymnosperm Williamsoniella (Bennettitales); both are candidate sister-groups to the angiosperms. (C and D reproduced, with
permission, from figs 1.8 and 1.11, respectively, of Soltis et al., 2005, and reprinted from Crane, 1985.) Scale bar = 100 lm.
into the flower; for example, Amborella (Fig. 1A, B) and
several other early-divergent angiosperms (e.g. Austrobaileya, Trimenia) exhibit a morphological continuum
between bracts and perianth (Endress, 2001b; Buzgo
et al., 2004). In others (e.g. Araceae, Cornaceae, Saururaceae) the inflorescence bracts are conspicuously petaloid
and hence perform at least one of the functions (pollinator
attraction) that are more typically performed by petals. For
example, in two genera (Houttuynia and Anemopsis) of
the perianth-less family Saururaceae, the inflorescence
bracts form a pseudocorolla, and the entire inflorescence
has a flower-like appearance (Tucker, 1981).
More significantly, many of the gymnospermous groups,
both extant and extinct, which are by definition considered
to lack a differentiated perianth, unequivocally possess
bract-like organs. Arber and Parkin (1907) coined the terms
‘anthostrobilus’ for the modern angiosperm flower, and
‘pro-anthostrobilus’ for the type of cone manifested not
only by the Mesozoic bennettite Williamsoniella (Fig. 1D)
but also by a hypothetical group of extinct stem-group
angiosperms that they termed ‘Hemiangiospermae’. They
noted that the bennettite cone possessed a series of sterile
leaf-like organs that they interpreted as an ‘undifferentiated
primitive perianth’. They also insightfully defined a major
subset of (derived) angiosperms as possessing ‘a euanthostrobilus, of which the distinctive features are the presence
of the special type of microsporophyll termed a stamen, and
of closed carpels’ (Arber and Parkin, 1907, p. 75). Thus,
if flowers are at least partly defined not by possession of
sepals and/or petals but by possession of the broader
category of leaf-like accessory reproductive structures, the
flower is no longer seen as a unique (and defining) attribute
Phylogenetic context of the flower
of the angiosperms, and when using the term ‘flowering
plants’ certain gymnospermous groups, especially extinct
taxa such as Bennettitales, should strictly be included (cf.
Crane, 1988). This conclusion is implicit in our own
(inevitably imperfect) definition of a flower: a determinate
axis bearing megasporangia that are surrounded by
microsporangia and are collectively subtended by at least
one sterile laminar organ. In formulating this definition,
the orientation of our discussion has, in practice, switched
from top-down to bottom-up. We note that, although we
do not necessarily exclude multiple origins of flower-like
structures (see below), our definition has converged on that
of Arber and Parkin (1907, p. 75), who characterized the
anthostrobilus as ‘a special form of amphisporangiate cone,
distinguished by the peculiar juxtaposition of the mega- and
microsporophylls, and by possessing a well-marked perianth’. We also note that Arber and Parkin primarily (and
almost uniquely) employed a bottom-up approach in what
became a benchmark study in floral evolution that remained
influential throughout the ensuing century.
Which are the (other) benchmark studies in
floral evolution?
Certain conceptual thresholds can readily be identified in
the study of flowers and flowering plants. In his benchmark
classification of flowering plants, Linnaeus (Linné, 1735)
emphasized the significance of the number and arrangement
of stamens and carpels. The possible equivalence of sepals,
petals, and stamen filaments (though not explicitly the
carpels) to modified leaves was greatly elaborated by
Goethe (1790) in an essentialist essay partly stimulated
by studying plant teratologies. However, Goethe’s ideas
about simple equivalence, summarized by the famous
phrase ‘all is leaf’, had no phylogenetic implications
(Lönnig, 1994). As noted by adherents of Zimmerman’s
telome theory (Zimmerman, 1930, 1938), such as Wilson
(1937), spore-bearing organs (i.e. sporangia) evolved
before leaves in early land plants. By contrast, Darwin’s
(1859) development of a credible evolutionary mechanism
to explain (albeit in a uniformly gradualistic manner) such
radical morphological transitions was followed by the
harnessing of Mendelian genetics to address the control
of such transitions by authors such as De Vries (1906) and
later Wardlaw (1965).
These new data sources informed some increasingly
sharp exchanges between proponents of the two main sets
of theories competing to explain the origin of the flower.
The more common euanthial theory that was pioneered by
Goethe (and is implicit in most of the definitions of a flower
collated in Table 1) postulates derivation from an unbranched, uniaxial structure, and hence interprets the flower
as a condensed sporophyll-bearing single axis with proximal microsporophylls (stamens) and distal megasporophylls
3475
(carpels) (Arber and Parkin, 1907; Arber, 1937). By
contrast, the more diverse pseudanthial theories all perceive the flower as having condensed from a multiaxial
structure (Wettstein, 1907; Melville, 1960; Eames, 1961;
Meeuse, 1975, 1987, Stuessy, 2004). Thus, the difference
between the two conflicting hypotheses relates more to the
nature of individual organs than to the flower as a whole.
Several authors have postulated secondary derivation of
flower-like structures from inflorescences (i.e. a secondary
pseudanthial origin in certain phylogenetic groups), based
on ontogenetic evidence. These studies relate to both
gymnosperms (Gnetales: Mundry and Stützel, 2004) and
angiosperms (e.g. alismatid monocots: many authors,
reviewed by Sokoloff et al., 2006). Multiple origins of
flower-like structures, both within angiosperms and other
seed plants, are implicit in these pseudanthial hypotheses.
Studies of flowers prompted by the various insights
outlined above were, to varying degrees, comparative, and
in some cases hypothesis-testing. However, they often (i)
focused on limited suites of morphological characters
considered a priori to be of particular importance, (ii) failed
to determine whether the characters of interest were ever
specified by a single genome (i.e. were observed in a single
individual), and/or (iii) unjustifiably equated extreme diversity of form with increased likelihood of multiple origins
of the feature in question. More importantly, they lacked
a unifying conceptual framework. This crucial prerequisite
has been provided, first by phylogeny reconstruction and
then by evolutionary-developmental genetics, epitomized
by the ABC model of floral whorl control (e.g. Coen and
Meyerowitz, 1991; Irish and Kramer, 1998; Kramer and
Irish, 1999; Lawton-Rauh et al., 2000; Cronk et al., 2002;
Theissen et al., 2002).
Which are the benchmark studies in
reconstructing seed-plant phylogeny?
The now ubiquitous tree motif routinely used to represent the supposed sequence of divergence of evolutionary
lineages was famously employed by Darwin (1859) and
elaborated by authors such as Haeckel (1894). However,
only with the early cladistic works of entomologists Hennig
(1966) and Brundin (1972) were we provided with the
rigorous conceptual framework needed to generate such
trees with a large degree of objectivity from matrices of
coded taxa, each explicitly scored for competing states of
a broad suite of characters. Central to this approach is the
concept of the congruence test of homology (e.g. Patterson,
1988). The preferred phylogeny for the scored taxa is the
one requiring fewest transitions between character-states.
We can then observe in the resulting tree(s) which characterstates delimit which taxonomic groups—in other words,
which prior statements of homology between species have
been upheld in the most-parsimonious tree(s). The
3476 Bateman et al.
particulate nature of the numerically scored character
states is preserved in the resulting trees, thereby simplifying various kinds of post hoc character analysis.
The cladistic approach was first used to compare the
major groups of seed-plants by Hill and Crane (1982). With
the assistance of rapidly improving computer hardware and
software, it spawned an increasing number of morphological phylogenies based on parsimony analyses during the
1980s and early 1990s. If we consider the development
of taxonomically broad morphological matrices for seedplants, four main lineages can be recognized, nucleating
around PR Crane (e.g. Hill and Crane, 1982; Crane, 1985,
1988), JA Doyle (e.g. Doyle and Donoghue, 1986, 1987,
1992; Doyle, 1996, 1998a, b, 2006; Hilton and Bateman,
2006), GW Rothwell (e.g. Rothwell and Serbet, 1994;
Rothwell and Nixon, 2006), and DW Stevenson (e.g.
Loconte and Stevenson, 1990, 1991; Nixon et al., 1994).
These studies placed as closest relatives of angiosperms, in
various paraphyletic combinations, several groups (most
of them wholly extinct) that possess reproductive organs
showing some flower-like properties, notably Gnetales,
Bennettitales (Fig. 1D), Pentoxylon, Caytonia (Fig. 1C),
and glossopterids. Such arrangements soon became known
collectively as the anthophyte hypothesis, implying that
the flower evolved only once and, hence, that all flowers
are fundamentally homologous.
By 1990, these morphological analyses were competing
with (and soon largely superseded by) phylogenetic studies
employing as characters the sequences of nucleotides (and
insertion–deletion mutations) in specific regions of the
three plant genomes: nuclear-chromosomal, plastid, and
mitochondrial. As the number of readily sequenced regions
(and hence the number of usable characters) increased
exponentially, driven by advances in sequencing technology, maximum parsimony was supplemented with more
mathematically complex methods of generating trees,
notably maximum likelihood and, latterly, Bayesian approaches (e.g. Page and Holmes, 1998). The current species richness and ecological pre-eminence of angiosperms
have caused them to be preferentially sampled for phylogenetic studies. The result is a framework of relationships
that is generally viewed as well-sampled and increasingly
(though by no means universally) as reliable (e.g. Soltis
et al., 2004, 2005), and underpins higher classifications
that are based on ‘natural’ monophyletic groups (e.g. APGII:
Angiosperm Phylogeny Group, 2003).
At present, increasing numbers of species have been
sequenced for the complete plastid and/or mitochondrial
genomes (e.g. Goremykin et al., 2005), and a few for the
entire nuclear genome (e.g. rice, Arabidopsis; Arabidopsis
Genome Initiative, 2000; Bennetzen 2002; Yu et al., 2002;
Yamada et al., 2003; International Rice Genome Sequencing Project, 2005). These sequence-based data not only
allow character-rich (if presently species-poor) phylogeny
reconstructions of extant species but also provide a yard-
stick for comparative studies that focus simultaneously on
changes in key developmental genes and the phenotypic
characters that they ‘control’—a growing discipline termed
evolutionary-developmental genetics (e.g. Cronk et al.,
2002). This approach is especially attractive, as it represents an alternative, and potentially more informative, test
of a priori homology statements that also explicitly links
genotype with the resulting phenotype, and thence ultimately with specific biological function(s). Thus, the major
phenotypic transitions first explored morphologically by
Goethe (1790), functionally by Darwin (e.g. 1859), and
genetically by De Vries (1906) can at last begin to be
examined for fundamental causation (e.g. Frohlich and
Parker, 2000; Bateman and DiMichele, 2002; VergaraSilva, 2003; Baum and Hileman, 2006; Hintz et al., 2006;
Theissen, 2006).
Despite their conflicting topologies (Fig. 2), these
different approaches share some common elements with
respect to seed-plant relationships. Angiosperm monophyly
is routinely inferred. Nonetheless, a few brave authors
generally viewed as mavericks, notably Meeuse (1976,
1987), have penned morphologically based (though not
matrix-based) arguments for polyphyly. Cladistic analyses,
both morphological and molecular, consistently place the
clade containing all extant angiosperm lineages (the
‘crown-group’ angiosperms: Fig. 3) on a long branch
with respect to the remaining seed plants. ‘Missing links’
(i.e. stem-group) angiosperms have been postulated from
the fossil record but have not hitherto obtained universal
acceptance (e.g. Archaefructus, discussed below). Furthermore, relationships among extant gymnosperms, and hence
homologies among their reproductive structures, remain
contentious. These issues can only be partly circumvented
by adopting a top-down approach but are fundamental to
any bottom-up approach.
Which kinds of phylogeny are of greatest value?
Why do tensions remain within the phylogenetic community regarding molecular versus morphological characters and extant versus extinct taxa? Over the past
two decades, molecular phylogenies have in practice
largely superseded morphological phylogenies. This shift
of emphasis has had profound consequences, yet it is
now rarely discussed. Arguments most commonly advanced against morphological rather than molecular phylogenetic analyses are:
(i) the limited number of characters available (and the
existence of an asymptote of the number of phylogenetically informative character states available as taxa
are successively added to a matrix: Bateman, 1992);
(ii) the high cost in time expended per unit character
coded (both in terms of actual character scoring and the
Phylogenetic context of the flower
3477
Fig. 2. Crude consensus of phylogenetic relationships recognized by morphological analyses using extant and extinct taxa (A) and sequence-based
analyses using only extant taxa (B), illustrating the dominance of paraphyly in the former and monophyly in the latter.
‘informal apprenticeship’ that must first be undertaken
in order to describe and code characters correctly);
(iii) an inevitable degree of subjectivity involved in
making a priori homology assessments (in other
words, in delimiting characters before each is in turn
divided into alternative character states);
(iv) the supposed comparatively high level of homoplasy
(this is caused by conflicting character states, which
are considered to mainly reflect similar responses to
similar pressures of directional or disruptive selection
in lineages that are in fact only distantly related:
Scotland et al., 2003), and;
(v) the potential developmental correlation of apparently
unrelated characters (e.g. pleiotropic expression of
a single critical mutation).
At least some observers perceive corresponding constraints
on molecular phylogenies:
(i) the limited number, and sporadic phylogenetic distribution, of taxa available, due to the inability to allow
molecularly recalcitrant extinct taxa to participate in
the tree-building procedure (a central issue of this
paper);
(ii) the fact that routinely sequenced regions of the three
genomes do not participate directly in the phenotypic
transitions that signal a macroevolutionary event
(Bateman, 1999);
(iii) an inevitable degree of subjectivity in aligning
nucleotides in matrices that are rich in insertion–
deletion events (in other words, in assigning some
states of some taxa to the correct character);
(iv) the availability of the same restricted set of a maximum of four (or arguably five: A, C, G, T, and absent)
states for each position/character artificially masks
much of the actual homoplasy (e.g. when a particular
A mutates to a T but later reverts to an A);
(v) the potential in expressed regions of the genome for
particular categories of nucleotide to behave differently (e.g. contrasting mutational rates in different
compartments and regions of the genome, first and
second versus third bases within codons, and radical
contrasts in the GC:AT ratio).
Additional phenomena that can negatively affect both
morphological and molecular matrices include:
(i) strong heterogeneity of rates of change within the
study group, encouraging the most rapidly changing
branches to falsely coalesce; this now thoroughly
researched, but still problematic, phenomenon is
termed long-branch attraction (e.g. Sanderson et al.,
2000; Felsenstein, 2004);
(ii) migration of genes (and the phenotypic characters
that they underpin) between lineages through hybridization, lateral gene transfer, and organelle capture,
and;
(iii) the necessity of rooting the tree, typically through
outgroup choice, in order to polarize characters and
thereby study character evolution (another central
issue of this paper: see below).
3478 Bateman et al.
and then mapped, thereby effectively generating tautologous
interpretations of evolution (Bateman, 1999).
How significant are optimization and outgroups
choice?
Fig. 3. Hypothetical phylogeny illustrating the relativistic nature of the
concepts of crown group and stem group, and the significance of the
subtending nodes in any attempt to reconstruct the nature of hypothetical
ancestors. The diagram also contrasts the node-based and apomorphybased approaches to delimiting monophyletic groups. Dashed branches
subtend extinct taxa, cross-strikes on the branch immediately below the angiosperm crown-group node indicate individual character-state transitions.
In addition, the main source of operator bias influencing
the resulting topology is a priori homology assessment
for morphological data, whereas for molecular data it is
selectivity among (i) available characters and (ii) treebuilding algorithms. Moreover, both categories of analysis
are prone to culling of ‘troublesome’ taxa from matrices,
and revised outgroup choices made in search of more
‘intuitively acceptable’ topologies.
Many phylogeneticists advocate a compromise approach
to the relative treatment of morphological and molecular
characters. In one frequently used protocol, morphological
characters are combined with the molecular characters prior
to tree building, but only after the initial morphological matrix
has been reduced to its bare essentials by culling characters
that evoke suspicion, most commonly due to the difficulty of
dividing a complex of continuous character into discrete
states. However, a recent detailed analysis by Wortley and
Scotland (2006) convincingly refutes this approach, demonstrating that the culled characters contain the same average
strength of phylogenetic signal as the supposedly superior
characters that survive the cull. The alternative, and more
commonly used, approach is generally termed ‘mapping’.
Here, the morphological characters are placed along the tips
of the phylogeny after it has been constructed. Mapping
prevents morphological characters from contributing in any
way to the tree-building exercise. Mapping can also dissuade
researchers from seeking (and then occasionally discovering)
new phylogenetically valuable characters. All too often, wellknown phenotypic characters of a priori interest are scored
The concept of character mapping leads naturally into
discussion of another area of phylogenetics that is pivotal to
the questions addressed by this paper, namely optimization.
If we wish to know what the first flower looked like, but
have not found it in the fossil record (such a discovery is
highly improbable, given the undoubted patchiness of the
fossil record of land-plants), we need to reconstruct that
flower conceptually. This is achieved using combinations
of characters found in species whose morphology has been
carefully described and whose phylogenetic relationships
have been rigorously inferred (this phrase is not oxymoronic; it is important to remember that even the most
rigorously reconstructed phylogeny remains wholly inferential). Provided we have access to a matrix that thoroughly
describes the morphology of all organs of the species under
scrutiny, then we can reconstruct the morphology of the
hypothetical ancestors that lie on each node of the cladogram. Optimization is most readily achieved by a simple
logical protocol that works downward through the tree
from the terminal taxa toward the outgroup node (Fig. 3)
(e.g. Maddison and Maddison, 2001), though other, more
complex, models can be applied, sometimes with advantageous results (Oakley and Cunningham, 2000; Polly, 2001;
Webster and Purvis, 2002; Crisp and Cook, 2005).
A most-parsimonious tree, derived from a particular data
matrix, yields similar information about the relationships
of the coded taxa and the relative lengths of the various
branches within the tree, irrespective of whether it is
unrooted or rooted. However, an unrooted tree lacks
polarity; it cannot be read in terms of transitions from
ancestral character states (plesiomorphies) to derived
character states (apomorphies). This in turn means that it
is not possible to optimize character states in order to
reconstruct the hypothetical ancestors occupying the nodes
of the tree. In earlier (and more experimental) phases in the
evolution of methods of phylogenetic analysis, several
conceptually distinct approaches to rooting were explored,
but most proved impractical and all appeared at least
partially tautologous.
Over the last two decades a single method, termed
outgroup comparison (e.g. Nixon and Carpenter, 1993), has
become ubiquitous, to the point where it is routinely
adopted without serious thought by almost all practising
phylogeneticists. It requires the analyst to select a priori
a set of comparable taxa (preferably species) that are of
interest and are suspected of being an inclusive, monophyletic group (in practice, the strongest guide to perceived
monophyly is a previous, and taxonomically broader,
Phylogenetic context of the flower
phylogenetic analysis; thus, the outgroup method is
vulnerable to accusations of logical tautology). Then, one
or more additional taxa, thought to lie phylogenetically
outside the chosen study group, and hence operationally
termed outgroups, are chosen to simultaneously root the
tree and polarize the scored characters. If multiple outgroups are chosen they constitute a test (albeit flawed) of
the monophyly of the ingroup, since an outgroup taxon that
is placed phylogenetically within the ingroup contradicts
ingroup monophyly. Clearly, the larger the numbers of
ingroup and outgroup taxa, the stronger is this test.
Outgroup choice leaves the analyst with a further
quandary. In most cases, the ingroup is chosen because it
is a morphologically and/or molecularly distinct aggregate
of species, and so is likely to differ considerably from even
its phylogenetically closest outgroups (hence, in the resulting trees, a monophyletic ingroup is likely to be subtended
by a relatively long branch). These differences make
character delimitation more difficult for both morphological
and molecular analysts. For the morphologist, the outgroup
is likely to lack structures found in the ingroup, possess
structures not found in the ingroup, or the two groups may
possess structures that are broadly similar yet sufficiently
different that their homology cannot be adequately assessed. For the molecular researcher, sequence alignment
becomes more challenging for many genic regions, and
base saturation becomes an ever-increasing risk. These
problems of strong ingroup–outgroup divergence can, in
theory, be averted by identifying one or more members of
the ingroup as operational outgroups, but the analyst is then
imposing a strong subjective overprint on the topology of
the resulting tree(s). Certainly, the polarity of characters,
and thus the optimizations that allow reconstruction of the
properties of the hypothetical ancestor of the ingroup,
would be strongly influenced by such a decision.
But before we contemplate implementing of an optimization procedure, we must first select our preferred
topology from among the very broad spectrum of landplant phylogenies generated since 1982.
What is the best way to choose among the
plethora of phylogenies?
One of our primary objectives here is to explore the relative
merits of maximizing sampling of taxa versus that of
characters per taxon in taxonomically broad (i.e. at least
Class level) phylogenetic analyses. Morphological studies
that include the best-understood fossils (i.e. conceptual
whole plants reconstructed from their component parts:
Chaloner, 1986; Bateman, 1992) offer the best opportunity
to maximize sampling of taxa that provide strongly
contrasting combinations of character states. By contrast,
recent molecular trees, some of which compare entire plastid genomes (e.g. Goremykin et al., 2003, 2004, 2005),
3479
offer the most obvious means of maximizing the number of
characters per taxon.
Given the continuing uncertainties regarding relationships among the major groups of land plants (discussed
below), and their potential influence on optimizations of
nodes, several branch-points distant from the target node(s),
we sought phylogenies that encompassed all such major
groups. In the case of morphological phylogenies, no
existing study convincingly stretched from the bryophytes
to the more derived angiosperms (eudicots sensu APGII:
Angiosperm Phylogeny Group, 2003). We therefore took the
controversial step of grafting a selected most-parsimonious
tree from our recent analysis of 48 lignophytes (angiosperms plus gymnosperms, plus progymnospermous pteridophytes as operational outgroups: Hilton and Bateman,
2006, fig. 10) onto the product of a recent parsimony
analysis of 52 coded taxa that focused on pteridophytes but
used the fossil non-vascular polysporangiophyte Aglaophyton as outgroup and just five taxa (one progymnosperm
plus three primitive pteridospermous gymnosperms and
the extant Pinus) as representative ‘placeholders’ for the
monophyletic lignophytes (Rothwell and Nixon, 2006, fig.
3). The resulting composite phylogeny contains 95 coded
taxa: 47 wholly extinct and 48 containing at least one extant
species (Fig. 4).
Selecting among the many broad-brush molecular trees
in order to produce Fig. 5 proved even more problematic.
The controversial analysis by Goremykin et al. (2003,
2004; see also Soltis et al., 2004; Martin et al., 2005) stood
out as being especially character-rich, since it was based on
nearly completely sequenced plastid genomes. The tradeoff is that the analysis lacked data from the other two plant
genomes (nucleus and mitochondrion), and was restricted
to <20 coded taxa; moreover, the taxa were selected as
much for their economic importance as their likely
phylogenetic significance. This left the trees open to
accusations of distortion by long-branch attraction (Soltis
et al., 2004; Stefanovic et al., 2004; Leebens-Mack et al.,
2005), though a more recent study suggests that likelihood
model mis-specification is a more probable source of
systematic error in the trees (Goremykin and Hellwig,
2006). The matrix of Soltis et al. (2002) encompassed
a similar number of coded taxa, albeit more evenly
distributed across the extant land-plant phylogeny, and
sequenced eight genic regions distributed among the
plastid (rbcL, atpB, psaA, psbB), mitochondrion (mtSSU,
cox1, atpA), and nucleus (18S rDNA).
At the other end of the spectrum of molecular characters
per coded taxon are analyses based on a single region but
with substantial taxon sampling. A good example is the
2538-taxon rbcL study of land plants by Källersjö et al.
(1998, 1999), subsequently reduced to an illuminating
series of analyses of various combinations of 80 taxa
sampled evenly across the phylogeny of extant land plants
by Rydin and Källersjö (2002). Matrices of intermediate
3480 Bateman et al.
Fig. 4. Composite morphological phylogeny of 95 taxa (47 fossil, in boldface) obtained by grafting (at arrow) a seed-plant tree of Hilton and Bateman
(2006, fig. 10) onto a pteridophyte tree of Rothwell and Nixon (2006, fig. 3a).
Phylogenetic context of the flower
3481
dimensions include the 560-taxon, three-region (rbcL,
atpB, 18S) analysis of angiosperms by Soltis et al. (2000,
2003a), the 105-taxon five-region (atpB, rbcL, atp1, matR,
18S) analysis of basal angiosperms and gymnosperms by
Qiu et al. (2000), and the subsequent 100-taxon, nineregion (atpB, matK, rbcL; atp1, matR, mtSSU, mtLSU; 18S,
26S) analysis of basal angiosperms and gymnosperms by
Qiu et al. (2005).
What is the best way to assess the quality of
a phylogeny?
Fig. 5. Composite sequence-based phylogeny of 87 extant taxa obtained by grafting (at arrows) a eudicot tree of Soltis et al. (2000)
onto a basal angiosperm phylogeny of Zanis et al. (2002, as summarized by Soltis et al., 2005, fig. 3.7), and this in turn onto a
pteridophyte-plus-gymnosperm phylogeny of Pryer et al. (2001, fig. 1).
When discussing the reliability of a published phylogeny,
three terms are typically employed: accuracy, resolution,
and robustness. From our philosophical viewpoint, the term
accuracy can legitimately be applied to the topology that
reflects the relationships of the taxa analysed only in very
rare cases when the analyst is attempting to reconstruct a
known and wholly dichotomous genealogy engendered by
mankind (e.g. Hillis et al., 1992; Oakley and Cunningham,
2000; contra Rokas and Carroll, 2005). Given that we can
never gain access to the ‘one true tree of life’, by definition
we cannot assess its accuracy, which is an absolute rather
than a relative property.
When a fully dichotomous phylogeny (i.e. one that lacks
polytomous nodes) is recovered from the matrix it is often
said to be well-resolved. Robustness extends this concept to
determining the relative strength of support for a particular
node (relationship) within the context of that particular
phylogeny. It is traditionally tested using now ubiquitous
data-resampling techniques to generate support values, but
even strong advocates of these techniques are being obliged
to acknowledge their limitations. For example: ‘With high
support for their tree, researchers can become confident in
incorrect topologies. Although the bootstrap method for
assessing confidence was originally suggested to provide
confidence intervals for tree branches (i.e. how well the data
at hand represent an underlying universe of data), it is now
well-recognized that this resampling method and others,
such as the jack-knife, provide, at best, a representation of
confidence [pertinent] only [to] the data at hand; even
random data can yield high bootstrap support. Furthermore,
bootstrap values decrease as the number of taxa [included]
increases, making it much more likely that high bootstrap
values will be obtained if few taxa are analyzed.’ (Soltis
et al., 2004, pp. 478–479). In other words, strong statistical
support for a particular topology (or, more likely, a set of
equally probable topologies)—that is, for a specific hypothesis of relationships—has little bearing on the unknowable property of its accuracy. This conclusion is borne out
by numerous studies wherein highly contrasting, yet reliably
strongly statistically supported, hypotheses of relationship
have been generated from the same substantial data matrix.
Such internal contradictions are achieved either by using
3482 Bateman et al.
contrasting tree-building algorithms or by subsampling the
matrix; for example, by removing ‘wildcard’ taxa that
demonstrably destabilize the topology (an act that often
passes unreported in the resulting publications: cf. Hilton
and Bateman, 2006), or by omitting specific categories of
characters (e.g. third bases in codons: Jeffroy et al., 2006),
or both (Philippe, 2006).
If accuracy is unknowable, and robustness unreliable,
what criteria are left by which to judge the credibility of
a particular inferred phylogeny? Most workers, including
ourselves, consider the key word to be congruence. This
term was originally coined to describe the relative behaviours of comparable characters (typically morphological
features) when simultaneously subjected to a parsimony
analysis, which aimed to minimize incongruence among
characters (termed homoplasy). But soon incongruence was
also used to describe any differences between multiple
topologies describing the relationships of the same range of
coded taxa.
The latter usage is well illustrated by an insightful, yet
infuriating, study on ‘resolving incongruence’ in phylogenetic relationships among eight yeast species by sequencing
106 nuclear genes (;2% of the number present in the
genome: Rokas et al., 2003). Despite the small number of
taxa analysed, the majority-rule consensus trees generated
from each gene collectively offered 20 alternative phylogenetic hypotheses, most rich in ‘strongly supported’ nodes
(defined by an arbitrary threshold of a bootstrap value
exceeding 70%). Over half of the gene trees required the
removal of at least two of the eight species to achieve
topological congruence. Moreover, none of the data
partitions or statistical patterns explored among the genes
allowed the relative performances of individual genes to be
predicted or cogently explained. Nonetheless, ‘concatenating’ (combining) all 106 genes ‘yielded a single tree with
100% bootstrap values at every branch’, so the authors
‘conclude[d] that it accurately represents the historical
relationships of these eight yeast taxa and will be referred
to hereafter as their species tree [they eventually concluded
that, on average, 20 genes would generate a reliable species
tree]. The maximum support for a single topology regardless of the method of analysis is strongly suggestive of the
power of large data sets in overcoming the incongruence
present in single-gene analyses’ (Rokas et al., 2003, p.
800). We find this conclusion extraordinary; in our view,
the incongruence has not been ‘overcome’ but rather obscured by the sheer volume of data and the failure to explain
the cause(s) of the profoundly conflicting phylogenetic
signals provided by the individual genes (for a re-evaluation
of this data-set see the final section of this paper).
Rather, we are most interested in assessing topological
congruence between trees generated from different categories of data, each scored for the same set of taxa (preferably
the same species, and ideally the same representative
individuals). There is certainly a strong case for comparing
phylogenetic signals from the three plant genomes, and
from exons and introns; these genic regions operate under
strongly contrasting molecular constraints (e.g. Page and
Holmes, 1998). Obtaining similar results from all (or at
least most) categories of data greatly increases our confidence (in the biological, rather than the statistical, sense)
that the relationships recovered are credible. But there is
a strong case for analysing genic regions separately before
combining them (e.g. Bateman, 1999).
Surely the most versatile category of phylogenetic data is
morphology sensu lato. Although restricted in total number
of characters, most of those characters are phylogenetically
informative (Wortley and Scotland, 2006) and each is potentially divisible into a large number of meaningful character states. Morphology informs not only on phylogenetic
relationships but also on function, and through function it
directly reflects various modes of natural selection. Detailed
knowledge of morphology, along with other biologically
relevant data such as symbiotic partners, habitat preference
and environmental tolerance, is essential to any evolutionary interpretation of the range of taxa under scrutiny. And
morphology maximizes taxon sampling by allowing full
integration of fossil species, which serve a vital role in
filling the vast morphological and molecular lacunae that
separate clades possessing extant representatives.
Thus, the congruence that we seek is similar topologies
generated from the same set of taxa by highly contrasting
types of data, including morphology. Where trees generated
from one or more categories of data (including morphology) disagree with trees generated from the majority of
categories, a causal explanation should be sought, based on
our knowledge of the biological properties of each category
of data. This is the simplest way to identify processes that
can confound reliable reconstruction of credible phylogenies.
For molecular data matrices, such processes include
lateral gene transfer between taxa (Bergthorsson et al.,
2004), lateral gene transfer among the three genomes
within a plant (Huang et al., 2005), hybridization (Linder
and Rieseberg, 2004), organelle capture (Rieseberg and
Wendel, 1993), chromosomally localized gene duplication
(Zahn et al., 2005), wholesale gene duplication through
polyploidy (De Bodt et al., 2005), lineage sorting (Doyle
et al., 1999; Degnan and Rosenberg, 2006), and codon bias.
As summarized by Frohlich (2006), codon bias describes
substantial differences in the relative frequencies of synonymous codons specifying particular amino acids. It can
reflect both mutational biases that affect overall GC content
and selection on individual codons (Kawai and Otsuka,
2004; Liu et al., 2004; Liu and Xue, 2005; Jeffroy et al.,
2006). Both factors primarily affect third codon positions in
general and transitions in particular, and are especially
problematic in highly expressed genes. This is because
selection among codons is hypothesized to reflect the
relative abundances of tRNAs that bear different anticodons
but synonymously insert the same amino acid; as a codon
Phylogenetic context of the flower
becomes more scarce, the synthesis of its protein product
is increasingly inhibited. This argument can be used to
justify removal of third positions from phylogenetic
analyses of highly expressed genes, including typical
plastid genes. Such process-based explanations are, in our
view, a necessary pre-requisite for selectively removing
certain kinds of information from a phylogenetic matrix
(see below).
By contrast, morphology can be undermined by convergence toward similar function(s) that is driven by directional or disruptive selection; examples include substantial
vegetative modifications associated with transitions from
terrestrial to aquatic habitats and from autotrophic to
heterotrophic nutrition (e.g. Bateman, 1996), and floral
transitions associated with switching pollinators (e.g.
Chase, 1999). Less frequently discussed but also problematic are multiple, closely juxtaposed paedomorphic transitions within a clade, where the consequent morphological
simplification can under certain circumstances be misconstrued by parsimony as primitiveness rather than as secondary simplification (Bateman, 1996).
Do some nodes in the land-plant phylogeny
merit particular emphasis?
In practice, it is congruence among phylogenetic studies
employing contrasting data matrices that has provided
a study of land-plant phylogeny with its key reference
nodes delimiting major monophyletic groups. Here, we
contrast some widely accepted ‘reference nodes’ with other
critical areas of land-plant phylogeny that ostensibly carry
much greater uncertainty. In order to effectively deploy a
bottom-up approach to understanding angiosperm origins,
it is necessary to begin the discussion at phylogenetic nodes
well below those of greatest interest.
Working upward through a crude consensus phylogeny
that is based only on extant groups of land-plants but
considering both morphological and molecular analyses
(Fig. 2), we immediately encounter the embryophytes,
a group that is diagnosed by the presence of archegonia and
antheridia and so encompasses all land plants from bryophytes onwards. Excluding the ‘bryophytes’ leaves the
monophyletic tracheophytes, which possess bona fide
xylem. This is followed by a basal dichotomy within the
‘pteridophytes’ between the microphyllous lycopsids and
megaphyllous euphyllophytes. Excluding the ‘moniliformopses’ (‘ferns’ sensu lato) from the euphyllophytes leaves
a monophyletic seed-bearing spermatophytes; this clade
encompasses four gymnospermous groups (Figs 4, 5), each
widely regarded as monophyletic, plus the similarly monophyletic angiosperms, which arguably are best diagnosed
by their possession of a triploid endosperm (however,
even this character is unreliable in early-divergent angiosperms; for example, the endosperm is diploid in Nuphar
3483
polysepalum: Williams and Friedman, 2002). Bypassing
a few, mostly small, basally divergent groups to which we
will presently return, 96% of extant angiosperm species
fall into two groups viewed as sister clades in a minority
of analyses: monocots and eudicots. Monocots must be
diagnosed by a gestalt of characters, each unreliable on its
own. They possess a single cotyledon and lack a vascular
cambium (both characters in common with a few earlydivergent angiosperms and eudicots). Other putative
monocot characters, such as trimerous flowers and sulcate
pollen, are actually common among early-divergent angiosperms (e.g. Rudall and Furness, 1997). Most core
eudicots are well defined by the possession of tricolpateor tricolpate-derived-pollen (i.e. pollen with three equatorial apertures), which is otherwise extremely rare (Furness
and Rudall, 2004).
There are four main areas of uncertain relationships in
such fossil-free trees:
(i) among the three main groups of bryophytes: liverworts, hornworts, and mosses;
(ii) among the five main groups of megaphyllous ‘ferns’:
Psilotales, Ophioglossales, Marattiales, leptosporangiates, and equisetophytes;
(iii) among the four main gymnosperm groups: cycads
(Cycadaceae sister to Stangeriaceae plus the relatively
diverse Zamiaceae), Ginkgo, conifers, and Gnetales
(the relatively species-rich Ephedra sister to Gnetum
plus Welwitschia), and;
(iv) among the six main groups of basally divergent
angiosperms: Amborella, Nymphaeales (perhaps
including the antipodean hydrophiles of the
Hydatellaceae; S Graham, pers. comm., 2006), Austrobaileyales (Austrobaileyaceae plus Schisandraceae
plus Trimeniaceae), Chloranthaceae, magnoliids (including Piperales/Aristolochiaceae and Laurales—the
only one of these ‘basal’ groups that exceeds 100
species), and Ceratophyllum.
Each of these four problematic groups (one bryophytic, one
pteridophytic, one gymnospermous, and one angiospermous) is highly morphologically diverse.
If we parse through the land-plant phylogeny once again,
this time including extinct taxa (and so confining our
attention to morphological analyses), a substantially more
complex story emerges (Fig. 4). The Early Devonian
Aglaophyton, which uniquely possesses near-isomorphic
sporophyte and gametophyte generations (e.g. Kenrick,
1994), is interpolated between the bryophytes and tracheophytes. The fossil rhyniophytes form a distinctively
vascularized clade that is sister to the eutracheophytes.
The lycopsids are subtended by the wholly extinct SiluroDevonian cooksonioids (probably paraphyletic) and zosterophylls (either monophyletic or paraphyletic: zosterophylls
plus lycopsids=lycophytes). The Devonian psilophytes
and (arguably) the Devono-Carboniferous stauropterids
3484 Bateman et al.
interpolate at the base of the euphyllophytes, while DevonoCarboniferous cladoxyls provide potentially crucial information on the phylogenetic position of equisetophytes
(Rothwell and Nixon, 2006). Devono-Carboniferous progymnosperms form an either monophyletic or, more likely,
paraphyletic group that is interpolated between the ferns
sensu lato and the spermatophytes, thereby permitting
recognition of the lignophytes (progymnosperms plus
spermatophytes).
Most critical of all from the viewpoint of inferring angiosperm origins is the immense diversity of gymnosperm
groups that span a long and phylogenetically crucial period
from the Late Devonian to the Cretaceous (see below).
Three major groups of extinct Devono-Carboniferous seedferns (hydraspermans, medullosans, and callistophytes)
together form a wholly extinct paraphyletic group of ‘core
pteridosperms’, placed immediately below the divergence
point of the cycads (Fig. 4). Paraphyletic or monophyletic
Triasso-Jurassic peltasperms diverge above the cycads.
Monophyletic Carbonifero-Permian cordaites form a sisterclade to the conifers, which now show multiple divergences
of fossil taxa below the ‘crown group’. And in several
studies, four derived gymnospermous groups, one PermoTriassic (glossopterids) and three late Triassic–early Cretaceous (Pentoxylon, bennettites, Caytonia: Fig. 1C, D), each
of which is resolved as sharing several attractive synapomorphies with extant angiosperms, form a paraphyletic
group immediately subtending the angiosperm clade (e.g.
Doyle, 2006; Hilton and Bateman, 2006).
Interestingly, it is only when considering relationships
among angiosperm groups that the fossil record ceases to be
central to the discussion. Much-debated fossils explored
at greater length below, such as Archaefructus (Sun et al.,
2002) and Sinocarpus (Leng and Friis, 2003) from the late
Early Cretaceous (late Hauterivian–Early Aptian) Jehol
biota (Zhou et al., 2003), were initially said to occupy
phylogenetic positions earlier than the first divergence
among extant angiosperm taxa (e.g. Sun et al., 1998).
However, to some observers they appear more derived on
closer examination, seemingly fitting comfortably within
the angiosperm ‘crown group’ (Friis et al., 2003, 2006;
Hilton and Bateman, 2006: see later discussion).
The node defining the crown group (Fig. 3) was
described by Frohlich (2006) as constituting the ‘basal
flower’, and its attributes as ‘the endpoint for any reasonable theory of flower origins.’ Certainly, on present
knowledge, the hypothetical ancestor of the crown-group
angiosperms can legitimately be reconstructed on the basis
of extant taxa alone, in what has become the standard topdown approach (e.g. Doyle and Endress, 2000; Endress
2001b). However, we believe that fresh insights are more
likely to arise from a bottom-up perspective on angiosperm
origins. If so, it is a mistake to over-emphasize this
particular node, which reflects the mere happenstance
survival of extant lineages.
Given that some nodes delimiting major clades are
widely accepted, there is an increasing temptation to
constrain the results of further phylogenetic analyses to
maintain those ‘known’ relationships (e.g. Doyle, 2006;
Frohlich, 2006), irrespective of levels of character support
within the matrix in question, and to focus attention on less
well-understood regions of the phylogeny. For example,
there have been no recent phylogenetic analyses, either
morphological or molecular, that contradict the widely
accepted monophyly of angiosperms; surely we can now
take that node as read and routinely programme it into our
tree-building exercises? In our view, this methodology is
an excellent servant but a poor master. It is most useful as
an experimental tool for exploring specific hypotheses
of relationship.
For example, using similar morphological matrices, both
Hilton and Bateman (2006) and Doyle (2006) tested the
effect of constraining their topology to enforce monophyly
of all extant gymnosperms, reflecting a topology recovered
from several recent molecular analyses of extant seedplants (Figs 2B, 5). Hilton and Bateman simply posed the
question: How much less parsimonious is the morphological analysis when constrained by that one critical molecularly defined node? By contrast, Doyle elected to base the
bulk of his excellent discussion of morphological characterstate transitions among seed-plants on the unparsimonious
topology that was constrained by the node that specified
extant gymnosperm monophyly and was derived from
wholly different (molecular) data matrices. Our preference
is to give the characters that have been painstakingly coded
within a particular matrix full freedom to express themselves. No node in land-plant phylogeny carries sufficient
certainty to override the parsimony principle that is rightly
fundamental to phylogeny reconstruction.
Why is it important that optimal taxon sampling
dissects long branches?
The weakness of adopting only a neobotanical, top-down
view of angiosperm origins becomes immediately apparent
when one continues down the land-plant phylogeny from
the node subtending the earliest divergence of extant
lineages. There is no widely accepted evidence of fossil
angiosperms older than the Early Cretaceous (;130 Ma),
while the earliest evidence of their molecular sister-group,
the gymnosperms, dates controversially from the Late
Carboniferous (;290 Ma; putative primitive walchian
conifers: Hernandez-Castillo et al., 2001) and certainly
from the earliest Permian (;280 Ma; Cycas-like sporophylls: Gao and Thomas, 1989). The intervening period
represents sufficient time not only for a great deal of
morphological innovation, which tends to occur very
rapidly on a geological time-scale, but also for extensive
molecular change, which is widely recognized to follow
Phylogenetic context of the flower
a broadly clock-like pattern (Bateman, 1999; Bateman and
DiMichele, 2002; Sanderson et al., 2004). In other words,
irrespective of whether one is considering molecular or
morphological trees (with or without fossil taxa), the node
subtending the last-diverging gymnosperms and the
adjacent, relatively derived node subtending the firstdiverging angiosperms are separated by an archetypal
long branch (Hilton and Bateman, 2006) (branch emphasized in Fig. 3).
We have already encountered some of the problems
presented by both phenotypic and genotypic long branches;
their tendency to attract each other to generate a false
statement of relationship, and their tendency to receive
maximum bootstrap/jack-knife values that can be misinterpreted as evidence of accuracy. In addition, they can
substantially weaken the effectiveness of outgroup comparison for rooting trees and polarizing characters, and pose
particular challenges to a posteriori character analysis.
Specifically, we cannot to infer the sequence in which the
many character-state transitions supporting that branch took
place, or determine whether they might be developmentally
correlated. Our a priori assertions of homology of contrasting states of a character on either side of that branch will be
less secure. Hypothetical ancestors reconstructed for the
crucial internal nodes located at either side of the long
branch will, by definition, be strongly divergent.
Fortunately, each of these problems can potentially be
alleviated by improved taxon sampling. Increasing the
number, and especially the phylogenetic spectrum, of taxa
included in an analysis is likely to shorten branches
supported by multiple character-state transitions, so that
we can infer the temporal sequence in which those
transitions occurred (Fig. 6). By dissociating previously
positively co-occurring character-state transitions we also
demonstrate that they are not directly developmentally or
pleiotropically correlated, which in turn falsifies any prior
hypothesis of saltational macroevolution that requires each
of those character-state transitions (such profound and
instantaneous heritable shifts in phenotype are the most
appropriate null hypothesis for multi-transition branches
according to Bateman and DiMichele, 1994, 2002).
Shortening the branch that separates a monophyletic
ingroup from the operational outgroups is especially desirable (Fig. 6). We have already discussed the critical role of
outgroup choice in rooting the tree and thereby polarizing
character-state changes, and the reasons why the branch
separating the outgroup(s) from the ingroup taxa will likely
be long relative to those among the ingroup taxa. One or
more added taxa that interpolate into that portion of the tree
therefore have a disproportionately greater effect in improving the reliability of the resulting phylogeny, particularly by reducing the uncertainties surrounding homology
assessment during the a priori character delimitation
process (this statement applies equally to phenotypic and
genotypic matrices).
3485
In an ideal world, we would score every taxon that ever
existed in the chosen clade. In practice, the best strategy is
to sample coded taxa that are hypothesized to be distributed
approximately evenly across the phylogeny under scrutiny.
Where the available taxa are, by contrast, clustered in
morphological and/or molecular space, increasing sampling
density will simply add taxa that possess similar characteristics to those already included in the analysis, and the
critical branches will not be significantly shortened; much
analytical effort will thus have been expended for little
interpretational reward. Beyond a certain density of sampling, this is inevitably the case in phylogenetically broad
studies that analyse only extant taxa. It therefore becomes
a powerful argument for pursuing analytical protocols
that can allow active, and preferably full, participation of
fossil taxa (Fig. 4).
What are the effects of culling taxa and/or
characters?
The above argument can also be reversed by deliberately
reducing the number of taxa included in a particular
analysis in order to see what proportion of the original
statements of relationship are retained in the reduced data
matrix. One might hope that most data-rich matrices,
molecular or morphological, would withstand this approach
robustly, but that is rarely the case for either morphological
(e.g. Hilton and Bateman, 2006; Rothwell and Nixon,
2006) or molecular (e.g. Rydin and Källersjö, 2002; Rydin
et al., 2002) matrices. However, most matrices yield
substantially altered relationships among the remaining
taxa after the removal of relatively few taxa.
This effect is most pronounced where it is the number
or identity of the operational outgroups that is altered. For
example, Hilton and Bateman (2006) used in their core
analysis three reconstructed progymnosperms to root their
morphological analysis of extant plus extinct seed plants
(Fig. 4). When only one (arguably the most derived) of the
three progymnosperms, Cecropsis, was identified as outgroup, the rooting of the tree was radically altered.
Specifically, the relationships of early-diverging taxa more
closely resembled the results of recent molecular analyses,
with angiosperms diverging below a monophyletic group of
extant gymnosperms. By contrast, perceived relationships
among the angiosperms, and among the extant gymnosperms, did not converge on those generated in molecular
studies; rather, the fundamental polarity of evolutionary
transitions was reversed. Moreover, only when Doyle
(2006) constrained his similar but more angiospermfocused morphological matrix to specify obligate monophyly of extant gymnosperms did he obtain a topology
among the coded angiosperm taxa that resembled that in
the most prominent molecular trees. In other words, forcing
Gnetales into a sister-group relationship with extant
3486 Bateman et al.
Fig. 6. Hypothetical phylogeny illustrating the use of phylogenies as
tests of correlation among characters (and thus of saltational hypotheses
of evolution). The initial analysis of outgroup species O and ingroup
species A–C yields a single fully resolved topology. Relatively long
branches subtend the ingroup (species A–C) and species C alone.
Simultaneous change in the five character-state transition characters
supporting each of these branches is assumed as the null hypothesis, and
then tested by re-analysing the matrix after adding four further species
(labelled D–G) that fortuitously do not perturb the previous topology.
Species D is attached to an irrelevant branch. Although species E is
connected to a relevant branch, it fails to dissociate the four correlated
character-state transitions. However, addition of species F separates
characters 1 and 2 from characters 3–5. This implies that the state
transitions occurred earlier in characters 1 and 2, and thereby falsifies that
part of the prior saltational hypothesis. Species G performs a similar
role on the branch separating outgroup O from the ingroup. Note that
the fossil status of three of the species (B, F, O; asterisked) has no bearing
on their analytical performance. Modified after Bateman and DiMichele
(1994, fig. 3).
conifers has profound topological effects on a clade that
diverges from a node several nodes distant from the taxa
that actually caused the topological shift.
Moving on to molecular data, Soltis et al. (2004, fig. 3)
were able to recover the much-maligned ‘monocots sister to
dicots, Amborella sister to Calycanthus’ topology obtained
from whole-plastid sequences by Goremykin et al. (2003,
2004) by reducing their own three-gene matrix to a similar
number of taxa. However, they were also able to obtain
from their data matrix their perception of the ‘correct’
topology—Amborella diverging before monocots which
in turn diverge before Calycanthus—simply by adding to
their analysis the orchid Oncidium. The Goremykin group
responded by adding to their whole-plastid analyses
data for Nymphaea (Goremykin et al., 2004), Acorus
(Goremykin et al., 2005), and Phalaenopsis (Chang
et al., 2006; Goremykin and Hellwig, 2006) with no
consequent modification to their original topology. Indeed,
a weakly supported ‘grasses-basal’ topology was also
obtained by Burleigh et al. (2006) in their bootstrap
consensus supertree, based on their gap-rich matrix of
69 taxa and 254 genes. Due to the large volumes of data
involved, all pertinent relationships have strong statistical
support. How then do we decide which statement of
relationships is the more probable?
Several other taxonomically broad phylogenetic studies
have explored the topological consequences of culling
particular categories of character. The majority have considered the relative effects in expressed regions of sequenced genomes of first and second versus third position
nucleotides in the codons that dictate the identity of the
resulting amino acids. For example, the detailed analysis of
the large plastid regions psaA plus psaB of 63 extant landplants by Magallón and Sanderson (2002, fig. 3) generated
substantially different topologies from parsimony analyses
using (i) positions 1+2, (ii) position 3 alone, and (iii)
positions 1+2+3. Analyses (ii) and (iii) placed horsetails
below ferns, which were in turn improbably placed below
lycopsids. Extant conifers were not monophyletic, with
Gnetales the earliest to diverge. And Amborella diverged
below rather than above Nymphaeales. Thus, analysis (i)
would reconstruct hypothetical ancestors radically different
from those of analyses (ii) and (iii) at the nodes on either
side of the crucial branch separating gymnosperms from
angiosperms. Adding insult to injury, maximum likelihood
topologies differed significantly from parsimony topologies
generated from the same matrix.
Similarly, the parsimony analysis of 31 extant vascular
land-plants and 13 genic regions by Burleigh and Mathews
(2004, fig. 7) revealed radically different placements between nuclear and plastid regions on the one hand, and
mitochondrial regions on the other when all sites were
included. However, when the most rapidly mutating sites
were excluded (actually constituting two-thirds of the
matrix), the three genomes yielded far more congruent
topologies that placed Gnetales as sister to Pinaceae, agreed
on extant gymnosperm monophyly, and placed Amborella
as the earliest diverging extant angiosperm. Predictably,
the authors concluded that the broad congruence of these
signals from the three genomic compartments outweighed
the contrasting signal when the number of characters used
was maximized. Thus, the supposed primary advantage of
molecular phylogenetic data—quantity of characters—was
pragmatically qualified by the understandable desire to
improve the perceived overall quality of signal obtained
from the data. Moreover, this decision was constructively
supported by an at least partial (and enhanced) understanding of the biological differences between the data types
retained and those discarded.
What are the pros and cons of monophyly?
The conventional ‘ladderized’ classification of land plants,
based largely on grades rather than clades (i.e. bryophytes,
pteridophytes, gymnosperms, angiosperms), was rapidly
overturned by morphological phylogenies that strongly
Phylogenetic context of the flower
emphasized recognition of monophyletic groups. Taxonomically well-sampled studies agreed that all major groups
other than angiosperms were non-monophyletic (Figs 2A,
4). However, this conclusion has often been challenged
by multi-gene molecular phylogenies using only extant
taxa, which increasingly resolve as monophyletic not only
extant angiosperms but also extant gymnosperms (cycads,
Gingko, conifers, and, controversially, Gnetales), extant
pteridophytes, and (less confidently) extant bryophytes
(Figs 2B, 5). Thus, high-level classifications of land plants
that emphasize monophyletic groups increasingly resemble
early, pre-cladistic models, encouraging an improbable
approchement between two previously disparate groups
of researchers: molecular phylogeneticists and those systematic morphologists who never accepted the cladistic
paradigm. Only some phylogenetic morphologists, particularly those with palaeobotanical interests like us, remain
unconvinced that these events constitute unequivocal
progress. What lies at the root of our continued scepticism?
We have already addressed one major set of concerns—
the profoundly deleterious consequences of the vast
phylogenetic lacunae that separate the major groups that
possess extant representatives: bryophytes versus pteridophytes versus cycads versus Ginkgo versus conifers versus
Gnetales versus basal angiosperms. These render less
certain a priori homology assessment and tree rooting via
outgroups, and increase the probability of generating
incorrect topologies through long-branch attraction, particularly when using sequence data.
The second motivation is that a broader knowledge of the
morphological diversity shown by land-plants since their
putative Silurian origin not only reinforces our perception
of the scale of the lacunae separating the extant ‘crown
groups’ but also emphasizes the improbability that the
molecular topologies specifying major-group monophyly
are correct. Monophyly of extant non-lycopsid pteridophytes means that the lineage leading to lignophytes
(progymnosperms plus seed-plants) diverged before the
divergences among the major eusporangiate groups (including the Psilotales and Equisetales) and before that
separating the eusporangiate and leptosporangiate taxa.
Similarly, monophyly of extant gymnosperms requires that
the lineage leading to the ‘crown group’ angiosperms
diverged before all of the diverse gymnosperm lineages
(e.g. Doyle, 1998a). This statement applies even to the
cycads, which are widely accepted as retaining many
plesiomorphic characteristics and occur in latest Pennsylvanian assemblages. This hypothesis of relationship demands a minimum of 160 my with no readily recognized
fossil angiosperms, and yet more time elapsed prior to their
well-defined mid-Cretaceous radiation (Friis et al., 2006).
Thus, we can either accept that no extant fern lies on the
lineage leading to spermatophytes and no extant gymnosperm lies on the lineage leading to angiosperms, despite
the extremely low combined probability that both state-
3487
ments are correct, or we can explore further the possibility
that sequence-based phylogenies are biased toward monophyly, presumably as a consequence of the aforementioned
lacunae in taxon sampling. Certainly, we believe it is critical that debates continue regarding the pros and cons of
specific topologies (and specific kinds of data).
The third set of issues concerning monophyly is central
to the topic of this essay. If we perceive extant land-plants
as constituting just five major taxonomic groups (in order of
successive divergence: bryophytes, lycopsids, non-lycopsid
pteridophytes, gymnosperms plus angiosperms; Fig. 2),
we have no evidence available to reconstruct the lengthy
sequences of character-state acquisitions (i.e. morphologically and molecularly long branches) between these
vastly disparate groups. In other words, we cannot to address
directly the most profound question in land-plant evolution.
Thus, monophyly of major groups is a boon to classification
but an anathema to those of us wishing to study the patterns
and processes of plant evolution. Given this observation,
are our concerns regarding the likelihood of majorgroup monophyly driven less by a dispassionate view of
existing data than by biased self-interest?
Which key characters best define the flower?
Understandably, most developmental geneticists have
come to view the Arabidopsis flower as archetypal, yet
presumably its standard complement of 4 sepals, 4 petals,
6 (4+2) stamens and 2 carpels is derived from the classic
5+5+5+[2–5] floral bauplan of the eudicot clade, which
encompasses ;75% of extant angiosperm species. This
clade is well-defined by both molecular and morphological
data, and possesses some relatively consistent synapomorphies, notably the possession of tricolpate- and tricolpatederived-pollen, and also (at least in core eudicots) a ‘typical’
floral groundplan of hermaphrodite pentamerous flowers
showing clear differentiation of the perianth into sepals and
petals, an androecium of two stamen whorls, and a synorganized gynoecium of two to five fused carpels.
Furthermore, a clear series of basally divergent extant
eudicots (the non-core eudicots sensu Angiosperm Phylogeny Group, 2003) allows bottom-up polarization of floral
character states (e.g. Soltis et al., 2003; Ronse Decraene,
2004), though even this approach is problematic. For
example, Wanntorp and Ronse Decraene (2005) cautioned
that the minute, dimerous and highly reduced flowers of
Gunnerales (the putative sister to the core eudicots in many
recent molecular phylogenies) are unlikely candidates for
the ‘ancestral’ eudicot flower.
By contrast, understanding the origin of the angiosperm
flower is a far more challenging task. The plethora of often
contradictory theories surrounding the origin of the flower
(e.g. Arber and Parkin, 1907; Wettstein, 1907; Melville,
1960; Eames, 1961; Meeuse, 1975, 1987; Hughes, 1976;
Burger, 1977) suggests that a comparative top-down study
3488 Bateman et al.
of extant angiosperms cannot provide an unequivocal
picture of floral evolution. As outlined above, floral
structures in early-divergent angiosperms are highly diverse
and often lack definitive features, including hermaphroditism, fully closed carpels, and a distinctly whorled arrangement of organs.
Certainly, the flower is less readily definable in groups
that diverged prior to the core eudicots. For example, it is
problematic to distinguish between sepals and petals in the
majority of monocots and early-divergent angiosperms
(Endress, 2001a, b). Endress (2001a) usefully disassembled the flower into components that occur in all seed
plants (ovules and microsporangia) and uniquely angiospermous elements (carpels). The carpel can be interpreted
either as an ovule-bearing leaf homologue (a megasporophyll) or as a composite organ consisting of an ovulebearing structure surrounded by a modified bract. Thus,
Endress argued that the ‘real’ angiosperm innovation is
post-genital carpel fusion, which led to further morphological innovations. However, he also acknowledged that in
many early-divergent extant angiosperms carpel closure
is caused not by bona fide fusion but rather by occlusion
via ‘secretion’ (presumably of mucilage).
Top-down comparisons of extant angiosperms with
putative stem-group fossils also fail to resolve the debate.
The case of the fossil aquatic plant Archaefructus, excavated from the Yixian Formation of north-east China,
nicely illustrates this point. When first discovered, Archaefructus was considered to be late Jurassic in age (Sun
et al., 1998) and was interpreted as a basal angiosperm
providing plesiomorphic morphological characters elucidating our understanding of angiosperm origins. Archaefructus was consequently hailed as a potential sister-taxon
to all extant angiosperms, based on a morphological
phylogenetic analysis (Sun et al., 2002). However, the
age of the Yixian Formation was subsequently reinterpreted
as late early Cretaceous (e.g. Barrett, 2000; Zhou et al.,
2003), significantly younger than originally claimed.
Moreover, Friis et al. (2003) reinvestigated these and other
associated fossils, leading to their own morphological
phylogenetic analysis. Their re-coding of the characters
suggested that Archaefructus is better interpreted as either
a magnoliid or even a basal eudicot, albeit one specialized
for an aquatic habitat. Like several extant early-divergent
angiosperms, Archaefructus lacks a perianth, and presents
an array of structures of questionable homology. Friis et al.
(2003) suggested that it had unisexual flowers consisting
of only two stamens (in male flowers) or one or two
carpels (in female flowers), rather than possessing the
hermaphrodite flowers with axially separated paired
carpels and stamens envisaged by Sun et al. (2002). Their
reinterpretation has been supported by subsequent palaeobotanical discoveries (Ji et al., 2004; Friis et al., 2006).
This ambivalent structure is consistent with that of
another aquatic wildcard taxon, Ceratophyllum, and some
aquatic monocots in the monocot order Alismatales, whose
origin and floral homologies have proved similarly difficult
to interpret (reviewed by Sokoloff et al., 2006), as have
those of the hydrophilic Hydatellaceae. However, unlike
Archaefructus, these are extant taxa, so that their homologies could potentially be explored using evolutionarydevelopmental genetic techniques (see below). Less
controversially, the similarly aged genus Sinocarpus is
also placed as a basal eudicot (Leng and Friis, 2003) and so
belongs among the ‘crown group’ angiosperms. In the
morphological analysis of Hilton and Bateman (2006),
adding Sinocarpus left the original topology unaltered, but
it was placed within the ‘crown group’ as sister to the
magnoliid family Piperaceae. By contrast (and perhaps
predictably), Archaefructus acted as a wildcard taxon,
destabilizing relationships among the basal extant groups
to generate a five-taxon polytomy.
Endress (2001a) suggested that the two most prominent
angiosperm floral elements, carpels and thecal stamen
organization, likely arose as key innovations in as-yet
undiscovered stem-group angiosperms. By contrast, features adapted to attract pollinators, such as a colourful
perianth and odour- and nectar-producing structures, probably evolved more than once within the angiosperm clade.
Scent production, sometimes associated with internal heat
generation, is believed by some to have evolutionarily
preceded colour (Thien et al., 2000). Based on fossil
evidence of insect faecal remains in bennettite strobili and
gymnosperm pollen in the gut contents of fossil insects,
Labandeira (1997, 1998) argued that insect pollination was
common before the first recognizable angiosperms appeared in the late Jurassic, at a time when foraging insects
sought pollen rather than nectar. Floral diversity among
extant early-divergent angiosperms probably reflects experimentation with insect mutualisms, especially with small
insects such as beetles, flies, and thrips (Thien et al., 2000;
Endress, 2001b). Much evidence, from both morphology
and developmental genetics, indicates multiple origins of
petals, either from bracts (termed bracteopetals) or from
stamens (termed andropetals). Only andropetals are commonly associated with a differentiated perianth (reviewed
by Kramer and Irish, 2000). Some early-divergent angiosperms lack a perianth entirely, including some Piperales
and most Chloranthaceae; both these groups are well
represented in the early Cretaceous fossil record (e.g. Friis
et al., 2000, 2006). These taxa tend to have more generalist
pollination strategies; for example, many Saururaceae
(Piperales) are pollinated by a diverse range of small
insects and a few by wind (Thien et al., 2000).
Why are palaeobotanists obsessed with
extinct gymnosperms?
Although we could perhaps consider ourselves fortunate to
have in the extant flora representatives of at least three and
Phylogenetic context of the flower
arguably four major lineages of gymnosperms—cycads,
Gingko, conifers, and Gnetales—these represent a modest
fraction of the total morphological diversity evident among
the gymnosperms if fossil groups are also considered (e.g.
Stewart and Rothwell, 1993). It is therefore feasible, and
even likely, that these groups plus the angiosperms arose as
independent lineages from within the plexus of wholly
extinct gymnosperms. Certainly, in morphological phylogenetic analyses rich in fossil taxa these extant groups
rarely emerge as sisters; rather, each is sister to at least one
wholly fossil lineage (reviewed by Hilton and Bateman,
2006) (Fig. 4).
Just as fossil progymnosperms are crucial for routinely
inferring a single origin of seed-plants, the combined fossil
and extant gymnosperms are critical for routinely inferring
a single origin of angiosperms. Here, at least, there is
general agreement between morphological and molecular
phylogenies (Fig. 2). However, by contrast, recent molecular assertions of monophyly of extant gymnosperms
profoundly alter our perception of seed-plant relationships
(cf. Figs 4, 5).
Most discussions have focused on the controversial
placements of the clade formed by the three extant genera
of Gnetales as either sister to extant conifers or even just
extant Pinaceae. However, the controversy surrounding
the phylogenetic placement of Gnetales [reviewed by
Burleigh and Mathews (2004), with more recent contributions from Burleigh et al. (2006), Hajibabaei et al. (2006),
and D Quandt (pers. comm., 2006)] has tended to obscure
the potential interest of the more plesiomorphic taxa such as
Ginkgo and especially cycads, which on morphological
phylogenetic evidence could legitimately be regarded as
extant representatives of the supposedly wholly extinct
pteridosperms (Hilton and Bateman, 2006). Extant cycads
and Ginkgo offer a unique opportunity to examine the
characteristics of early-diverging spermatophytes (Brenner
et al., 2003). However, surprisingly few modern studies
exist of these ‘living fossils’, and those that do rarely employ
a bottom-up comparative approach. One notable exception
is the ontogenetic study by Mundry and Stützel (2003),
who reinterpreted the male sporangiophores of Zamia as
pinnate, with synangia on reduced leaflets, and hence suggested that cycad male sporangiophores may be better
homologized with the radial synangial groups observed in
some Carboniferous medullosan pteridosperms than with
the simple sporangiophores that characterize extant conifers.
For those of us familiar with fossil gymnosperms, the
implication that extant angiosperms diverged prior to the
separation of extant cycads and Ginkgo appears strongly
incongruent, both with morphological phylogenies and
with first appearances of relevant taxa in the fossil record.
Admittedly, as noted by Doyle (2006), ‘the position of
cycads is one of the more weakly supported aspects of
molecular phylogenies (cf. Magallón and Sanderson, 2002;
Soltis et al., 2002)’. Nevertheless, extant gymnosperm
3489
monophyly would require that angiosperms diverged from
the other seed plants by the end of the Carboniferous (;290
Ma), when the only likely candidates for ancestor in the
fossil record are various pteridosperm lineages (Fig. 4).
This necessitates an extraordinarily long angiosperm ghost
lineage of some 160 my to reach the point in time when
the first widely accepted angiosperm fossils appear, in the
early Cretaceous.
However, accepting at face value this almost interminable ghost lineage constitutes an unnecessarily nihilistic
view of the implications of such phylogenies. Although the
molecular placement of extant Gnetales adjacent to or
within extant conifers has led to widespread triumphing
of the demise of the anthophyte hypotheses (and of the
implicit pseudanthial origin of the gnetalean flower),
many morphological cladistic analyses (Fig. 4) still recover
the remainder of the anthophyte clade, containing not
only the angiosperms and the Mesozoic Caytonia, Pentoxylon, and bennettites (recently credibly reinterpreted by
Rothwell and Stockey, 2002: Fig. 1C, D) but also the
Carboniferous–Permian glossopterids (recently credibly
reinterpreted by Doyle, 2006). Rather than agree with
Frohlich’s statement that ‘the underpinnings are removed
from the anthophyte theory’ (Frohlich, 2006), we prefer his
view that ‘one must be careful not to discard an entire
scenario if a separable component is [more correctly,
‘appears’] wrong’.
More broadly, morphological cladistic analyses provide
a credible series of stem-groups to link the angiosperm
crown-group to Carboniferous pteridosperms (Doyle, 2006;
Hilton and Bateman, 2006). They also have the potential to
elucidate how the various components of the angiosperm
flower accrued, since each of these stem-groups possesses
some flower-like features. In the great shell-game of angiosperm origins, it appears increasingly unlikely that the pea
lies underneath the Gnetalean shell; however, the other
anthophyte shells remain resolutely in play. They are a clear
priority for more intensive research, preferably based on
fossil assemblages that combine contrasting preservation
states.
By now, it should be clear that our preferred definition of
the flower is considerably more inclusive than our preferred
definition of angiosperms. Defining a flower as a determinate axis terminating in megasporangia that are surrounded
by microsporangia and are subtended by at least one sterile
laminar organ (Table 1) effectively encompasses the reproductive structures of several anthophytes. Consequently,
accepting the currently most popular phylogenetic placement of Gnetales—adjacent to or within extant conifers—
would require at least two independent origins of the flower
among extant taxa, and additional origins if wholly extinct
taxa are also considered. Our preferred characters for delimiting the angiosperms focus on the carpel and its contents; specifically, we require both a closed (though not
necessarily fused) carpel preventing direct contact between
3490 Bateman et al.
the microspore wall and the megaspore wall, and the occurrence of double fertilization leading to formation of functional endosperm (both features are absent from Gnetales,
as they are from some early-divergent angiosperms).
Are clades such as angiosperms best defined
using taxa or characters?
Placing character-state transitions within the explicit context of a cladogram helps to clarify the key issue of
recognizing and delimiting natural (monophyletic) groups.
Three approaches are available, though in practice
individual phylogeneticists rarely state which approach
they prefer.
A node-based group omits all of the characters on the
branch subtending the basal node of the exclusive set of
coded taxa that constitute the group (Fig. 3). Thus, by definition, it can be delimited only by the taxa that it encompasses, rather than by unifying synapomorphies (consequently,
this is not a practical approach to delimiting monophyletic
groups). Any taxon subsequently added to the matrix that
proves to be interpolated within the original subtending
branch will, by definition, be excluded from that group.
Conversely, a stem-based group includes, without prejudice, all of the character-state changes on the branch
subtending the basal node. If that branch is long, many
ostensibly diagnostic characters are available. Any taxon
subsequently added to the matrix that proves to be
interpolated within the original subtending branch will,
by definition, be included in that group.
The third approach bisects the subtending branch by
selecting an acquisition of a single apomorphic character
state as being diagnostic of that group (e.g. for angiosperms
one could opt to prioritize double fertilization leading to
a triploid functional endosperm; Fig. 3). This meets our
requirement to readily diagnose the clade, and means that
an additional taxon attaching itself directly to that branch
might or might not join that clade, depending on whether or
not it possessed the key diagnostic character state. It is also
the only one of the three approaches to explicitly allow
delimitation (via autapomorphies) of groups represented in
the analysis by only one coded taxon (i.e. operationally
monotypic). Of course, the requirement could be that clade
membership by a taxon requires possession of not one
but two or more of the synapomorphies allocated to the
subtending branch (e.g. adding to the triploid functional
endosperm a requirement for a proembryo that is not tiered
and a secondary suspensor that is nonetheless partly derived
from the primary suspensor cell, or simply the presence
of a bona fide embryo sac; for further details of these
characters see Doyle, 2006).
Of the three approaches, we regard specifying apomorphies as being the most logical, stable, and widely
applicable. However, it does require the proponents of
each analysis to strongly justify their subjective choice of
diagnostic character state(s), effectively constituting a
posteriori character weighting.
Do some categories of character merit particular
emphasis?
Morphological characters
Ever since Linnaeus (1735) chose to prioritize floral
features over vegetative features, and more precisely
stamens over carpels and both over tepals, most botanical
systematists have been tempted to prioritize certain selected
characters (or, more precisely, in monophyletic classifications, apomorphic states of characters) for classifying plants
and, latterly, for determining their phylogenetic relationships. With the exception of a widely held preference for
characters readily divisible into discrete states, morphological cladistics has encouraged a more egalitarian perspective on characters; weighting, if practised at all, has
generally been post hoc rather than ad hoc. Indeed, post
hoc comparisons of relative levels of homoplasy in different organs have encouraged some phylogeneticists to
counsel against routinely prioritizing selected organs in
general and flowers in particular, instead perceiving
egalitarianism among organs (and characters) as one of
the greatest strengths of the cladistic method (Bateman and
Simpson, 1998).
Most analysts have transferred their emphasis from
morphological to molecular data when constructing phylogenies. Nonetheless, the majority still prefer one or more
readily recognized phenotypic character(s) for delimiting
clades in the resulting trees. Even then, tensions can
emerge; for example, some of the most effective groupdelimiting morphological synapomorphies are not readily
recognized in fossils (these include the double fertilization
and resulting triploid endosperm, and the reduced proembryo, that were highlighted in the previous section as
potentially delimiting the angiosperms).
Point mutations versus large-scale mutations
Perceptions of the various processes of mutation have
undergone a radical overhaul during the previous decade.
Studies have overturned the long-held fundamental genetic
principles that both mutation and recombination are random
factors (e.g. Jin and Bennetzen, 1994; Tsunoyama et al.,
2001). Moreover, typical mutation rates have been shown
to be one to two orders of magnitude higher than previously
thought (;2 per genome per generation: Denver et al.,
2004), and to be subject to reversal to the plesiomorphic
condition over a very small number of generations (Lolle
et al., 2005; Weigel and Jürgens, 2005). In addition,
unexpectedly high frequencies and heritabilities have
been demonstrated for methylated ‘epialleles’ (e.g. Kalisz
and Purugganan, 2004).
Phylogenetic context of the flower
Arguing for a fundamental contrast in the nature of
morphological phylogenetic and molecular phylogenetic
data, Bateman (1999, p. 446) provocatively stated that ‘the
vast majority of morphological character-state transitions
occur during speciation events, whereas the vast majority of
molecular character-state transitions occur between them’.
The main exception to this rule envisaged by Bateman
(1999) and Bateman and DiMichele (2002) was the minute
percentage of mutations (often simple point mutations) that
are both non-lethal and enable direct, substantial modification of the phenotype. These would have greatest effect
when affecting key developmental genes; the mutation
alters the amino acid(s) generated, which in turn alters the
behaviour (or concentration) of the protein sufficiently to
ultimately cause substantial modifications of the resulting
phenotype. Such events are especially likely to prompt
shifts in the timing (heterochrony) and/or spatial expression
(heterotopy) of developmental events, and be expressed in
multiple parts of the organism (i.e. pleiotropically). When
fed into a molecular phylogenetic analysis such mutations
disappear from view, immediately becoming needles in a
nucleotide haystack (though this is not necessarily the case
when they become the foci of evolutionary-developmental
studies; see below).
Although the vast majority of DNA characters are point
mutations, many analysts have been drawn preferentially to
larger-scale mutations, involving insertions, deletions,
inversions, and other genomic rearrangements (King,
1993; Rokas and Holland, 2000; Levin, 2002) that affect
expressed regions of the genomes. One of the attractions of
such characters (termed ‘rare genomic changes’ by
Burleigh and Mathews, 2004) was the reasonable assumption of an extremely low likelihood that such mutations
would show homoplasy (i.e. would have multiple origins or
revert to the previous condition), though this assertion was
based on the aforementioned assumptions of randomness.
One such mutation that has at least partly stood the test of
time is the acquisition of the inverted repeat in the
chloroplast genome on the spine of the land-plant phylogeny, which occurred between the divergence of the
lycopsids and that of the remaining extant pteridophytes
(Fig. 2) (Raubeson and Jansen, 1992a). This region,
constituting ;20% of the plastid genome, was not only
duplicated but also reversed, so that it is transcribed in the
opposite direction from that of the original copy. Interestingly, the duplicated region is partly or wholly absent
from all extant conifers but present in all extant Gnetales
studied to date (Strauss et al., 1988; Raubeson and Jansen,
1992b; Raubeson et al., 2004), implying a Gnetales-sister
topology. By contrast, the absence of functional ndh genes
from the plastid genomes of both Gnetales and Pinaceae
supports the Gnepine hypothesis (Chaw et al., 2000;
Burleigh and Mathews, 2004). Several other genomic
rearrangements serve simply to emphasize the distinctness
of Gnetales from all extant conifers.
3491
The impression gained over the last few years is that rare
genomic changes are neither as rare, nor as reliably unique
(i.e. immune to homoplasy), as was previously suspected.
Nonetheless, this observation cannot adequately justify the
rapid disappearance of these useful characters from most
phylogenetic discussions.
Gene duplications and gene expression:
evolutionary-developmental genetics
Another category of rare genomic change, which is proving
to be less rare than previously believed, is various modes of
gene duplication, which have understandably attracted
much attention recently (e.g. Lynch, 2002). Naturally,
much of this attention has focused on the limited number
of developmental genes that underpin the ABCDE model of
floral organ control (examples of A=AP1/SQUA, B=DEF/
GLO, Bs=ABS, C=AG/PLE, D=AG-like, E=SEP: Theissen
et al., 2002). In ‘typical’ eudicots, sepals reflect expression
of A genes, petals A+B+E, stamens B+C+E, carpels
C+E(6Bs), and ovules Bs+D+E. Typical monocots (and
other early-divergent angiosperms) lack a distinct A-only
sepal whorl, and A-function is wholly absent from extant
gymnosperms, where both male and female cones exhibit
C/D expression, but males exhibit B expression whereas
females exhibit Bs expression (Theissen et al., 2002;
De Bodt et al., 2003; Zhang et al., 2004; Krizek and
Fletcher, 2005; Hintz et al., 2006; G Theissen, pers. comm.,
2006).
Current evidence suggests that B- and C/D-function gene
lineages diverged prior to the node subtending extant
angiosperms and extant gymnosperms, ;400–300 Ma, as
did the two main clades in the C/D-function AG group
(Zhang et al., 2004) and the AGL6 versus AGL2–4+AGL9
split in the SEP group (Zahn et al., 2005). Many of the
remaining key divergences have been attributed to that
most troublesome branch that links the divergence point
of angiosperms and extant gymnosperms to the node
subtending the extant angiosperms. Somewhere along this
branch are placed the divergence of the C and D functions
(Kramer et al., 2004), the two main clades of B-function
genes, DEF/AP3 and GLO/PI (Theissen and Becker, 2004)
(a divergence dated to 260 6 30 Ma by Kim et al., 2005),
and the two main clades within the SEP group, AGL2–4 and
AGL9 (Zahn et al., 2005). Molecular clock estimates
indicate that these duplications could have been contemporaneous, leading to suggestions of a crucial polyploidy
event on the stem-lineage leading the extant angiosperms
(De Bodt et al., 2005; Zahn et al., 2005).
These gene families show extensive evidence of duplications among the extant angiosperms (Kim et al., 2004;
Kramer et al., 2004), which can often be linked to
morphological shifts. For example, Litt and Irish (2003)
suggested that the restriction of A-function euAP1 genes to
core eudicots could indicate co-option of new mechanisms
3492 Bateman et al.
of floral development that are correlated with the ‘fixation’
of floral structure in the eudicot clade, most notably the
strong distinction between petals and sepals. Within the
petaloid monocots, the exceptional differentiation among
the tepals of orchids is reflected in an unusual diversity of
B-class genes (Tsai et al., 2004; Xu et al., 2006). Within
dipsacalean eudicots, multiple duplications in CYC-family
genes have been implicated in various phylogenetic shifts
in floral morphology and symmetry (Howarth and Donoghue,
2005), while the subtleties of expression of such genes among
perianth members have been well-documented in various
Brassicaceae (S Zachgo, pers. comm., 2006).
Polyploidy is perhaps the easiest category of gene
duplication to detect, since every nuclear gene is copied
simultaneously during the whole-genome duplication process (e.g. Linder and Rieseberg, 2004; De Bodt et al.,
2005). In the case of allopolyploidy, the hybridization
event that accompanies genome duplication generates
a unique combination of genes, albeit at the expense of
introducing into the phylogeny a reticulation event that
links two previously independent lineages. For example,
despite its small genome size, the eudicot model organism
Arabidopsis retains evidence of no less than three
duplications of the entire nuclear genome: one in the
mid-Cenozoic following the main angiosperm radiation,
one in the late Cretaceous during the radiation, and one
broadly contemporaneous with the estimated divergence
time of the monocots, basal dicots, and earliest eudicots, in
the late Jurassic–early Cretaceous (De Bodt et al., 2005).
Admittedly, the estimated timings of these events carry
large error bars, a statement that applies equally to singlegene duplications.
Any or all of these events could have aided taxonomic
diversification by allowing functional diversification within
specific gene families, particularly those involved in
transcriptional regulation and/or signal transduction (Blanc
and Wolfe, 2004a, b; Kellogg, 2004, 2006; Maere et al.,
2005); categories that include key developmental genes
such as the MADS-box clusters (Zahn et al., 2005).
Wholesale gene duplication remains an attractive paradigm,
because it allows the subsequent accumulation of the large
amount of genetic diversity necessary to explain the ‘slow
fuse’ that preceded the extraordinarily rapid morphological
(and thus taxonomic) diversification of the angiosperms.
Fewer key developmental genes survive small-scale duplication events, as co-adapted sets of genes widely distributed on the chromosomes will become dissociated.
A classic study of the potential significance of duplication in nuclear gene families was conducted by Frohlich
and Parker (2000) (see also Frohlich, 2002, 2006), who
sequenced the low-copy nuclear gene LEAFY (=FLO) in
a phylogenetically well-chosen set of place-holder gymnosperm and angiosperm taxa, plus two pteridophyte outgroups. The resulting trees were consistent with extant
gymnosperm monophyly, but also revealed the presence of
two copies of LEAFY in most of the gymnosperms studied
(the exception was the Gnetalean Gnetum). The copy that is
absent from pteridophytes, angiosperms, and Gnetum was
named Needly. Assuming extant gymnosperm monophyly,
the most parsimonious explanation of the phylogenetic
distribution of copies of LEAFY s.l. would be a single
duplication event, which generated Needly, occurring
between the divergence of the angiosperms from the
gymnosperms and the crown-group node subtending the
extant gymnosperms; this was in turn followed by a loss of
Needly from Gnetum. In other words, Needly never
occurred in the angiosperm lineage. However, the molecular phylogenies, including the amino acid-based phylogeny derived from LEAFY itself, suggest that the duplication
of LEAFY preceded the divergence of extant angiosperms
and extant gymnosperms; gymnosperm LEAFY is depicted
as sister to angiosperm LEAFY rather than to gymnosperm
Needly. Acceptance of this topology requires a less parsimonious explanation of the gene duplication itself. The
original duplication is now perceived as having occurred
below the divergence point of the angiosperms, with the
Needly copy suffering parallel losses (strictly, deactivations) in Gnetum and, more importantly, early in the
independent life of the angiosperm lineage.
It was this interpretation that, in turn, prompted the
‘mostly male’ theory of angiosperm origins, arguing that
the deactivation in angiosperms of Needly, which specifies
femaleness in gymnosperms, allowed a switch from ancestral monoecious cones to bisexual flowers by immediate (i.e.
saltational sensu Bateman and DiMichele, 2002) ectopic
(broadly, heterotopic) expression of ovules on a fundamentally male shoot. This intriguing hypothesis is eminently
testable through further evolutionary-developmental study
(Frohlich, 2006). However, we note that there is no obvious
method of demonstrating that the Needly copy was indeed
present in the shared ancestor of angiosperms and extant
gymnosperms; this key element of the hypothesis remains
an inference because of the growing perception of extant
angiosperms and extant gymnosperms as monophyletic
sister-groups.
Baum and Hileman (2006) recently built, on groundwork
laid by Theissen and coworkers (Theissen et al., 2002;
Theissen and Becker, 2004), an elegant evolutionary model
designed to explain the determinate growth and bisexuality
of the flower, and the origin of petals. It boils down to
a sequence of four key steps:
(i) Evolution of a bisexual axis via a gynomonoecious
intermediate, through homeotic conversion of distal
microsporophylls (stamens) into megasporophylls
(carpels) within the pollen cone. This was explained
by differences in maximal expression levels of Band C-class organ identity genes and competition
among their gene products. Specifically, the B-class
MADS-box genes that, along with C-class genes, are
Phylogenetic context of the flower
necessary for expressing maleness generate proteins
that must operate in combination with E-class SEPALLATA genes. Femaleness requires the presence of
C-class expression but the absence of B-class expression. Any concentration of C-function proteins in the
apex of a male cone could complex excessive amounts
of SEPALLATA protein so that little remains to interact
with the B-function proteins, thereby allowing the
terminal portion of the cone to cross the critical
threshold into femaleness.
(ii) Evolution of floral axis compression and determinacy. Here, C-class genes became negative regulators
of the meristem maintenance gene WUSCHEL, which
maintains the indeterminacy of the apical meristem.
As a result of the ensuing switch to determinacy,
apical growth ceases and the pattern of initiation of
organ primordial grades from a helix to a spiral,
allowing their relative positions to become a critical
factor in determining their respective developmental
fates.
(iii) Evolution of petaloid perianth by sterilization of outer
stamens. This is postulated to have occurred when
WUSCHEL was co-opted as co-regulator of C-class
genes, causing B-gene expression to extend closer to
the axial apex than C-gene expression and thus
creating a zone where expression of petals (effectively
hybrid organs) was feasible.
(iv) Evolution of the classic perianth of core eudicots. The
perianth became strongly dimorphic and relatively
invariant, as B-class function became increasingly
dependent on UFO co-regulation.
We echo Frohlich (2006) in recognizing that both of these
hypotheses of the origin of the angiosperm flower are
elegant and testable. However, they also emphasize the fact
that the increasing numbers of gene duplication studies are
revealing ever-more complex patterns. The term ‘single
copy nuclear gene’ has been replaced by ‘low copy nuclear
gene’ as it has become increasingly clear that both
duplications and losses occur more commonly in most
developmentally expressed gene families than was previously supposed (Page, 2000; Barker and Pagel, 2005;
Frohlich, 2006). As well as potentially undermining the
ensuing phylogenetic analyses, this perception of increasing complexity also increases the risk that true sister-genes
(i.e. those derived from the most recent duplication event
in the lineage) will be overlooked among the plethora of
their relatives.
On the other hand, plants generally do not appear to
support more than two copies of LEAFY. Some polyploids
in Brassicaceae possess two copies but others rapidly lose
the second copy, due to a low inherent probability of one
copy being co-opted for a novel function. This could reflect
either drift (Baum et al., 2005) or the unusual gradational
(rather than on–off) mode of expression of LEAFY, which
3493
allows the possibility of major shifts in phenotype through
epigenetic threshold effects, and permits strong selection
against possession of two copies (Albert et al., 2002;
Frohlich, 2006). Similarly, considerable pleiotropy is
evident in LEAFY expression in eudicots. In addition to
influencing the initiation and timing of the transition from
vegetative to reproductive growth, LEAFY is required to
render pea leaves compound but not those of tomato
(reviewed by Frohlich, 2006). Within angiosperms, similarly profound phylogenetic transitions in the location and
effect of expression have been observed in every wellstudied family of key genes.
Gene duplications as erstwhile tools for assessing
error in molecular phylogenies
One of the best-documented examples of gene duplication
involves the phytochrome (PHY) gene family, which occurs
as three to five copies in most angiosperms. PHYA and
PHYC are monophyletic families that have subtly different
roles in generating photoreceptors that influence seed
germination and seedling development in most angiosperms, whereas only one related lineage occurs in extant
gymnosperms, suggesting that a duplication event occurred
immediately below the angiosperm crown-group node
(Mathews and Sharrock, 1997; Mathews and Donoghue,
1999). The trees simultaneously generated from PHYA and
PHYC by Mathews and Donoghue (2000, fig. 1) are more
congruent in both topology and comparative branch lengths
than those generated in most other studies of duplicate
genes, but even here only slightly over half of the nodes are
shared by the two topologies. Thus, even a relatively
congruent result contains a large proportion of conflicting
statements of relationship, in the sense that the contrasting
statements cannot both reflect the species tree (though it is
of course perfectly possible, and indeed likely, that neither
gene tree accurately reflects the species tree).
We have not come across any studies that use the
subsequent history of duplicated genes to estimate the
minimum level of congruence shown by the subsequent
mutational histories of the duplicates. It seems to us that
these cases could be exploited far more effectively,
applying to duplicate genes broadly the same logic that is
applied to studies of twin children in biomedical and
psychologicial research.
Gene duplications as erstwhile tools for cutting long
branches
Cases such as the PHY duplication can, in theory, free
sequence-based analyses from the tyranny of outgroup
rooting, especially in cases (the majority) where the chosen
outgroups are strongly divergent and thus prone to longbranch attraction. Instead, they allow duplicate gene rooting
of phylogenetic trees, using one or other of two distinct
philosophies. As described by Mathews and Donoghue
3494 Bateman et al.
(2000, p. S51), ‘under the reciprocal outgroup view,
sequences of one of the gene copies are viewed as
outgroups for the other, and vice versa, and a rooted species
tree is derived by consensus of the two gene subtrees. In
contrast, under the minimum events view, the best-rooted
species tree is the one that minimizes additional duplications and losses, lineage sorting, and lateral transfer events
in the gene tree.’ (in the case of Frohlich and Parker’s
LEAFY study described above, sequence parsimony has
effectively been prioritized over minimizing duplication/
loss events). However, it is difficult to compare duplicated
copies of the gene with unduplicated copies. One suggested
solution, termed ‘uninode coding’ (Simmons et al., 2000),
effectively reconstructs hypothetical ancestors at internal
nodes of the gene tree that precede the inferred duplication
event (note that it is the ancestral gene sequence that is
being reconstructed, not the ancestral morphology). Unfortunately, this approach is particularly questionable in cases
where concerted evolution, lineage sorting, lateral transfer,
and/or extensive duplication or losses are suspected
between the duplicated gene lineages (Mathews and
Donoghue, 2000; Simmons et al., 2000). Moreover,
accumulating data are showing each of these processes to
be more frequent than was previously believed.
Even more relevant to this essay is the assertion by
Mathews and Donoghue (2000, p. S51) that ‘in effect,
a duplication occurring along the branch to the ingroup
would bisect [our italics] the long branch connecting the
ingroup with outgroups’. As summarized by Frohlich
(2006), ‘long branches can be cut by gene duplications
(Mathews and Donoghue, 2000), and this may be the only
way to cut long branches of crucial importance in understanding plant evolution. A lineage of organisms that
persists over a long geologic interval, and does not generate
any surviving sister groups during that interval, cannot have
its long branch cut by additional taxon sampling of
unduplicated genes. But if a gene duplication event occurs
during this interval, and if both gene lineages survive to the
present, then the long branch is cut at the point in time when
the duplication event occurred.’ A similar argument could
perhaps be made for the timing of the loss (or, more
accurately, deactivation) of particular copies of key developmental genes, such as the independent losses of the
Needly copy of LEAFY postulated in the angiosperm and
gnetalean lineages by Frohlich and Parker (2000).
However, does this method truly ‘cut’ or ‘bisect’
a molecular long branch, analogous to the way adding
a taxon to an analysis can cut a morphological long branch
by inserting an additional node marking a divergence event
in the species tree (Fig. 6)? Our view is that these two
approaches are not truly analogous, in the sense that placing
a gene duplication (or deactivation) event on a branch does
not allow us to directly separate events on that branch (other
than the gene duplication itself) into those that preceded the
duplication event from those that occurred subsequently.
The branch is not truly cut, and certainly not bisected, but
rather it simply acquires an additional synapomorphy. The
attractive concept of cutting the branch might arguably be
rescued, at least in part, by Frohlich’s rationale (Frohlich,
2006) that ‘the long branch is cut at the point in time when
the gene duplication occurred’. He predicted that many
gene duplications would prove beneficial, thereby stimulating, and so immediately antedating, evolutionary radiations (cf. Bateman, 1999; Kellogg, 2006). This in turn
should mean that, based on the somewhat unreliable ticking
of the molecular clock, little sequence divergence should
separate the duplication event from subsequent species
divergences. The products are conventionally termed
paralogous and orthologous gene copies, respectively;
a better terminology may be in-paralogue for the products of duplication within the clade of interest and outparalogue for duplication events that precede the
crown-group node (cf. Remm et al., 2001; O’Brien et al.,
2005; Kellogg, 2006).
However, this hypothesis assumes not only that the
duplication will prove immediately beneficial to the
evolutionary lineage but also that a representative percentage of the products of the ensuing radiation will benefit
sufficiently to survive to the present day. This assumption
may have validity when, for example, it is applied to
relatively recent radiations within the eudicot angiosperms,
but when compared with the much greater high-level
morphological diversity evident in the fossil record, the
frequency of survivorship to the present of lineages from
deeper radiations appears decidedly poor. Moreover, the
currently preferred molecular topologies, which encapsulate monophyly of extant non-lycopsid pteridophytes and
monophyly of extant gymnosperms, mean that very few of
the long branches in the topology are capable of yielding
critical information on angiosperm origins even if they are
somehow ‘cut’. Specifically, the emphasis is placed on just
two branches separating three nodes: the divergence point
of extant non-lycophyte pteridophytes from extant seed
plants, that of extant gymnosperms from angiosperms, and
that located at the base of extant angiosperms (Fig. 2b).
Post hoc homology assessment
We conclude that a phylogeny provides a valuable guide to
evolutionary-developmental studies, by focusing attention
on those taxa best placed phylogenetically to allow comparative studies of key phenotypic characters and the genes
that control them (e.g. Albert et al., 2005). For their part, the
results of such studies constitute a simultaneous homology
test of corresponding features of the organism and its
genome. This is an especially valuable post hoc homology
test, in that it is not dependent on the congruence test
inherent in simultaneously pitting the phylogenetic signal
from each character against every other character when the
phylogeny is being algorithmically constructed (Patterson,
Phylogenetic context of the flower
1988). Moreover, evolutionary-developmental genetic investigation offers tests that directly and causally links the
genotypic and genotypic realms. Perhaps this will prove to
be the category of characters worthy of greatest emphasis?
Nonetheless, even with such sophisticated tools at our
disposal, the long branch separating the latest diverging
extant gymnosperms from the earliest divergent extant
angiosperms remains a recalcitrant barrier to confidently
reconstructing the first flower.
What is the preferred research programme for
the next decade? A plea for pluralism
A recurrent theme in this extended essay has been the
tensions surrounding the unavoidable decision of whether
to prioritize taxon sampling or character sampling in
phylogenetic analyses. This argument has mainly been
pursued in debates regarding the relative merits of morphological and molecular phylogenetic data, although
recently they have been revived in a different context:
character-rich whole genomes versus fewer characters
derived from multiple genic regions of more taxa (cf.
Goremykin et al., 2003, 2004; Martin et al., 2005; Soltis
et al., 2005)—an approach that, in its most minimalistic
form oriented toward sequence-based specimen identification, has recently become known as DNA bar-coding (e.g.
Tautz et al., 2003; Savolainen et al., 2005). Despite our
reservations concerning some recent optimistic predictions
regarding the likely effectiveness of this approach, we
choose to view this debate constructively, as evidence that
molecular phylogenetic analyses are becoming more sophisticated and more biologically informed. In particular,
morphologists will be encouraged that, in molecular circles,
data quality is making a welcome resurgence against
simplistic arguments that over-emphasize data quantity.
In the course of this debate, Martin et al. (2005, pp. 208–
209) argued forcefully that both character-rich (here termed
‘genomic’) and relatively taxon-rich (here termed ‘polygenic’) research programmes should progress in parallel.
We heartily agree, but would further argue that a third
(currently relatively poorly resourced) phylogenetic research programme—one that maximizes taxonomic sampling (‘morphologic’)—remains essential, not just in the
taxonomically broad studies discussed here but at all levels
in the phylogenetic–taxonomic hierarchy. Only better taxon
sampling can improve our understanding of the sequence of
acquisition of the functional phenotypic character states
that are the raw material of adaptation, or permit improved
reconstructions of the hypothetical ancestors that occupy
key nodes in the phylogeny. And only morphological
analyses make full use of reconstructed fossil plants,
benefiting from their unique combinations of characters
that bridge evolutionary lacunae and their implicit statements regarding the timing of specific evolutionary events.
Such dating allows estimation of the relative extents in
3495
contrasting topologies of gaps (‘ghost lineages’) in the
fossil record (e.g. Doyle, 1998a), and permits calibration of
molecular clocks (e.g. Won and Renner, 2003; Sanderson
et al., 2004). Techniques for reconstructing hypothetical
ancestors merit further exploration. Although parsimonybased optimizations remain attractive for their simplicity,
there is merit in exploring likelihood-type techniques that,
for example, weight towards the more common state
observed in the vicinity of the node to be reconstructed
(cf. Frohlich, 2006).
We would argue that the most significant advances of the
last decade in understanding seed-plant relationships have
not been made as a consequence of the volume of available
data per se. Rather, they are the result of what has been
learnt about the biological constraints on, and evolutionary
implications of, each of the various categories of data that
has been applied to phylogenetic problems. This trend is
likely to continue through the next decade. Hopefully, the
present fashion for combining different categories of data in
increasingly data-rich simultaneous analyses without first
analysing them separately will fade, since such analyses
preclude deeper understanding of the properties of each
individual data category (admittedly, few taxonomically
broad studies have combined morphological and molecular
data in simultaneous analyses: Nandi et al., 1998; Doyle
and Endress, 2000; Pryer et al., 2001; Rothwell and Nixon,
2006). Moreover, we are more persuaded by the weight
of evidence implicit in congruent topologies obtained
from different categories of data experiencing different
constraints. When meaningful discrepancies have been
identified, we can then seek the underlying cause, and
our fundamental understanding of the data is genuinely
enhanced (e.g. Jeffroy et al., 2006).
Note that progressing the three phylogenetic approaches
in parallel is not synonymous with progressing them in
isolation. Pragmatically, better co-ordination of taxonomic
sampling would be helpful, to allow several different
categories of data to be gathered not only from the same
higher taxon, but from the same reference individual of
a single species. Then we can at last be certain that the data
reflect, either directly or indirectly, the same integrated
genome(s).
Even more important are the manifold opportunities for
reciprocal illumination between contrasting data streams
(e.g. Crane et al., 2004). For example, we are confident that
whole-genome sequencing will provide invaluable yardsticks for process-based interpretations. They will help to
determine the frequency of lateral gene transfer, both
between the three genomes within a species and among
the same category of genome between species. Such studies
will usefully inform others that use a more restricted range
of candidate genes, helping to optimize choices of (i)
region(s) to be sequenced, (ii) approaches to filtering the
resulting data, and (iii) methods of building the trees.
Increasingly sophisticated chromosome mapping will
3496 Bateman et al.
inform not only phylogeny reconstruction but also
evolutionary-developmental genetics, which will act as an
external homology test linking studies of candidate genes
and morphogenesis (Kellogg, 2006). We concur with
Frohlich’s (2006) prediction that complementation studies
will demonstrate more subtlety in exploring differences in gene function than do the currently prevalent
over-expression studies, and that paralogues will be more
effectively compared if the range of transformable model
organisms can be expanded to include species that have
phylogenetic placements closer to the key divergences and
that permit reciprocal transformation (Baum, 2002). Most
importantly, we hope that it will become more widely
accepted that studies intended to improve our understanding of the relationships of taxa without simultaneously
improving our understanding of character evolution have
relatively little utility.
One prediction we make with confidence is that the
number of concepts broadly encompassed by the muchused term ‘homology’ will continue to increase. Concepts
applied to morphology have frequently been contrasted
with those applied to sequence alignment, but now we also
face the challenges posed by deeper knowledge of the
control of phenotype by genotype. These have long been
confounded to a degree by pleiotropy and epigenetic
phenomena. However, we can now demonstrate, with
some confidence, cases of ‘homocracy’, where organs
demonstrably share orthologous gene expression but nonetheless are inferred via phylogenies to have independent
origins (e.g. K Geuten, pers. comm., 2006).
Our knowledge of molecular evolution (and the associated rapidly growing areas of bioinformatics) is insufficient
to allow us to gaze insightfully deep into our crystal ball.
Nonetheless, we are confident that integrating new categories of data into various aspects of phylogeny reconstruction will be one of the major challenges of the next
decade. Taxonomically ever-wider surveys of expressed
sequence tags offer one obvious source of data that may
prove challenging to code (e.g. Kellogg, 2006), while
researchers wishing to link gene phylogenies to precise
functions of particular gene copies in particular species will
be obliged to pay more attention to the interactions of genes
in functional networks (e.g. Barker and Pagel, 2005),
operating within the expanding realms of epigenetics and
proteomics. Improved tests are needed for recognizing,
and differentiating contrasting modes of, selection at the
molecular level, along with more sophisticated modelling
of the ‘ancestral’ and ‘derived’ proteins.
Returning to more traditional data sources, we suspect
that the preferred methods for analysing morphological data
on the one hand and molecular data on the other, which
have in practice diverged substantially during the previous
decade, will continue to diverge during the next decade.
Parsimony will remain the preferred technique for building
morphological trees (see also Rothwell and Nixon, 2006).
Both a priori homology statements and a posteriori
character evolution will continue to be assessed by the
analyst on a character by character basis, which permits
intimate knowledge of the data. By contrast, sequencebased trees will routinely be generated from vast tracts of
data via mathematical models (currently likelihood and/or
Bayesian; Palmer et al., 2004). Increasingly complex
algorithms and automated computerized routines will
determine both prior homology assessment (i.e. alignment)
and subsequent character analysis (e.g. first and second
versus third positions in codons; also, sorting genic
regions—and codon positions within those regions—into
contrasting mutation rate categories). In both morphological and molecular analyses, confidence estimates of nodes
will increasingly utilize measures such as the decay index
that, unlike resampling techniques such as the bootstrap,
jackknife, and posterior probability, do not require absolute
upper bounds to their values (Frohlich, 2006).
Indeed, there is a downside to rapid increases in the
number of characters available, which in practice prevent
the analyst from exploring the behaviour and significance
of individual characters, either implicitly or using more
explicit methods such as sequential character removal
(Davis et al., 1993; Frohlich, 2006). Rather, specific
characters routinely become lost in a statistical mélange,
blurring reciprocal illumination between characters and
topologies and complicating attempts to determine the
functions of, and factors that control, particular phenotypic
features. Certainly, when seeking to define the evasive
threshold beyond which a set of gene trees can be viewed as
delivering the desired species tree, we would favour use of
process-based explanations of incongruence rather than
a statistically based justification of perceived congruence.
Fortunately, when molecular studies address deeper
issues, seeking not only inferred relationships but also the
genetic changes that permit phenotypic shifts, a more
intimate relationship between analyst and data is restored.
Moreover, the ever-expanding volumes of sequence data
available, increasingly including whole genomes (Margulies
et al., 2005; Kellogg, 2006; Service, 2006), will encourage
analysts to re-code those bases as amino acids. The consequent radical reduction in number of phylogenetically
informative characters is more than compensated for by the
greatly increased number of potential states per character
(as individual characters, amino acids behave far more like
morphological characters than do nucleotides).
Pluralistic approaches that are carefully integrated to
address the same suite of high-profile hypotheses, among
them the origin of the flower, should reap rich rewards.
However, even then, a healthy level of critical evaluation
should be applied to the resulting evolutionary trees. In an
enjoyably provocative essay, Frohlich (2006) argued that
one major problem compromising our understanding of
angiosperm origins ‘is the deadening hand of wonderful,
classic research. This phrasing is not oxymoronic. Superb
Phylogenetic context of the flower
studies sometimes become enthroned as classics of scientific research, to the point that no one looks at the subject
again for decades, although analytic tools improve and
scientific questions change. I think that this has been the
case in the study of the morphology of living plants.’ We
would make two observations in the context of this
statement. Firstly, most genuinely classic morphological
studies date from the nineteenth and early twentieth
centuries; modern studies are far more likely to cite more
recent syntheses that paraphrase, and frequently misrepresent, the earlier primary literature. In short, several recent
‘discoveries’ have in truth been rediscoveries. Secondly, we
perceive the main advances achieved in understanding the
origins of flowers over the last two decades as being more
explicit means of stating and exploring hypotheses—
hypotheses of relationship, hypotheses of homology, and
hypotheses of character-state transitions. The botanical
community has erected an invaluable framework of overlapping, and potentially integrated, analytical approaches
that offers the promise of better answers to some of
botany’s longest-standing questions.
However, we would also argue strongly that, for the
many reasons elucidated above, scepticism should be
applied to the conclusions of any study that strongly
advocates one particular hypothesis of relationship over
its competitors, irrespective of the nature of the underlying
data. Rather than attempting to ‘resolve incongruence’
between data sets (Rokas et al., 2003; Martin et al., 2005;
Soltis et al., 2005; Jeffroy et al., 2006) (cf. Figs 4, 5), it
might be better to explore its causes while heeding the
cautionary note that it sounds.
A particularly apt molecular case study is the exemplary
re-analysis by Jeffroy et al. (2006) of the 106-gene matrix
of Rokas et al. (2003) for eight yeast species (discussed
above in the context of assessing the quality of a phylogeny). Jeffroy et al. cogently argued that genome-scale
matrices are likely to progressively overcome topological
incongruence caused by (i) stochastic errors and (ii)
violations of the assumption of orthology by processes
such as gene duplication, lateral gene transfer, and lineage
sorting. (Admittedly, in our view, the ‘buffering’ of these
violations by sheer volume of data is no substitute for their
accurate identification and subsequent amelioration.)
Rather, Jeffroy et al. identified as most problematic
systematic errors caused by heterogeneity of nucleotide
composition, rate variation across lineages, and/or withinsite rate variation. Together, these factors can lead to
incorrect but highly statistically supported trees (Phillips
et al., 2004; Philippe et al., 2005; H Philippe, pers. comm.,
2006).
Most of the among-gene incongruence reported for the
yeast data by Rokas et al. (2003) was attributed by Jeffroy
et al. (2006) to stochastic error. However, they identified
strong incongruence separating trees generated by maximum parsimony and those generated using Bayesian
3497
inference that increased when the 106 genes were concatenated. Most of this systematic incongruence was attributable to nucleotide compositional bias, which had a much
stronger effect (especially under parsimony) when the
matrix was coded as nucleotides rather than amino acids.
This was due largely to a combination of mutational bias
and mutational saturation at third-codon positions, primarily affecting transitions rather than transversions. Thus,
Jeffroy et al. ultimately viewed the vast number of
characters available in phylogenomic data-sets primarily
as facilitating the progressive elimination of much of the
initial data while still retaining sufficient information to
yield a resolved and statistically robust tree. Discarding
third positions, and coding sequence data as amino acids
(preferably for analysis by Bayesian or likelihood methods), was the treatment recommended for large data-sets
that had been accumulated to explore deep phylogenetic
divergences. Increasing taxon sampling was especially
recommended as a means of better detecting multiple
substitutions, combined with probabilistic methods that
minimize the harmful effects of model mis-specification
(Steel, 2005). Thus, new statistical tests are being developed that seek to identify and quantify such misspecification (Goremykin and Hellwig, 2006). Jeffroy
et al. (2006, p. 230) understandably concluded by advocating that the phylogenetic community should place
greater research ‘emphasis on the development and refinement of objective methods aimed at detecting and removing
the part of the data containing a high level of nonphylogenetic signal’, an argument reinforced by H Philippe
(pers. comm., 2006).
The alternative approach to character culling is to retain
the more problematic components of the data matrix but
attempt to compensate for their negative effects. One
example is the analysis by Kawai and Otsuka (2004) of
rbcL sequences for 13 representative extant land-plants
(sadly excluding cycads and Ginkgo) plus a charophyte
outgroup. Rather than discarding characters when treebuilding they differentially treated contrasting categories of
base position in order to accommodate third-position bias
attributed to selection, via a factor termed the ‘empirical
relative base change probability’. This protocol resulted in
identical NJ (neighbour-joining) and UPGMA (unweighted
pair group method with arithmetic means) trees that yielded
expected relationships (Kawai and Otsuka, 2004, fig. 4)
with one notable exception: Gnetales were placed firmly
below the divergence of extant conifers and angiosperms,
and were calculated to have diverged from other seed plants
in the earliest Permian. This topology undermines not only
the anthophyte hypothesis but also its currently widely
favoured replacement, the monophyletic extant gymnosperms hypothesis. Interestingly, this placement was also
obtained by D Quandt and co-workers (pers. comm., 2006),
based on the highly conserved P8 region of the plastid
trnL intron, which suggested retention in Gnetales of the
3498 Bateman et al.
ancestral splicing system found in bryophytes and ferns.
Once again, the best defence available to proponents of
these hypotheses against the Kawai and Otsuka topology
may be the sparse taxon sampling evident in the study.
We also caution that, contrary to frequent assertions, our
inability to access the ‘one true tree’ means that topological
conflicts cannot genuinely be ‘resolved’, they can only be
explored.
More generally, it is clear that an experimental approach
to cladistics remains highly desirable—i.e. an approach that
tests the relative merits of particular combinations of taxa,
characters, and topologies (e.g. Magallón and Sanderson,
2002; Rydin and Källersjö, 2002; Rydin et al., 2002;
Doyle, 2006; Frohlich, 2006; Hilton and Bateman, 2006;
Jeffroy et al., 2006; Rothwell and Nixon, 2006). A topdown, sequence-dominated, group-oriented perspective
presently holds sway. It has proved fairly effective at
exploring the eudicots, a relatively recently evolved group
that consequently today retains much of the taxonomic
diversity that it has possessed throughout its entire history.
However, travelling backward through time to the base of
the eudicots carries us only a modest percentage of the
phylogenetic distance back to the earliest angiosperm.
During the remainder of our top-down journey, it becomes
less certain that the flowers encountered are single composite organs. The long-standing debates regarding euanthial versus pseudanthial origins, most of which assume
a single origin of the flower, become simplistic, suggesting
that homologies are better tested (and the flower is better
defined) at the lower hierarchical level of individual categories of floral organ (Endress, 2001a; Friis et al., 2006).
We believe that it is equally important to pursue
a bottom-up approach to exploring the origin(s) of the
angiosperms. Increasingly popular molecular hypotheses
implying monophyletic extant gymnosperms and monophyletic extant non-lycopsid pteridophytes mean than any
such approach must begin no later than the bryophytes,
which likely originated as early as the late Ordovician
(Wellman and Grey, 2000). Patchy representation of each
of these three groups in the extant flora means that the
bottom-up approach requires utilization of comparative
data not only from derived flower-bearing seed plants such
as angiosperms and Gnetales but also from ‘living fossils’
such as cycads and Ginkgo, and from the many pertinent
groups of bona fide fossils. In this context, we applaud the
clarity and modernity of the classic discussion of angiosperm origins offered a century ago by Arber and Parkin
(1907).
We conclude that any scientific hypothesis will become
the aforementioned ‘deadening hand’ if it is prematurely
elevated to the status of a widely accepted, incontrovertible
‘truth’. In our opinion, no single aspect of our understanding of the origin(s) of flowers yet merits that exalted status.
Nonetheless, we are far from discouraged. Thoughtful
researchers are now formulating hypotheses that overall
are better informed and more sophisticated than any of their
recent predecessors, and the range of tools at our disposable
suitable to test these hypotheses is continually expanding.
We merely caution that, as new techniques are adopted, the
older techniques, and the conceptual insights that underpin
them, should not be casually discarded. We are confident
that they will be needed again in the future.
Acknowledgements
We are grateful to David Baum, Jim Doyle, Gar Rothwell and,
especially, Mike Frohlich for offering previews of excellent manuscripts
that are frequently cited in this essay, and Günter Theissen for
a constructive review. We also thank Yohan Pillon for providing the
material, and Mathew Box for capturing the SEM images, of the
Amborella flowers featured in Fig. 1. JH acknowledges financial
support from the Nuffield Foundation (NAL/00883/G) and the Royal
Society.
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