Molecular Phylogenetics and Evolution 61 (2011) 413–424
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Molecular Phylogenetics and Evolution
journal homepage: www.elsevier.com/locate/ympev
Giants and dwarfs: Molecular phylogenies reveal multiple origins of annual
spurges within Euphorbia subg. Esula
Božo Frajman ⇑, Peter Schönswetter
Institute of Botany, University of Innsbruck, Sternwartestrasse 15, A-6020 Innsbruck, Austria
a r t i c l e
i n f o
Article history:
Received 9 February 2011
Revised 7 June 2011
Accepted 13 June 2011
Available online 25 June 2011
Keywords:
Bayesian inference
Character state reconstruction
Parsimony
Phylogeny
Taxonomy
a b s t r a c t
Euphorbia (Euphorbiaceae) comprises over 2150 species and is thus the second-largest genus of flowering
plants. In Europe, it is represented by more than 100 species with highest diversity in the Mediterranean
area; the majority of taxa belong to subgenus Esula Pers., including about 500 taxa. The few available phylogenetic studies yielded contrasting results regarding the monophyly of subg. Esula, and the phylogenetic relationships among its constituents remain poorly understood. We have sampled DNA
sequences from the nuclear ribosomal internal transcribed spacer (ITS) and the plastid trnT-trnF region
from about 100, predominantly European taxa of subg. Esula in order to infer its phylogenetic history.
The plastid data support monophyly of subg. Esula whereas the ITS phylogeny, which is generally less
resolved, is indecisive in this respect. Although some major clades have partly incongruent positions in
the ITS and plastid phylogenies, the taxonomic content of the major terminal clades is congruent in both
trees. As traditional sectional delimitations are largely not corroborated, an improved classification is
proposed. Character state reconstruction illustrates that the annual life form developed independently
several times in different clades of subgenus Esula from perennial ancestors, and that several morphological traits used in previous classifications of Euphorbia developed in parallel in different lineages.
Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction
Euphorbia (Euphorbiaceae) is with over 2150 species (Bruyns
et al., 2006) the second-largest genus of flowering plants, outsized
only by Astragalus (Mabberley, 2008). Distributed worldwide and
varying in habit from prostrate annuals to 20 m tall trees, the spurges achieve their greatest diversity in arid areas of Africa and
Madagascar, where many of them are cactus-like succulents (Turner,
1998). In Europe, Euphorbia is represented by more than 100 species (Smith and Tutin, 1968) with highest diversity in the Mediterranean area. The majority of European taxa belong to subgenus
Esula Pers. (Smith and Tutin, 1968), which largely corresponds to
Euphorbia subg. Paralias (Raf.) Prokh. (Prokhanov, 1949) or Euphorbia sect. Tithymalus Boiss. (Boissier, 1862), sometimes treated at
generic level as Tithymalus Gaertn. (e.g., Scopoli, 1772; Chrtek
and Křísa, 1992). The subgenus Esula includes roughly 500 herbaceous perennials, annuals, shrubs, small trees and succulents naturally occurring on all continents except Australia and Antarctica,
but achieving its greatest diversity in northern temperate regions
(Bruyns et al., 2006; Steinmann and Porter, 2002). Most species
have alternate, exstipulate and (sub)sessile cauline leaves and terminal pleiochasial inflorescences. The stem growth terminates
⇑ Corresponding author. Fax: +43 5125072715.
E-mail addresses: [email protected] (B. Frajman), [email protected] (P. Schönswetter).
1055-7903/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.ympev.2011.06.011
with the development of a terminal cyathium, mostly surrounded
by a whorl of ray-leaves. The latter subtend a fascicle of three to
many dichotomously branching rays, bearing several dichasially
arranged cyathia, subtended by raylet leaves. The involucral glands
lack petaloid appendages and are of different forms, such as suborbicular to transversely ovate, two-horned, or with truncate to
emarginate outer margins. Ovaries are three-locular, and seeds
usually bear a caruncle (Smith and Tutin, 1968; Turner, 1998;
Steinmann and Porter, 2002).
Even if Euphorbia is one of the richest genera in number of taxa,
only a few studies have addressed phylogenetic relationships within this genus. Steinmann and Porter (2002) inferred the phylogeny
of the tribe Euphorbieae, with the majority of the sampled taxa
belonging to Euphorbia, using nuclear ribosomal internal transcribed spacer (ITS) sequences and the coding plastid region ndhF.
They have shown that although Euphorbia subg. Esula as traditionally circumscribed (e.g., Wheeler, 1943) is polyphyletic, most taxa
form a clade (referred to as ‘‘clade B’’ by Steinmann and Porter,
2002), including also some African succulent species from subgenus Tirucalli (Boiss.) S. Carter. This clade has relatively high support
(86% bootstrap) in the ndhF tree, but no support in the ITS tree.
Later, Bruyns et al. (2006) inferred the phylogeny of southern African
spurges using ITS and plastid psbA-trnH sequences. With partly
different taxon sampling they corroborated the main results of
Steinmann and Porter (2002). Similar results were obtained by
Park and Jansen (2007) and Zimmermann et al. (2010), who used
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B. Frajman, P. Schönswetter / Molecular Phylogenetics and Evolution 61 (2011) 413–424
a partly different set of taxa and the latter also another plastid region (ndhF and trnL-trnF, respectively). When analysed in a Bayesian framework, clade B received maximal support (posterior
probability, PP 1) in the ITS tree (Bruyns et al., 2006; Zimmermann
et al., 2010). The exact phylogenetic position of this group, however, remains unclear (Zimmermann et al., 2010). Based on the results of their phylogenetic analyses, Bruyns et al. (2006) proposed a
new subgeneric classification for Euphorbia, in which they assigned
all taxa of clade B to subg. Esula, thus including also some succulent
members, formerly considered part of subg. Tirucalli.
None of the previous phylogenetic studies included a sufficient
number of taxa from subg. Esula to be able to draw conclusions
about interspecific relationships and to compare the phylogenetic
assemblages with traditional (sub)sectional delimitations of, e.g.,
Boissier (1862) or Prokhanov (1949). Steinmann and Porter
(2002) showed that some subsections of Boissier’s sect. Tithymalus
are polyphyletic and several taxa do not belong to subg. Esula. They
concluded that out of all of Boissier’s subsections only Decussatae
Boiss., Oppositifoliae Boiss., Carunculares Boiss., Galarrhaei Boiss.,
Esulae Boiss., and Myrsiniteae Boiss., as well as some taxa from subsect. Pachycladae Boiss. and subg. Tirucalli can be considered members of subg. Esula (sect. Tithymalus subsect. Osyrideae Boiss. was
not included in their studies!). However, they were neither able
to draw conclusions regarding the monophyly of these subsections
nor about the relationships among them, and these questions remain unsolved to date.
Molecular phylogenies can provide a framework to trace the
evolution of morphological characters through the evolutionary
history of organisms (e.g., Escobar García et al., 2009; Huelsenbeck
et al., 2003; Maddison and Maddison, 2010; Schäffer et al., 2010).
Morphological characters traditionally applied for the (sub)sectional classification of Euphorbia subg. Esula are mostly derived
from the plants’ reproductive organs. Especially the shape of the
nectarial glands on the cyathial margin, presence and shape of
tubercules on the capsules as well as seed ornamentation played
an important role in classification (e.g., Boissier, 1862; Prokhanov,
1949). Important vegetative characters include leaf arrangement
and venation, as well as life form. Annual species with bicornate
nectaries were assigned to sect. Cymatospermum (Prokh.) Prokh.,
although Prokhanov (1949) expressed his doubts about the naturalness of this group. It has often been assumed that annuals generally evolve from perennial ancestors (e.g., Stebbins, 1957; see
also Tank and Olmstead, 2008), but also the opposite has been
observed in groups like Castilleja (Orobanchaceae; Tank and
Olmstead, 2008).
The aim of our study is to disentangle the phylogenetic history
of Euphorbia subg. Esula using DNA sequences of nuclear ribosomal
ITS and the plastid trnT-trnF region from 99 predominantly European taxa. In particular, we (1) address the question of monophyly
of the subgenus using available and new sequence data. Using
character state reconstruction, we (2) trace the development of life
forms (annual vs. perennial) as well as the evolution of morphological traits used in previous classifications of Euphorbia. In addition,
(3) we assess the traditional sectional and subsectional assemblages for monophyly and (4) propose an improved sectional classification for E. subg. Esula. Finally (5), we summarise ecology and
morphological characteristics of the inferred groups.
2. Materials and methods
2.1. Plant material
We sampled 99, mostly European taxa from Euphorbia subg.
Esula from all of Boissier’s (1862) subsections currently included
in subg. Esula, with exception of subsect. Osyrideae (Steinmann
and Porter, 2002; Bruyns et al., 2006). In the ITS data set we also
included all sequences belonging to clade B from Steinmann and
Porter (2002) and Bruyns et al. (2006). Based on these studies we
also selected the outgroup taxa from their clades A, C and D. Voucher data and GenBank accession numbers are presented in Tables
S1 and S2 in the Supplementary material.
2.2. DNA isolation, PCR and sequencing
Extraction of total genomic DNA from herbarium specimens or
silica-gel dried material was performed following the modified
CTAB-protocol of Tel-Zur et al. (1999). Prior to extraction with
high-salt CTAB buffer the ground tissue was washed three times
with wash buffer containing sorbitol to remove polysaccharides.
Amplification of ITS, purification of PCR products, cycle-sequencing
and subsequent electrophoresis followed Schönswetter and
Schneeweiss (2009). The plastid trnT-trnF region (trnTUGUtrnLUAA-trnFGAA intergenic spacers including the trnLUAA intron;
from here on referred to as trnTF) was amplified using the primer
pair a and f (Taberlet et al., 1991). The PCR reaction mix contained
9 ll of ReadyMix (Sigma–Aldrich), 13 ll water, 1 ll BSA (10 mg/
ml; Promega), 0.5 ll of each primer (10 lM), 0.5 ll of MgCl2
(25 lM), and 0.5–1 ll of total genomic DNA of unknown concentration. We used the following PCR conditions: 5 min at 95 °C, followed by 35 cycles of 30 s at 94 °C, 30 s at 48 °C and 4 min at 65 °C,
followed by a final 10 min extension period at 65 °C. Purification of
PCR products and cycle sequencing were performed as for ITS,
using the primers a, c and f, in some cases also b and d (Taberlet
et al., 1991).
2.3. Contig assembly, sequence alignment and phylogenetic analyses
Contigs were assembled and edited using Staden (Staden et al.,
1998). Base polymorphisms were coded using the NC-IUPAC ambiguity codes. Sequences were manually aligned using QuickAlign
(Müller and Müller, 2003), mostly without major problems. The
alignments are available from B. Frajman.
Maximum parsimony (MP) analyses as well as MP bootstrap
(MPB) analyses of both data sets were performed using PAUP
4.0b10 (Swofford, 2002). The most parsimonious trees were
searched heuristically with 1000 replicates of random sequence
addition, TBR swapping, and MulTrees on. The swapping was performed on a maximum of 1000 trees (nchuck = 1000). All characters were equally weighted and unordered. The data set was
bootstrapped using full heuristics, 1000 replicates, TBR branch
swapping, MulTrees option off, and random addition sequence
with five replicates. Euphorbia balsamifera, E milii, E. obesa, and
E. pulcherrima were used as outgroups in ITS, and E. ipecacuanhae,
E. obesa, and E. pulcherrima in trnTF analyses, based on previous
studies (Steinmann and Porter, 2002; Bruyns et al., 2006).
Combinability of the trnTF and ITS data sets (pruned to taxa sequenced for both regions) was assessed in a parsimony framework
using the incongruence length difference (ILD) test implemented in
PAUP 4.0b10 (Swofford, 2002) employing 1000 partition replicates,
each with 10 random sequence addition replicates saving no more
than 500 trees per replicate and TBR branch swapping.
Bayesian analyses were performed employing MrBayes 3.1
(Ronquist and Huelsenbeck, 2003), using the parallel version (Altekar
et al., 2004) at the computer cluster Bioportal at the University of
Oslo (http://www.bioportal.uio.no/) applying the substitution
models proposed by the Akaike information criterion implemented
in MrAIC.pl 1.4 (Nylander, 2004; Table 1). Values for all parameters, such as the shape of the gamma distribution, were estimated
during the analyses. The settings for the Metropolis-coupled Markov
chain Monte Carlo (MC3) process included four runs with four
chains each (three heated ones using the default heating scheme),
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run simultaneously for 10,000,000 generations each, sampling
trees every 1000th generation using default priors. The PP of the
phylogeny and its branches was determined from the combined
set of trees, discarding the first 1001 trees of each run as burn-in.
As the relationships at deeper nodes in the ITS tree were poorly
resolved and to some extent conflicting with the trnTF tree, we
used SplitsTree4 4.10 (Huson, 1998; Huson and Bryant, 2006) to
generate a NeighbourNet network (Bryant and Moulton, 2004) in
order to display conflicts in the ITS data. The NeighborNet method
computes a set of incompatible splits, which are represented in the
split network by edges in non-parallel positions (Huson and
Bryant, 2008). We applied the UncorrectedP method to compute
the proportion of positions at which two sequences differ. Ambiguous
base codes were treated as missing states (we also applied the options ‘‘average’’ and ‘‘match’’ for the ambiguous bases, but the
resulting networks did not differ substantially; not shown). As
the parsimony and Bayesian as well as the NeighbourNet analyses
resulted in ambiguous position of the outgroup taxa, we also computed a NeighbourNet network for the Euphorbia ITS data set from
Steinmann and Porter (2002), using their alignment (provided by
Steinmann), pruning the outgroup taxa.
2.4. Life forms, morphological traits and character states
reconstruction
Assignment of morphological traits and life forms to each species is based on our own observations of living and/or herbarium
specimens (see also Frajman and Jogan, 2007), in some cases complemented with descriptions from the literature (mainly Boissier,
1862; Hegi and Beger, 1924; Prokhanov, 1949; Smith and Tutin,
1968; but also Benedí et al., 1997; Chrtek and Křísa, 1992; Heubl
and Wanner, 1996; Norton, 1900; Radcliffe-Smith, 1982). We
scored the following traits (character states in brackets): (1) life
form (annual; perennial), (2) indumentum (absent; present), (3)
leaf arrangement (alternate; opposite; decussate), (4) leaf venation
(pinnate; palmate), (5) shape of nectarial glands on the cyathia
(transversely ovate, outer margin convex; truncate, outer margin
truncate or shallowly concave; bicornate, horns dilated; bicornate,
horns not dilated, slender; semilunate, crescentic), (6) presence of
bracts among the male flowers (present; absent), (7) capsule surface (smooth; granulate, with small papillae; tuberculate, with
wart-like processes of different lengths; winged, with two narrow
wings along each keel), (8) pericarp (indurated; spongy), and (9)
seed surface (smooth; punctate-rugulose; sulcate, furrowed; pitted; vermiculate-rugose, wrinkled; faveolate; definitions partly
from Heubl and Wanner, 1996).
In a few cases, we could not unambiguously assign a character
state due to the plasticity of some characters. In general, the prevailing character states were assigned (e.g., taxa that are in general
glabrous, but can occasionally have some trichomes, were treated
as ‘‘glabrous’’ in our analyses; occasionally occurring single individuals with deviating life form were neglected). As we did not assign character states to the outgroup taxa, the reconstructed
character states only rely on the ingroup taxa. Chromosome num-
bers were taken from Benedí et al. (1997), Bennet and Leitch
(2010), Chrtek and Křísa (1992), Fedorov (1969), Goldblatt and
Johnson (1979), Hans (1973), Moore (1973), Smith and Tutin
(1968), and Urbatsch et al. (1975). We reconstructed ancestral
states for the characters using Mesquite (Maddison and Maddison,
2010), with the ‘‘Trace Character Over Trees’’ module applying the
parsimony reconstruction method over all trees derived from the
MrBayes analyses, discarding the first 1001 trees of each run as
burn-in.
3. Results
3.1. Phylogenetic relationships
The number of terminals, included characters, parsimony informative characters, percentage of parsimony informative characters, number and lengths of MP trees, consistency and retention
indices for both DNA regions, as well as the model of evolution
proposed by MrAIC and used in MrBayes analyses are presented
in Table 1.
Monophyly of Euphorbia subg. Esula is strongly supported by
the trnTF sequences (100% MPB, PP 1; Fig. 1), whereas the ITS sequences are not informative in this respect (Fig. 2). Relationships
at deeper nodes are generally poorly resolved in the ITS tree as
compared to the plastid tree (Figs. 1–3). The inferred ITS phylogenies differ to some extent between parsimony and Bayesian inference methods, but this mostly concerns weakly supported nodes
(MPB < 70% and/or PP < 0.95). For instance, the parsimony analysis
of the ITS data set infers sect. Helioscopia as sister of the outgroup
taxa E. milii and E. pulcherrima with 67% MPB, and in the Bayesian
tree they are positioned within the outgroup with PP 0.93. On the
other hand, conflicts between the two DNA regions are evident
(Fig. 3) and were detected also by the ILD test (P = 0.001), therefore
we did not proceed with the analyses of the concatenated data sets.
The taxon composition of the main terminal clades, furnished
with sectional names in Figs. 1 and 2 and whose circumscription
is defined in the Section 4.4, is largely congruent between the inferred phylogenies (note that some taxa were included only in
the ITS data set). However, the relationships among the terminal
clades differ to some extent between plastid and ITS trees
(Fig. 3a). Especially the position of sect. Conicocarpae is ambiguous
as it is resolved as sister to sect. Helioscopia by the Bayesian analysis of the ITS data with moderate support (PP 0.96), whereas in
the trnTF tree it is sister to sect. Myrsiniteae with strong support
(100% MPB, PP 1). The ITS NeigbourNet network (Fig. 3b) indicates
that sect. Conicocarpae shares splits with sections Myrsiniteae and
Helioscopia. In the trnTF tree (Fig. 3a), sect. Myrsiniteae and Conicocarpae are most closely related to sections Aphyllis, Carunculares,
Esula, Paralias, Patellares, and Peplus (94% MPB, PP 1), whereas in
the ITS tree (Fig. 3a) there is no support for such a relationship.
In the ITS NeigbourNet network (Fig. 3b), both are in intermediate
position between sect. Helioscopia and sections allied to sect. Esula.
The relationships among other sections are congruent in both
trees, or at least not conflicting: sections Paralias and Peplus are
Table 1
Matrix and phylogenetic analyses statistics for the two DNA regions analysed as well as substitution models proposed by MrAIC and used in the Bayesian analyses.
Region
trnTF
ITS
Number of terminals
Number of included characters
Number/percentage of parsimony informative characters (within the ingroup)
Length of MP trees
Consistency index (CI; excluding uninformative characters)
Retention index (RI)
Substitution model
104
2531
275 (251)/10.9% (9.9%)
698
0.824 (0.739)
0.963
HKY + C
135
840
319 (308)/38.0% (36.7%)
1741
0.397 (0.367)
0.866
HKYI + C
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Fig. 1. Bayesian consensus phylogram of trnT-trnF sequences sampled in mostly European representatives of Euphorbia subg. Esula. Numbering of multiple accessions per
taxon corresponds to Tables S1 and S2 in the Supplementary material. Numbers above branches are MPB values >50%, those below branches PP values >0.90. Reconstruction
of the life form is indicated by branch style: annual, thick black; perennial, thin black; ambiguous, grey. The classification proposed in this paper is indicated in the rightmost
column.
sisters, and sister to the sections Aphyllis, Esula, Oppositifoliae, and
Patellares, relationships among the latter being poorly resolved.
Monophyly of sect. Carunculares is not supported by the ITS data
(Figs. 2 and 3); in the trnTF data set only E. serrata was included.
B. Frajman, P. Schönswetter / Molecular Phylogenetics and Evolution 61 (2011) 413–424
417
Fig. 2. (a and b). Bayesian consensus phylogram of ITS sequences sampled in mostly European Euphorbia subg. Esula. Numbering of multiple accessions per taxon corresponds
to Tables S1 and S2 in the Supplementary material. The dashed branch in (a) was resolved by parsimony analysis. Numbers above branches are MPB values >50%, those below
branches PP values >0.90. Reconstruction of the life form is indicated by branch style: annual, thick black; perennial, thin black; ambiguous, grey. Character states are
indicated by symbols, and chromosome numbers taken from the literature are listed. Classifications by Smith and Tutin (1968), Boissier (1862), and Prokhanov (1949) are
indicated by symbols, and the one proposed in this paper is indicated in the rightmost column.
3.2. Life forms, morphological traits and character states
reconstruction
Assignment of morphological traits and life forms to each species is shown in Fig. 2. The inferred ancestral character states for
all sections are congruent between the ITS and trnTF phylogenies,
or at least not conflicting (single vs. more character states inferred
for a certain clade; Fig. 4). Three characters (leaf arrangement, presence of bracts among the male flowers, pericarp) are not presented
in Figs. 2 and 4, as one character state is specific for a single section:
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Fig. 2 (continued)
decussate leaf arrangement and spongy pericarp only for sect.
Lathyris, opposite leaves only for sect. Oppositifoliae, and absence
of bracts between male flowers only for sect. Myrsiniteae. They were
consequently reconstructed as ancestral for that particular section.
B. Frajman, P. Schönswetter / Molecular Phylogenetics and Evolution 61 (2011) 413–424
419
Fig. 3. Relationships among the main groups/sections of Euphorbia subg. Esula. (a) Summary diagrams of Bayesian analyses of ITS (left) and trnT-trnF (right) datasets derived
from the trees presented in Figs. 1 and 2. Nodes with support MPB < 70% and < 0.95 PP are collapsed. Numbers above branches indicate MPB values, those below branches PP
values. (b) NeighbourNet network of ITS sequences.
Fig. 4. Summary diagrams of Bayesian analyses of ITS (left) and trnT-trnF (right) datasets from mostly European representatives of Euphorbia subg. Esula. Nodes with support
MPB <70% and <0.95 PP are collapsed. Reconstructed character states are indicated by symbols, ordered from left to right as in the legend. In ambiguous cases character states
are shown, if one character state was reconstructed for a certain branch in >90% of all trees where this branch was present. In all other cases, ambiguous character states are
indicated by question marks.
Although it is not possible to infer whether the ancestor of subg.
Esula was perennial or annual (Figs. 1 and 4), it is clear that the annual life form developed several times and within several sections
of subg. Esula independently (thick branches in Figs. 1 and 2). The
reconstruction of eight morphological traits indicates that the
ancestor of subg. Esula was likely glabrous, with pinnately veined
leaves, bicornate nectarial glands, bracts present among the male
flowers, smooth capsules and smooth seeds (Fig. 4). The results
of the analysis are not conclusive regarding the ancestral state of
leaf arrangement (decussate vs. alternate) or pericarp type (spongy
vs. indurated).
4. Discussion
4.1. Monophyly of Euphorbia subg. Esula and limited utility of ITS for
inferring the evolutionary history of Euphorbia
The strong support for monophyly of Euphorbia subg. Esula by
plastid trnTF sequences (Fig. 1) and non-informativeness of the
ITS sequences in this respect is in agreement with the results of
Steinmann and Porter (2002), and partly of Bruyns et al. (2006).
In the latter study the Bayesian analyses of the ITS data set yielded
strong support for the monophyly of subg. Esula, whereas in our
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study the support is not high (PP 0.93; Fig. 2) In the parsimony
analyses the outgroup taxa E. milii and E. pulcherrima from clades
C and D of Steinmann and Porter (2002), respectively, are nested
in subg. Esula with low support (67% MPB; dashed branch in
Fig. 2a). However, support for this incongruence, which is inferred
by both inference methods, does not surpass cut-off levels of
MPB > 70% and/or PP > 0.95, applied here for recognition of wellsupported branches and identification of significant conflicts in
the trees.
Conflicting signal in the ITS data set is also indicated by a
square in the central part of a NeigbourNet network (Fig. S3 in
the Supplementary material) constructed with the ITS alignment
from Steinmann and Porter (2002). Their clade B, corresponding
to subg. Esula, shares a set of splits with clade A, represented in
our study by the outgroup taxa E. obesa and E. balsamifera. Contradictory results regarding the monophyly and position of subg.
Esula obtained by various analyses of ITS data sets might partly
be explained by different taxon sampling, likely in combination
with biological processes such as hybridisation or lineage sorting
(Wendel and Doyle, 1998; Slowinski and Page, 1999), coupled
with specific properties of the ITS region, e.g., incomplete concerted evolution (Álvarez and Wendel, 2003). Moreover, relatively
strong divergence of ITS sequences of taxa from different subgenera likely causes alignment problems and results in increased
homoplasy. Other nuclear DNA regions such as low-copy nuclear
genes or more conserved regions such as 18S and 28S nrDNA
should be preferably used to elucidate the phylogenetic relationships among major clades of Euphorbia in order to establish better-resolved nuclear DNA phylogenies that can provide a firm
basis to test hypotheses regarding evolution and biogeography
of the genus.
4.2. Conflicting relationships in plastid and ITS sequences among
different groups of Euphorbia subg. Esula
Relationships among the clades at deeper nodes in the ITS tree
are poorly resolved, whereas the trnTF tree offers better resolution
among the major clades. For simplicity, in the following we use the
sectional names defined in the last section of the Discussion and
shown in Figs. 1–4. The topologies in both trees are to some extent
incongruent (Fig. 3a); especially the position of sect. Conicocarpae
is ambiguous, appearing most closely related either to sect. Helioscopia (ITS) or to sect. Myrsiniteae (trnTF). The ITS NeigbourNet network (Fig. 3b) indicates conflicting splits in subg. Esula, and
sections Conicocarpae and Myrsiniteae have intermediate position
between sect. Helioscopia and a group including sect. Esula and allied sections. A set of relatively long, thus strongly weighted, parallel splits leading to sect. Helioscopia, however, clearly indicates
its divergence. Sect. Conicocarpae, positioned between the sections
Helioscopia and Myrsiniteae, shares more morphological characteristics with sect. Myrsiniteae (e.g., palmately veined, glaucous
leaves, similar ecology; Figs. 2a and 4, see also Sections 4.4.1–
4.4.3) than with sect. Helioscopia.
Different processes can be responsible for incongruent phylogenetic patterns (see Wendel and Doyle, 1998; Slowinski and Page,
1999), classified as interlineage (hybridisation, lateral gene transfer between organismal lineages) or intralineage (incomplete lineage sorting, orthology/paralogy conflation). Stochastic or
systematic errors, such as failure of phylogenetic models and
methods to converge on the correct solution, may further complicate the situation. Nuclear ribosomal DNA that is present in multiple copies in the genome is subject to different processes that can
be responsible for conflicting phylogenetic signals, e.g., differential
and incomplete homogenisation of the multiple copies by concerted evolution within and among different lineages (Álvarez
and Wendel, 2003). Concerted evolution, following hybridisation,
or differential sorting of ancestral polymorphisms could be responsible for conflicting splits indicated by the NeighbourNet network,
high homoplasy observed in our ITS data set (Table 1), and incongruences between the ITS and trnTF phylogenies. It is difficult to
distinguish between hybridisation and incomplete lineage sorting
(e.g., Frajman et al., 2009), but the intermediate position of section
Conicocarpae between sections Myrsiniteae and Helioscopia in the
ITS NeighbourNet network might indicate its hybrid origin, the
ancestor of sect. Myrsiniteae serving as the maternal parent (plastids are maternally inherited in Euphorbia; Corriveau and Coleman,
1988; Zhang et al., 2003).
Relationships among other sections are not conflicting, but display different support levels in both phylogenies (Figs. 1–3). Both
nuclear and plastid sequences support the common origin of sections Aphyllis, Carunculares, Esula, Oppositifoliae (only included in
the ITS tree), Paralias, Patellares and Peplus. Also in the Neighbour-Net network (Fig. 3b) they share several common splits,
E. serrata from sect. Carunculares being most divergent. Morphologically, the members of these sections differ from sect. Helioscopia,
which always exhibits convex nectarial glands, by having mostly
bicornate to semilunate nectarial glands with truncate to concave
outer margin, and from sections Myrsiniteae and Conicocarpae by
having mostly non-glaucous, pinnately veined leaves (palmately
veined in sect. Paralias; Fig. 2).
4.3. Multiple origins of annual life form in the evolution of Euphorbia
subg. Esula
Prokhanov (1949) expressed doubt concerning the naturalness
of sect. Cymatospermum, in which he included several annual spurges with more or less bicornate nectaries. The taxa included (Fig. 1)
are indeed a heterogeneous assemblage of annuals (see Fig. 2 for
different character states). Using both a phylogenetic framework
and ancestral character state reconstruction, we clearly show that
the annual life form developed in several lineages of subg. Esula
independently from perennial ancestors (Figs. 1 and 2). Nine shifts
from perennials to annuals in five sections can be observed in the
plastid tree (Fig. 1), and one more in the ITS tree (Fig. 2). Similar to
the annual life form within subg. Esula, also succulence developed
several times in the evolution of Euphorbia (Bruyns et al., 2006;
Steinmann and Porter, 2002; Zimmermann et al., 2010). Extended
taxon sampling will likely reveal the occurrence of annuals in other
sections as well. It remains ambiguous, however, whether the
ancestor of subg. Esula was annual or perennial (Figs. 1 and 4).
Character state reconstruction including members from other subgenera is needed to resolve this question.
Phylogenetic studies in several other plant groups have revealed that previously co-classified annuals have in fact developed
several times independently from their perennial ancestors (e.g.,
Astragalus: Liston and Wheeler, 1994; Veronica: Albach et al.,
2004; but see Tank and Olmstead, 2008). As reported for other
groups (Andreasen and Baldwin, 2001; Müller and Albach, 2010;
Smith and Donoghue, 2008), the branches leading to annual species of Euphorbia are often relatively longer as compared to their
perennial sister taxa (e.g., E. pterococca vs. E. hirsuta, E. peplus/
E. peploides vs. E. brachycera, E. falcata vs. other members of the
sect. Conicocarpae, E. terracina vs. E. dendroides; Fig. 2). In all these
and other cases, the annuals differ substantially in habit and
growth height from the closely related perennials (e.g., E. hirsuta is
usually about seven times taller than its sister species E. pterococca).
Different life form and divergent overall habit were likely the
reason why some closely related species (e.g., E. dendroides and
E. terracina) were never classified together, even if they share
several morphological traits and exhibit the same chromosome
base number (see Fig. 2).
B. Frajman, P. Schönswetter / Molecular Phylogenetics and Evolution 61 (2011) 413–424
4.4. Relationships within the major clades, evolution of morphological
traits and taxonomic implications
The taxonomic composition of the well-supported major clades
is congruent between the ITS and trnTF phylogenies (Figs. 1 and 2;
some taxa were included only in the ITS data set). Various, only
partly compatible sectional and subsectional classifications of
Euphorbia subg. Esula have been proposed in the past (e.g., Boissier,
1862; Prokhanov, 1949; Fig. 2). The most recent (sub)sectional
revision of extra-tropical Eurasian members of Euphorbia was proposed by Geltman (2007), but it was neither based on a phylogenetic framework nor was there a clear overview of the taxa
included. Our results clearly show that most of the infrageneric
groups proposed by Geltman (2007) are unnatural, often polyphyletic assemblages. Exceptions are sections Helioscopia and Myrsiniteae. The sectional classifications, largely incongruent with
phylogenetic history (see Fig. 2), likely resulted from the plasticity
and parallel evolution of different morphological characters (Figs. 2
and 4), and the use of only a few characters, such as seed surface
for classification. Chromosome numbers are apparently of only
limited classificatory value in Euphorbia (Fig. 2), as noted already
by Hans (1973). Most sections have various chromosome numbers,
exceptions being section Myrsiniteae with 2n = 20, and the sister
sections Peplus and Paralias with mostly 2n = 16 chromosomes.
Polyploidisation played a negligible role in the evolution of the
three before-mentioned sections, but was important in most others. Polyploid series can be observed in several annual taxa (e.g.,
E. exigua, E. falcata, E. helioscopia) and in different perennial members of sect. Esula. For several species, multiple chromosome numbers have been reported, which might be due to inaccurate counts
(Hans, 1973). Erroneous determinations might play a role as well.
A revision of Slovenian spurges revealed that 20% out of almost 900
Euphorbia specimens from different herbaria were wrongly determined (Frajman and Jogan, 2007). In addition, deviating counts
for the same species might be also due to the presence of various
cytotypes, some of which possibly act as cryptic species. A sound
caryological revision of subg. Esula is certainly needed to substantiate further discussion about chromosome evolution in this group.
Below we summarise the composition of the sections and the
internal relationships, as well as their morphology (see also
Fig. 2), distribution and ecology. Ancestral character states for each
group are shown in Fig. 4. The inferred states of all sections are
congruent, or at least not conflicting, between ITS and trnTF trees.
The proposed sectional classification will certainly need to be
amended, as, although all subsections of Boissier (1862) shown
to belong to subg. Esula were sampled, only one fifth of all members of subg. Esula were included in our analyses. With addition
of other taxa in future studies, new sections might need to be
established. However, all sections recognised here have strong support in our phylogenies and it is not likely that additional analyses
with denser taxon or DNA sampling would collapse these clades.
4.4.1. Euphorbia sect. Helioscopia Dumort., Fl. Belg. 87. 1827. Type: E.
helioscopia L.
Most taxa included in this clade have also in the past been
classified together (sect. Helioscopia, sect. Tulocarpa (Raf.) Prokh.,
sect. Tithymalus (Scop.) Boiss. subsect. Galarrhaei Boiss.; see
Fig. 2). An exception is E. mellifera, previously classified in sect.
Balsamis Webb and Berth. Section Helioscopia comprises annual
and perennial herbs or shrubs with highest diversity in Europe,
the Mediterranean, and temperate Asia. Its members are glabrous
or pubescent, have alternate, pinnately veined leaves, transversely ovate nectarial glands with convex outer margin and
bracts between the male flowers. Capsules are either smooth or
tuberculate, with indurated pericarp, and the seeds are mostly
smooth, rarely punctate-rugulose or faveolate (see Fig. 2a).
421
Euphorbia helioscopia, E. apios, E. pterococca, and E. hirsuta form
a sequence of basal branches in the plastid tree, and the first
three, together with E. carniolica, also in the ITS tree. The relationships among other well-supported clades within sect. Helioscopia are mostly unresolved and their taxonomic constitution is
partly incongruent between both trees. Traditional subsectional
classifications (e.g., Prokhanov, 1949) are mostly not supported
by molecular data. Steinmann and Porter (2002) suggested that
the main morphological character to distinguish this section from
other members of subg. Esula could be tuberculate ovaries,
whereas members of other sections have smooth (or slightly
granulate) ovaries. This suggestion is not entirely supported by
our data, as some members of sect. Helioscopia (e.g., E. akenocarpa, E. helioscopia, E. villosa) have smooth capsules as well. Another character serving to distinguish sect. Helioscopia from
other members of subg. Esula is the shape of the nectarial glands,
which are transversely ovate with convex outer margin in sect.
Helioscopia, and mostly of other shapes, with truncate to concave
outer margin in other clades (some members of sect. Aphyllis can
also have a convex outer margin of the nectarial glands; Fig. 2).
An additional distinguishing character with limited discriminatory power is the type of the indumentum: several members of
sect. Helioscopia are pubescent with unicellular trichomes. Taxa
belonging to other clades are mostly glabrous, with the exception
of sect. Patellares bearing multicellular trichomes and some other
taxa from other clades that can be sparsely pubescent (e.g., E.
esula, E. herniariifolia, E. salicifolia). Ecologically, members of this
section are fairly heterogeneous, but many of them are relatively
mesophilic as compared to the other sections with the exception
of sect. Patellares.
4.4.2. Euphorbia sect. Conicocarpae (Prokh.) Frajman, comb. nov.
Basionym: Tithymalus Gaertn. sect. Conicocarpus Prokh., Sist. Obzor
Moloch. Sr. Azii 155. 1933. Type: E. humilis C.A. Mey
This clade consists of mostly perennial glabrous taxa, characterised by palmately veined, glaucous, equifacial (isolateral) leaves,
and truncate nectarial glands, sometimes with two, occasionally
bifid or dilated, horns. Capsules are shallowly sulcate, smooth to
granulate (e.g., E. nicaeensis, E. segueriana), and seeds mostly
smooth (see Fig. 2a for exceptions). Most members of this clade
are relatively thermophilic.
This clade largely corresponds to sect. Murtekias (Raf.) Prokh.
subsect. Conicocarpae Prokh., but includes some taxa from other
sections (e.g., E. falcata). Inclusion of the members of this clade into
sect. Paralias Dumort or sect. Tithymalus subsect. Esulae Boiss., as
traditionally classified (see Fig. 2), is not supported by molecular
data. We suggest treating this clade as an independent section
Conicocarpae. Prokhanov (1949) assigned E. segueriana as the type,
but his choice is predated by Wheeler (1943), who designated
E. humilis C.A. Mey as lectotype.
Euphorbia falcata and E. pithyusa are successively sister species
to the other taxa in this section and might merit subsectional recognition. Relationships among the other taxa are somewhat conflicting between both trees. Contrary to previous suggestions
(e.g., Fischer et al., 2008), E. saxatilis is not most closely related to
E. triflora and E. kerneri, and E. herzegovina is not conspecific with
E. barrelieri as assumed in the past (e.g., Trinajstić, 2007). Neither
Euphorbia segueriana and E. niciciana nor E. glareosa and E. nicaeensis are most closely related. Consequently, they should be treated
as independent species rather than as subspecies as suggested by
Smith and Tutin (1968). More detailed studies with broader geographic sampling suggest that the evolutionary history of this clade
is even more complicated, and incongruences are observed between the plastid and ITS data sets (Frajman and Schönswetter,
unpubl.).
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B. Frajman, P. Schönswetter / Molecular Phylogenetics and Evolution 61 (2011) 413–424
4.4.3. Euphorbia sect. Myrsiniteae (Boiss.) Lojac., Fl. Sicula 2, 2: 345.
1904. Type: E. myrsinites L.
This clade contains E. myrsinites and closely related taxa, which
have also traditionally been recognised as a separate section. Its
members are characterised by palmately veined, glaucous, equifacial leaves, and nectarial glands with short, often dilated and lobed
horns. Bracts between male flowers are absent, representing a
synapomorphy for this section. Capsules are smooth to granulate,
and seeds are smooth to vermiculate-rugose. The species of sect.
Myrsiniteae mostly grow in dry, exposed habitats and are in this
respect similar to most members of sect. Conicocarpae.
4.4.4. Euphorbia sect. Carunculares (Boiss.) Tutin in Feddes Repert. 79:
55. 1968. Type: E. serrata L.
In the ITS tree (Fig. 2b) E. serrata and E. calyptrata form a polytomy with the clade including sections Aphyllis, Esula, Oppositifoliae, Paralias, Patellares, and Peplus. They have both been grouped in
subsect. Carunculares by Boissier (1862), including species with
mostly roughly serrate, palmately veined leaves, and truncate to
two-horned nectarial glands, but their monophyly is not supported
by the ITS data (Figs. 2b and 3b). Further analyses are needed to
clarify the phylogenetic relationship between the two taxa and
their allies. Prokhanov (1949) classified E. serrata and E. calyptrata
in sect. Chylogala (Fourr.) Prokh., the type being E. bungei Boiss. As
E. bungei is not included in our analyses, we follow the classification by Tutin.
Taxa of the following sections (Patellares to Peplus; 4.4.5–4.4.10)
are characterised by mostly alternate (not in sect. Oppositifoliae),
bifacial, pinnately veined (not in sect. Paralias) leaves, crescent,
semilunate to bicornate nectarial glands with mostly slender
horns, and smooth to granulate capsules.
4.4.5. Euphorbia sect. Patellares (Prokh.) Frajman, comb. and stat. nov.
Basionym: Euphorbia subsect. Patellares Prokh. in Komarov, Fl. USSR
14: 743. 1949. Type: E. amygdaloides L.
This clade comprises perennial species characterised by connate
raylet leaves and pubescent–villous indumentum, composed of relatively long, multicellular hairs. Its members were included in sect.
Esula subsect. Patellares by Prokhanov (1949). We propose its treatment as an independent section Patellares, considering its clear
morphological differentiation as well as its monophyly, clearly distinct from sect. Esula as circumscribed here. All members of sect.
Patellares are relatively mesophilic.
4.4.6. Euphorbia sect. Oppositifoliae (Boiss.) Baikov, Molochan Severn.
Azii 114. 2007. Type: E. inderiensis Kar. et Kir.
Euphorbia turczaninowii was included in subsect. Oppositifoliae
by Boissier (1862), comprising annuals with opposite leaves distributed in Central Asia. As E. turczaninowii, the only representative
of the sect. Oppositifoliae in our ITS analysis, is phylogenetically
divergent from its sister sect. Aphyllis, and there are several morphological differences between the two groups (Fig. 2), we treat
sect. Oppositifoliae as an independent section.
4.4.7. Euphorbia sect. Aphyllis Webb and Berthel., Hist. Nat. Iles
Canaries 3: 253. 1846-47. Type E. aphylla Brouss. ex Willd
This clade is a heterogeneous assemblage of annual and perennial species that were in the past, due to their different habit and
diverse morphology, classified into several (sub)sections (Fig. 2).
Most of the Macaronesian dendroid spurges (included only in the
ITS analyses) as well as E. dendroides were by Boissier (1862) classified within subsect. Pachycladae, and their characteristics were
discussed by Molero et al. (2002). The other taxa included in this
clade were always classified in other sections (see Fig. 2); therefore
their phylogenetic alliance to E. dendroides was unexpected. It is
difficult to specify their common characteristics (but see, e.g., the
common characteristics of E. terracina and E. dendroides in
Fig. 2b) and several different (sub)sections might need to be recognised within this group. Further analyses, including more taxa as
well as plastid data for the Macaronesian group, are needed to clarify the relationships among the taxa included and to provide a solid
basis for a sectional revision.
4.4.8. Euphorbia sect. Esula Dumort., Fl. Belg. 87. 1827. Type: E. esula L
This clade contains perennial taxa with deeply sulcate capsules
and smooth seeds, often growing in dry grasslands. Most of them
were already in the past classified in this section (see Fig. 2). Members of sect. Patellares as well as E. terracina (see above) are not
most closely related to E. esula and its allies, although they were often classified together (Fig. 2, see also Geltman, 2007).
Relationships among the taxa are partly conflicting between ITS
and plastid phylogenies. Plastid data suggest that E. tshuensis from
Altai and E. valliniana, a narrow endemic from the southwestern
Alps form the basal-most branches of the clade, whereas in the
ITS tree there is no support for such relationships. However, E. valliniana and E. variabilis from the southeastern Alps as well as E.
kraussiana and E. genistoides from South Africa (the latter two are
not included in the trnTF data set) have isolated positions in the
Bayesian tree (Fig. 2). Several taxa from this section form polyploid
series (Fig. 2b), and some of them have been reported to hybridise,
whereas hybrids have not been reported from other clades (Hegi
and Beger, 1924; Chrtek and Křísa, 1992). Low phylogenetic resolution within the group, especially in the ITS tree (Fig. 2b), as well as
partly incongruent positions of some taxa in ITS and trnTF trees,
might result from reticulation and polyploidisation events, which,
together with concerted evolution, might have blurred the phylogenetic signal (Álvarez and Wendel, 2003).
Euphorbia lamprocarpa from Steinmann and Porter (2002;
GenBank number AF537545), as well as E. polychroma from Wurdack
et al. (2005; GenBank number AY794606), traditionally classified
within sect. Helioscopia, are included in sect. Esula in our study.
Inspection of the herbarium voucher of E. lamprocarpa revealed
that it actually belongs to E. virgata s.l., whereas the herbarium
voucher of E. polychroma was not available at NCU as indicated
(Wurdack et al., 2005). The obvious misplacement of Wurdack’s
accession of ‘‘E. polychroma’’ indicates that it is certainly misidentified.
4.4.9. Euphorbia sect. Paralias Dumort., Fl. Belg.: 87. 1827. Type:
E. paralias L.
This clade comprises glabrous and glaucous annuals (E. segetalis) and perennials with imbricate, isolateral, palmately veined
leaves (Figs. 2b and 4), characteristics not found in any other section of the clade including section Esula and allies (Fig. 1–3). The
species constituting sect. Paralias have similar habitat preferences;
most of them, with exception of E. segetalis, grow in coastal, often
saline areas. Section Paralias in our circumscription is less speciesrich than traditionally circumscribed (see Fig. 2).
4.4.10. Euphorbia sect. Peplus Lázaro, Comp. Fl. Españ. 282. 1896.
Type: E. peplus L.
It contains annual and perennial taxa with shallowly sulcate
capsules, which are mostly two-winged on the keels (not in
E. brachycera). The taxa included were never assumed to be closely
related (see Fig. 2) and are dissimilar in habit, but winged capsules
might be synapomorphic for this clade, and were probably secondarily lost in E. brachycera (Figs. 2b and 4).
4.4.11. Euphorbia sect. Lathyris Dumort., Fl. Belg. 87. 1827. Type: E.
lathyris L.
This section includes a single species, E. lathyris, which is morphologically unique within subg. Esula, having decussate leaf
B. Frajman, P. Schönswetter / Molecular Phylogenetics and Evolution 61 (2011) 413–424
arrangement and spongy pericarp, characteristics not found in any
other member of the subgenus (see Fig. 2). It is an annual to biennial species with linear to oblong-lanceolate, pinnately veined bifacial leaves, and nectar glands with two, mostly clavate, dilated
horns. It was already in the past included in its own (sub)section
and our data support this. The exact phylogenetic position of
E. lathyris within subg. Esula is, however, not clear. It is resolved
as weakly supported sister to all other members of subg. Esula in
the trnTF tree (Fig. 1) and unresolved within the ITS tree (Fig. 2).
In the ITS NeigbourNet network (Fig. 3b) it shares several common
splits with sect. Esula and allied sections, as well as with sect.
Myrsiniteae. The comparatively long branch leading to E. lathyris
(Figs. 1 and 2) might cause problems in phylogenetic inference,
preventing the method from converging on the ‘‘true’’ topology
(e.g., Chang, 1996; Ruano-Rubio and Fares, 2007). Increased substitution rates have also been observed in annual lineages of several
other plant groups, likely connected to their shorter generation
times as compared to perennials (Andreasen and Baldwin, 2001;
Müller and Albach, 2010).
For a more comprehensive taxonomic revision of the subgenus
further analyses are needed, including additional taxa, more extensive geographic sampling, and additional DNA regions. More extensive preliminary studies of certain groups (e.g., the E. villosa group in
sect. Helioscopia, sect. Myrsiniteae, and sect. Conicocarpae) indicate
that traditional classifications do not reflect the evolutionary
relationships inferred by phylogenetic studies (Frajman and
Schönswetter, unpubl.). Detailed studies, currently underway in
the framework of the Euphorbia Planetary Biodiversity Inventory
project, will likely provide answers to several remaining questions
regarding the evolution and diversification of Euphorbia subg. Esula.
Acknowledgments
}, W. Gutermann,
We are most grateful to T. Bačič, A. M. Csergo
L. Schratt-Ehrendorfer, W. Till, R. Vilatersana and M.M. Wernisch
for collecting some of the plant samples used, and to W. Till
(WU) for providing us with specimens, as well as to J. England
(RSA) for providing a photo of a herbarium specimen. Many thanks
are due to V. Steinmann for the ITS alignment of Euphorbia and to
P. Berry as well as one anonymous reviewer for their constructive
comments on previous versions of the manuscript. The Govern de
les Illes Baleares provided a collection permit (REC 121/2009) for
E. fontqueriana.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ympev.2011.06.011.
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