Molecular Phylogenetics and Evolution 61 (2011) 413–424
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Molecular Phylogenetics and Evolution
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Giants and dwarfs: Molecular phylogenies reveal multiple origins of annual spurges within Euphorbia subg. Esula ⇑ Bozˇo Frajman , Peter Schönswetter
Institute of Botany, University of Innsbruck, Sternwartestrasse 15, A-6020 Innsbruck, Austria article info abstract
Article history: Euphorbia (Euphorbiaceae) comprises over 2150 species and is thus the second-largest genus of flowering Received 9 February 2011 plants. In Europe, it is represented by more than 100 species with highest diversity in the Mediterranean Revised 7 June 2011 area; the majority of taxa belong to subgenus Esula Pers., including about 500 taxa. The few available phy- Accepted 13 June 2011 logenetic studies yielded contrasting results regarding the monophyly of subg. Esula, and the phyloge- Available online 25 June 2011 netic 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 Keywords: from about 100, predominantly European taxa of subg. Esula in order to infer its phylogenetic history. Bayesian inference The plastid data support monophyly of subg. Esula whereas the ITS phylogeny, which is generally less Character state reconstruction Parsimony resolved, is indecisive in this respect. Although some major clades have partly incongruent positions in Phylogeny the ITS and plastid phylogenies, the taxonomic content of the major terminal clades is congruent in both Taxonomy 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 morpholog- ical traits used in previous classifications of Euphorbia developed in parallel in different lineages. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction with the development of a terminal cyathium, mostly surrounded by a whorl of ray-leaves. The latter subtend a fascicle of three to Euphorbia (Euphorbiaceae) is with over 2150 species (Bruyns many dichotomously branching rays, bearing several dichasially et al., 2006) the second-largest genus of flowering plants, outsized arranged cyathia, subtended by raylet leaves. The involucral glands only by Astragalus (Mabberley, 2008). Distributed worldwide and lack petaloid appendages and are of different forms, such as subor- varying in habit from prostrate annuals to 20 m tall trees, the spur- bicular to transversely ovate, two-horned, or with truncate to ges achieve their greatest diversity in arid areas of Africa and emarginate outer margins. Ovaries are three-locular, and seeds Madagascar, where many of them are cactus-like succulents (Turner, usually bear a caruncle (Smith and Tutin, 1968; Turner, 1998; 1998). In Europe, Euphorbia is represented by more than 100 spe- Steinmann and Porter, 2002). cies (Smith and Tutin, 1968) with highest diversity in the Mediter- Even if Euphorbia is one of the richest genera in number of taxa, ranean area. The majority of European taxa belong to subgenus only a few studies have addressed phylogenetic relationships with- Esula Pers. (Smith and Tutin, 1968), which largely corresponds to in this genus. Steinmann and Porter (2002) inferred the phylogeny Euphorbia subg. Paralias (Raf.) Prokh. (Prokhanov, 1949)orEuphor- of the tribe Euphorbieae, with the majority of the sampled taxa bia sect. Tithymalus Boiss. (Boissier, 1862), sometimes treated at belonging to Euphorbia, using nuclear ribosomal internal tran- generic level as Tithymalus Gaertn. (e.g., Scopoli, 1772; Chrtek scribed spacer (ITS) sequences and the coding plastid region ndhF. and Krˇísa, 1992). The subgenus Esula includes roughly 500 herba- They have shown that although Euphorbia subg. Esula as tradition- ceous perennials, annuals, shrubs, small trees and succulents nat- ally circumscribed (e.g., Wheeler, 1943) is polyphyletic, most taxa urally occurring on all continents except Australia and Antarctica, form a clade (referred to as ‘‘clade B’’ by Steinmann and Porter, but achieving its greatest diversity in northern temperate regions 2002), including also some African succulent species from subge- (Bruyns et al., 2006; Steinmann and Porter, 2002). Most species nus Tirucalli (Boiss.) S. Carter. This clade has relatively high support have alternate, exstipulate and (sub)sessile cauline leaves and ter- (86% bootstrap) in the ndhF tree, but no support in the ITS tree. minal pleiochasial inflorescences. The stem growth terminates Later, Bruyns et al. (2006) inferred the phylogeny of southern African spurges using ITS and plastid psbA-trnH sequences. With partly
⇑ Corresponding author. Fax: +43 5125072715. different taxon sampling they corroborated the main results of E-mail addresses: [email protected] (B. Frajman), peter.schoenswetter@- Steinmann and Porter (2002). Similar results were obtained by uibk.ac.at (P. Schönswetter). Park and Jansen (2007) and Zimmermann et al. (2010), who used
1055-7903/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2011.06.011 414 B. Frajman, P. Schönswetter / Molecular Phylogenetics and Evolution 61 (2011) 413–424 a partly different set of taxa and the latter also another plastid re- and Porter, 2002; Bruyns et al., 2006). In the ITS data set we also gion (ndhF and trnL-trnF, respectively). When analysed in a Bayes- included all sequences belonging to clade B from Steinmann and ian framework, clade B received maximal support (posterior Porter (2002) and Bruyns et al. (2006). Based on these studies we probability, PP 1) in the ITS tree (Bruyns et al., 2006; Zimmermann also selected the outgroup taxa from their clades A, C and D. Vou- et al., 2010). The exact phylogenetic position of this group, how- cher data and GenBank accession numbers are presented in Tables ever, remains unclear (Zimmermann et al., 2010). Based on the re- S1 and S2 in the Supplementary material. sults of their phylogenetic analyses, Bruyns et al. (2006) proposed a new subgeneric classification for Euphorbia, in which they assigned 2.2. DNA isolation, PCR and sequencing all taxa of clade B to subg. Esula, thus including also some succulent members, formerly considered part of subg. Tirucalli. Extraction of total genomic DNA from herbarium specimens or None of the previous phylogenetic studies included a sufficient silica-gel dried material was performed following the modified number of taxa from subg. Esula to be able to draw conclusions CTAB-protocol of Tel-Zur et al. (1999). Prior to extraction with about interspecific relationships and to compare the phylogenetic high-salt CTAB buffer the ground tissue was washed three times assemblages with traditional (sub)sectional delimitations of, e.g., with wash buffer containing sorbitol to remove polysaccharides. Boissier (1862) or Prokhanov (1949). Steinmann and Porter Amplification of ITS, purification of PCR products, cycle-sequencing (2002) showed that some subsections of Boissier’s sect. Tithymalus and subsequent electrophoresis followed Schönswetter and are polyphyletic and several taxa do not belong to subg. Esula. They Schneeweiss (2009). The plastid trnT-trnF region (trnTUGU- concluded that out of all of Boissier’s subsections only Decussatae trnLUAA-trnFGAA intergenic spacers including the trnLUAA intron; Boiss., Oppositifoliae Boiss., Carunculares Boiss., Galarrhaei Boiss., from here on referred to as trnTF) was amplified using the primer Esulae Boiss., and Myrsiniteae Boiss., as well as some taxa from sub- pair a and f (Taberlet et al., 1991). The PCR reaction mix contained sect. Pachycladae Boiss. and subg. Tirucalli can be considered mem- 9 ll of ReadyMix (Sigma–Aldrich), 13 ll water, 1 ll BSA (10 mg/ bers of subg. Esula (sect. Tithymalus subsect. Osyrideae Boiss. was ml; Promega), 0.5 ll of each primer (10 lM), 0.5 ll of MgCl2 not included in their studies!). However, they were neither able (25 lM), and 0.5–1 ll of total genomic DNA of unknown concen- to draw conclusions regarding the monophyly of these subsections tration. We used the following PCR conditions: 5 min at 95 °C, fol- nor about the relationships among them, and these questions re- lowed by 35 cycles of 30 s at 94 °C, 30 s at 48 °C and 4 min at 65 °C, main unsolved to date. followed by a final 10 min extension period at 65 °C. Purification of Molecular phylogenies can provide a framework to trace the PCR products and cycle sequencing were performed as for ITS, evolution of morphological characters through the evolutionary using the primers a, c and f, in some cases also b and d (Taberlet history of organisms (e.g., Escobar García et al., 2009; Huelsenbeck et al., 1991). et al., 2003; Maddison and Maddison, 2010; Schäffer et al., 2010). Morphological characters traditionally applied for the (sub)sec- 2.3. Contig assembly, sequence alignment and phylogenetic analyses tional classification of Euphorbia subg. Esula are mostly derived from the plants’ reproductive organs. Especially the shape of the Contigs were assembled and edited using Staden (Staden et al., nectarial glands on the cyathial margin, presence and shape of 1998). Base polymorphisms were coded using the NC-IUPAC ambi- tubercules on the capsules as well as seed ornamentation played guity codes. Sequences were manually aligned using QuickAlign an important role in classification (e.g., Boissier, 1862; Prokhanov, (Müller and Müller, 2003), mostly without major problems. The 1949). Important vegetative characters include leaf arrangement alignments are available from B. Frajman. and venation, as well as life form. Annual species with bicornate Maximum parsimony (MP) analyses as well as MP bootstrap nectaries were assigned to sect. Cymatospermum (Prokh.) Prokh., (MPB) analyses of both data sets were performed using PAUP although Prokhanov (1949) expressed his doubts about the natu- 4.0b10 (Swofford, 2002). The most parsimonious trees were ralness of this group. It has often been assumed that annuals gen- searched heuristically with 1000 replicates of random sequence erally evolve from perennial ancestors (e.g., Stebbins, 1957; see addition, TBR swapping, and MulTrees on. The swapping was per- also Tank and Olmstead, 2008), but also the opposite has been formed on a maximum of 1000 trees (nchuck = 1000). All charac- observed in groups like Castilleja (Orobanchaceae; Tank and ters were equally weighted and unordered. The data set was Olmstead, 2008). bootstrapped using full heuristics, 1000 replicates, TBR branch The aim of our study is to disentangle the phylogenetic history swapping, MulTrees option off, and random addition sequence of Euphorbia subg. Esula using DNA sequences of nuclear ribosomal with five replicates. Euphorbia balsamifera, E milii, E. obesa, and ITS and the plastid trnT-trnF region from 99 predominantly Euro- E. pulcherrima were used as outgroups in ITS, and E. ipecacuanhae, pean taxa. In particular, we (1) address the question of monophyly E. obesa, and E. pulcherrima in trnTF analyses, based on previous of the subgenus using available and new sequence data. Using studies (Steinmann and Porter, 2002; Bruyns et al., 2006). character state reconstruction, we (2) trace the development of life Combinability of the trnTF and ITS data sets (pruned to taxa se- forms (annual vs. perennial) as well as the evolution of morpholog- quenced for both regions) was assessed in a parsimony framework ical traits used in previous classifications of Euphorbia. In addition, using the incongruence length difference (ILD) test implemented in (3) we assess the traditional sectional and subsectional assem- PAUP 4.0b10 (Swofford, 2002) employing 1000 partition replicates, blages for monophyly and (4) propose an improved sectional clas- each with 10 random sequence addition replicates saving no more sification for E. subg. Esula. Finally (5), we summarise ecology and than 500 trees per replicate and TBR branch swapping. morphological characteristics of the inferred groups. 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 2. Materials and methods Oslo (http://www.bioportal.uio.no/) applying the substitution models proposed by the Akaike information criterion implemented 2.1. Plant material in MrAIC.pl 1.4 (Nylander, 2004; Table 1). Values for all parame- ters, such as the shape of the gamma distribution, were estimated We sampled 99, mostly European taxa from Euphorbia subg. during the analyses. The settings for the Metropolis-coupled Markov Esula from all of Boissier’s (1862) subsections currently included chain Monte Carlo (MC3) process included four runs with four in subg. Esula, with exception of subsect. Osyrideae (Steinmann chains each (three heated ones using the default heating scheme), B. Frajman, P. Schönswetter / Molecular Phylogenetics and Evolution 61 (2011) 413–424 415 run simultaneously for 10,000,000 generations each, sampling bers were taken from Benedí et al. (1997), Bennet and Leitch trees every 1000th generation using default priors. The PP of the (2010), Chrtek and Krˇísa (1992), Fedorov (1969), Goldblatt and phylogeny and its branches was determined from the combined Johnson (1979), Hans (1973), Moore (1973), Smith and Tutin set of trees, discarding the first 1001 trees of each run as burn-in. (1968), and Urbatsch et al. (1975). We reconstructed ancestral As the relationships at deeper nodes in the ITS tree were poorly states for the characters using Mesquite (Maddison and Maddison, resolved and to some extent conflicting with the trnTF tree, we 2010), with the ‘‘Trace Character Over Trees’’ module applying the used SplitsTree4 4.10 (Huson, 1998; Huson and Bryant, 2006)to parsimony reconstruction method over all trees derived from the generate a NeighbourNet network (Bryant and Moulton, 2004)in MrBayes analyses, discarding the first 1001 trees of each run as order to display conflicts in the ITS data. The NeighborNet method burn-in. computes a set of incompatible splits, which are represented in the split network by edges in non-parallel positions (Huson and 3. Results Bryant, 2008). We applied the UncorrectedP method to compute the proportion of positions at which two sequences differ. Ambiguous 3.1. Phylogenetic relationships base codes were treated as missing states (we also applied the op- tions ‘‘average’’ and ‘‘match’’ for the ambiguous bases, but the The number of terminals, included characters, parsimony infor- resulting networks did not differ substantially; not shown). As mative characters, percentage of parsimony informative charac- the parsimony and Bayesian as well as the NeighbourNet analyses ters, number and lengths of MP trees, consistency and retention resulted in ambiguous position of the outgroup taxa, we also com- indices for both DNA regions, as well as the model of evolution puted a NeighbourNet network for the Euphorbia ITS data set from proposed by MrAIC and used in MrBayes analyses are presented Steinmann and Porter (2002), using their alignment (provided by in Table 1. Steinmann), pruning the outgroup taxa. Monophyly of Euphorbia subg. Esula is strongly supported by the trnTF sequences (100% MPB, PP 1; Fig. 1), whereas the ITS se- 2.4. Life forms, morphological traits and character states quences are not informative in this respect (Fig. 2). Relationships reconstruction at deeper nodes are generally poorly resolved in the ITS tree as compared to the plastid tree (Figs. 1–3). The inferred ITS phyloge- Assignment of morphological traits and life forms to each spe- nies differ to some extent between parsimony and Bayesian infer- cies is based on our own observations of living and/or herbarium ence methods, but this mostly concerns weakly supported nodes specimens (see also Frajman and Jogan, 2007), in some cases com- (MPB < 70% and/or PP < 0.95). For instance, the parsimony analysis plemented with descriptions from the literature (mainly Boissier, of the ITS data set infers sect. Helioscopia as sister of the outgroup 1862; Hegi and Beger, 1924; Prokhanov, 1949; Smith and Tutin, taxa E. milii and E. pulcherrima with 67% MPB, and in the Bayesian 1968; but also Benedí et al., 1997; Chrtek and Krˇísa, 1992; Heubl tree they are positioned within the outgroup with PP 0.93. On the and Wanner, 1996; Norton, 1900; Radcliffe-Smith, 1982). We other hand, conflicts between the two DNA regions are evident scored the following traits (character states in brackets): (1) life (Fig. 3) and were detected also by the ILD test (P = 0.001), therefore form (annual; perennial), (2) indumentum (absent; present), (3) we did not proceed with the analyses of the concatenated data sets. leaf arrangement (alternate; opposite; decussate), (4) leaf venation The taxon composition of the main terminal clades, furnished (pinnate; palmate), (5) shape of nectarial glands on the cyathia with sectional names in Figs. 1 and 2 and whose circumscription (transversely ovate, outer margin convex; truncate, outer margin is defined in the Section 4.4, is largely congruent between the in- truncate or shallowly concave; bicornate, horns dilated; bicornate, ferred phylogenies (note that some taxa were included only in horns not dilated, slender; semilunate, crescentic), (6) presence of the ITS data set). However, the relationships among the terminal bracts among the male flowers (present; absent), (7) capsule sur- clades differ to some extent between plastid and ITS trees face (smooth; granulate, with small papillae; tuberculate, with (Fig. 3a). Especially the position of sect. Conicocarpae is ambiguous wart-like processes of different lengths; winged, with two narrow as it is resolved as sister to sect. Helioscopia by the Bayesian anal- wings along each keel), (8) pericarp (indurated; spongy), and (9) ysis of the ITS data with moderate support (PP 0.96), whereas in seed surface (smooth; punctate-rugulose; sulcate, furrowed; pit- the trnTF tree it is sister to sect. Myrsiniteae with strong support ted; vermiculate-rugose, wrinkled; faveolate; definitions partly (100% MPB, PP 1). The ITS NeigbourNet network (Fig. 3b) indicates from Heubl and Wanner, 1996). that sect. Conicocarpae shares splits with sections Myrsiniteae and In a few cases, we could not unambiguously assign a character Helioscopia. In the trnTF tree (Fig. 3a), sect. Myrsiniteae and Conico- state due to the plasticity of some characters. In general, the pre- carpae are most closely related to sections Aphyllis, Carunculares, vailing character states were assigned (e.g., taxa that are in general Esula, Paralias, Patellares, and Peplus (94% MPB, PP 1), whereas in glabrous, but can occasionally have some trichomes, were treated the ITS tree (Fig. 3a) there is no support for such a relationship. as ‘‘glabrous’’ in our analyses; occasionally occurring single indi- In the ITS NeigbourNet network (Fig. 3b), both are in intermediate viduals with deviating life form were neglected). As we did not as- position between sect. Helioscopia and sections allied to sect. Esula. sign character states to the outgroup taxa, the reconstructed The relationships among other sections are congruent in both character states only rely on the ingroup taxa. Chromosome num- 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 104 135 Number of included characters 2531 840 Number/percentage of parsimony informative characters (within the ingroup) 275 (251)/10.9% (9.9%) 319 (308)/38.0% (36.7%) Length of MP trees 698 1741 Consistency index (CI; excluding uninformative characters) 0.824 (0.739) 0.397 (0.367) Retention index (RI) 0.963 0.866 Substitution model HKY + C HKYI + C 416 B. Frajman, P. Schönswetter / Molecular Phylogenetics and Evolution 61 (2011) 413–424
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 Monophyly of sect. Carunculares is not supported by the ITS data Patellares, relationships among the latter being poorly resolved. (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 all sections are congruent between the ITS and trnTF phylogenies, reconstruction or at least not conflicting (single vs. more character states inferred for a certain clade; Fig. 4). Three characters (leaf arrangement, pres- Assignment of morphological traits and life forms to each spe- ence of bracts among the male flowers, pericarp) are not presented cies is shown in Fig. 2. The inferred ancestral character states for in Figs. 2 and 4, as one character state is specific for a single section: 418 B. Frajman, P. Schönswetter / Molecular Phylogenetics and Evolution 61 (2011) 413–424
Fig. 2 (continued) decussate leaf arrangement and spongy pericarp only for sect. of bracts between male flowers only for sect. Myrsiniteae. They were Lathyris, opposite leaves only for sect. Oppositifoliae, and absence 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. 4. Discussion Esula was perennial or annual (Figs. 1 and 4), it is clear that the an- nual life form developed several times and within several sections 4.1. Monophyly of Euphorbia subg. Esula and limited utility of ITS for of subg. Esula independently (thick branches in Figs. 1 and 2). The inferring the evolutionary history of Euphorbia reconstruction of eight morphological traits indicates that the ancestor of subg. Esula was likely glabrous, with pinnately veined The strong support for monophyly of Euphorbia subg. Esula by leaves, bicornate nectarial glands, bracts present among the male plastid trnTF sequences (Fig. 1) and non-informativeness of the flowers, smooth capsules and smooth seeds (Fig. 4). The results ITS sequences in this respect is in agreement with the results of of the analysis are not conclusive regarding the ancestral state of Steinmann and Porter (2002), and partly of Bruyns et al. (2006). leaf arrangement (decussate vs. alternate) or pericarp type (spongy In the latter study the Bayesian analyses of the ITS data set yielded vs. indurated). strong support for the monophyly of subg. Esula, whereas in our 420 B. Frajman, P. Schönswetter / Molecular Phylogenetics and Evolution 61 (2011) 413–424 study the support is not high (PP 0.93; Fig. 2) In the parsimony or differential sorting of ancestral polymorphisms could be respon- analyses the outgroup taxa E. milii and E. pulcherrima from clades sible for conflicting splits indicated by the NeighbourNet network, C and D of Steinmann and Porter (2002), respectively, are nested high homoplasy observed in our ITS data set (Table 1), and incon- in subg. Esula with low support (67% MPB; dashed branch in gruences between the ITS and trnTF phylogenies. It is difficult to Fig. 2a). However, support for this incongruence, which is inferred distinguish between hybridisation and incomplete lineage sorting by both inference methods, does not surpass cut-off levels of (e.g., Frajman et al., 2009), but the intermediate position of section MPB > 70% and/or PP > 0.95, applied here for recognition of well- Conicocarpae between sections Myrsiniteae and Helioscopia in the supported branches and identification of significant conflicts in ITS NeighbourNet network might indicate its hybrid origin, the the trees. ancestor of sect. Myrsiniteae serving as the maternal parent (plas- Conflicting signal in the ITS data set is also indicated by a tids are maternally inherited in Euphorbia; Corriveau and Coleman, square in the central part of a NeigbourNet network (Fig. S3 in 1988; Zhang et al., 2003). the Supplementary material) constructed with the ITS alignment Relationships among other sections are not conflicting, but dis- from Steinmann and Porter (2002). Their clade B, corresponding play different support levels in both phylogenies (Figs. 1–3). Both to subg. Esula, shares a set of splits with clade A, represented in nuclear and plastid sequences support the common origin of sec- our study by the outgroup taxa E. obesa and E. balsamifera. Contra- tions Aphyllis, Carunculares, Esula, Oppositifoliae (only included in dictory results regarding the monophyly and position of subg. the ITS tree), Paralias, Patellares and Peplus. Also in the Neigh- Esula obtained by various analyses of ITS data sets might partly bour-Net network (Fig. 3b) they share several common splits, be explained by different taxon sampling, likely in combination E. serrata from sect. Carunculares being most divergent. Morpholog- with biological processes such as hybridisation or lineage sorting ically, the members of these sections differ from sect. Helioscopia, (Wendel and Doyle, 1998; Slowinski and Page, 1999), coupled which always exhibits convex nectarial glands, by having mostly with specific properties of the ITS region, e.g., incomplete con- bicornate to semilunate nectarial glands with truncate to concave certed evolution (Álvarez and Wendel, 2003). Moreover, relatively outer margin, and from sections Myrsiniteae and Conicocarpae by strong divergence of ITS sequences of taxa from different subgen- having mostly non-glaucous, pinnately veined leaves (palmately era likely causes alignment problems and results in increased veined in sect. Paralias; Fig. 2). 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 relation- 4.3. Multiple origins of annual life form in the evolution of Euphorbia ships among major clades of Euphorbia in order to establish bet- subg. Esula ter-resolved nuclear DNA phylogenies that can provide a firm basis to test hypotheses regarding evolution and biogeography Prokhanov (1949) expressed doubt concerning the naturalness of the genus. of sect. Cymatospermum, in which he included several annual spur- ges with more or less bicornate nectaries. The taxa included (Fig. 1) 4.2. Conflicting relationships in plastid and ITS sequences among are indeed a heterogeneous assemblage of annuals (see Fig. 2 for different groups of Euphorbia subg. Esula different character states). Using both a phylogenetic framework and ancestral character state reconstruction, we clearly show that Relationships among the clades at deeper nodes in the ITS tree the annual life form developed in several lineages of subg. Esula are poorly resolved, whereas the trnTF tree offers better resolution independently from perennial ancestors (Figs. 1 and 2). Nine shifts among the major clades. For simplicity, in the following we use the from perennials to annuals in five sections can be observed in the sectional names defined in the last section of the Discussion and plastid tree (Fig. 1), and one more in the ITS tree (Fig. 2). Similar to shown in Figs. 1–4. The topologies in both trees are to some extent the annual life form within subg. Esula, also succulence developed incongruent (Fig. 3a); especially the position of sect. Conicocarpae several times in the evolution of Euphorbia (Bruyns et al., 2006; is ambiguous, appearing most closely related either to sect. Helios- Steinmann and Porter, 2002; Zimmermann et al., 2010). Extended copia (ITS) or to sect. Myrsiniteae (trnTF). The ITS NeigbourNet net- taxon sampling will likely reveal the occurrence of annuals in other work (Fig. 3b) indicates conflicting splits in subg. Esula, and sections as well. It remains ambiguous, however, whether the sections Conicocarpae and Myrsiniteae have intermediate position ancestor of subg. Esula was annual or perennial (Figs. 1 and 4). between sect. Helioscopia and a group including sect. Esula and al- Character state reconstruction including members from other sub- lied sections. A set of relatively long, thus strongly weighted, par- genera is needed to resolve this question. allel splits leading to sect. Helioscopia, however, clearly indicates Phylogenetic studies in several other plant groups have re- its divergence. Sect. Conicocarpae, positioned between the sections vealed that previously co-classified annuals have in fact developed Helioscopia and Myrsiniteae, shares more morphological character- several times independently from their perennial ancestors (e.g., istics with sect. Myrsiniteae (e.g., palmately veined, glaucous Astragalus: Liston and Wheeler, 1994; Veronica: Albach et al., leaves, similar ecology; Figs. 2a and 4, see also Sections 4.4.1– 2004; but see Tank and Olmstead, 2008). As reported for other 4.4.3) than with sect. Helioscopia. groups (Andreasen and Baldwin, 2001; Müller and Albach, 2010; Different processes can be responsible for incongruent phyloge- Smith and Donoghue, 2008), the branches leading to annual spe- netic patterns (see Wendel and Doyle, 1998; Slowinski and Page, cies of Euphorbia are often relatively longer as compared to their 1999), classified as interlineage (hybridisation, lateral gene trans- perennial sister taxa (e.g., E. pterococca vs. E. hirsuta, E. peplus/ fer between organismal lineages) or intralineage (incomplete line- E. peploides vs. E. brachycera, E. falcata vs. other members of the age sorting, orthology/paralogy conflation). Stochastic or sect. Conicocarpae, E. terracina vs. E. dendroides; Fig. 2). In all these systematic errors, such as failure of phylogenetic models and and other cases, the annuals differ substantially in habit and methods to converge on the correct solution, may further compli- growth height from the closely related perennials (e.g., E. hirsuta is cate the situation. Nuclear ribosomal DNA that is present in multi- usually about seven times taller than its sister species E. pterococca). ple copies in the genome is subject to different processes that can Different life form and divergent overall habit were likely the be responsible for conflicting phylogenetic signals, e.g., differential reason why some closely related species (e.g., E. dendroides and and incomplete homogenisation of the multiple copies by con- E. terracina) were never classified together, even if they share certed evolution within and among different lineages (Álvarez several morphological traits and exhibit the same chromosome and Wendel, 2003). Concerted evolution, following hybridisation, base number (see Fig. 2). B. Frajman, P. Schönswetter / Molecular Phylogenetics and Evolution 61 (2011) 413–424 421
4.4. Relationships within the major clades, evolution of morphological Euphorbia helioscopia, E. apios, E. pterococca, and E. hirsuta form traits and taxonomic implications a sequence of basal branches in the plastid tree, and the first three, together with E. carniolica, also in the ITS tree. The rela- The taxonomic composition of the well-supported major clades tionships among other well-supported clades within sect. Helios- is congruent between the ITS and trnTF phylogenies (Figs. 1 and 2; copia are mostly unresolved and their taxonomic constitution is some taxa were included only in the ITS data set). Various, only partly incongruent between both trees. Traditional subsectional partly compatible sectional and subsectional classifications of classifications (e.g., Prokhanov, 1949) are mostly not supported Euphorbia subg. Esula have been proposed in the past (e.g., Boissier, by molecular data. Steinmann and Porter (2002) suggested that 1862; Prokhanov, 1949; Fig. 2). The most recent (sub)sectional the main morphological character to distinguish this section from revision of extra-tropical Eurasian members of Euphorbia was pro- other members of subg. Esula could be tuberculate ovaries, posed by Geltman (2007), but it was neither based on a phyloge- whereas members of other sections have smooth (or slightly netic framework nor was there a clear overview of the taxa granulate) ovaries. This suggestion is not entirely supported by included. Our results clearly show that most of the infrageneric our data, as some members of sect. Helioscopia (e.g., E. akenocar- groups proposed by Geltman (2007) are unnatural, often polyphy- pa, E. helioscopia, E. villosa) have smooth capsules as well. An- letic assemblages. Exceptions are sections Helioscopia and Myrsini- other character serving to distinguish sect. Helioscopia from teae. The sectional classifications, largely incongruent with other members of subg. Esula is the shape of the nectarial glands, phylogenetic history (see Fig. 2), likely resulted from the plasticity which are transversely ovate with convex outer margin in sect. and parallel evolution of different morphological characters (Figs. 2 Helioscopia, and mostly of other shapes, with truncate to concave and 4), and the use of only a few characters, such as seed surface outer margin in other clades (some members of sect. Aphyllis can for classification. Chromosome numbers are apparently of only also have a convex outer margin of the nectarial glands; Fig. 2). limited classificatory value in Euphorbia (Fig. 2), as noted already An additional distinguishing character with limited discrimina- by Hans (1973). Most sections have various chromosome numbers, tory power is the type of the indumentum: several members of exceptions being section Myrsiniteae with 2n = 20, and the sister sect. Helioscopia are pubescent with unicellular trichomes. Taxa sections Peplus and Paralias with mostly 2n = 16 chromosomes. belonging to other clades are mostly glabrous, with the exception Polyploidisation played a negligible role in the evolution of the of sect. Patellares bearing multicellular trichomes and some other three before-mentioned sections, but was important in most oth- taxa from other clades that can be sparsely pubescent (e.g., E. ers. Polyploid series can be observed in several annual taxa (e.g., esula, E. herniariifolia, E. salicifolia). Ecologically, members of this E. exigua, E. falcata, E. helioscopia) and in different perennial mem- section are fairly heterogeneous, but many of them are relatively bers of sect. Esula. For several species, multiple chromosome num- mesophilic as compared to the other sections with the exception bers have been reported, which might be due to inaccurate counts of sect. Patellares. (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 deter- 4.4.2. Euphorbia sect. Conicocarpae (Prokh.) Frajman, comb. nov. mined (Frajman and Jogan, 2007). In addition, deviating counts Basionym: Tithymalus Gaertn. sect. Conicocarpus Prokh., Sist. Obzor for the same species might be also due to the presence of various Moloch. Sr. Azii 155. 1933. Type: E. humilis C.A. Mey cytotypes, some of which possibly act as cryptic species. A sound This clade consists of mostly perennial glabrous taxa, character- caryological revision of subg. Esula is certainly needed to substan- ised by palmately veined, glaucous, equifacial (isolateral) leaves, tiate further discussion about chromosome evolution in this group. and truncate nectarial glands, sometimes with two, occasionally Below we summarise the composition of the sections and the bifid or dilated, horns. Capsules are shallowly sulcate, smooth to internal relationships, as well as their morphology (see also granulate (e.g., E. nicaeensis, E. segueriana), and seeds mostly Fig. 2), distribution and ecology. Ancestral character states for each smooth (see Fig. 2a for exceptions). Most members of this clade group are shown in Fig. 4. The inferred states of all sections are are relatively thermophilic. congruent, or at least not conflicting, between ITS and trnTF trees. This clade largely corresponds to sect. Murtekias (Raf.) Prokh. The proposed sectional classification will certainly need to be subsect. Conicocarpae Prokh., but includes some taxa from other amended, as, although all subsections of Boissier (1862) shown sections (e.g., E. falcata). Inclusion of the members of this clade into to belong to subg. Esula were sampled, only one fifth of all mem- sect. Paralias Dumort or sect. Tithymalus subsect. Esulae Boiss., as bers of subg. Esula were included in our analyses. With addition traditionally classified (see Fig. 2), is not supported by molecular of other taxa in future studies, new sections might need to be data. We suggest treating this clade as an independent section established. However, all sections recognised here have strong sup- Conicocarpae. Prokhanov (1949) assigned E. segueriana as the type, port in our phylogenies and it is not likely that additional analyses but his choice is predated by Wheeler (1943), who designated with denser taxon or DNA sampling would collapse these clades. E. humilis C.A. Mey as lectotype. Euphorbia falcata and E. pithyusa are successively sister species 4.4.1. Euphorbia sect. Helioscopia Dumort., Fl. Belg. 87. 1827. Type: E. to the other taxa in this section and might merit subsectional rec- helioscopia L. ognition. Relationships among the other taxa are somewhat con- Most taxa included in this clade have also in the past been flicting between both trees. Contrary to previous suggestions classified together (sect. Helioscopia, sect. Tulocarpa (Raf.) Prokh., (e.g., Fischer et al., 2008), E. saxatilis is not most closely related to sect. Tithymalus (Scop.) Boiss. subsect. Galarrhaei Boiss.; see E. triflora and E. kerneri, and E. herzegovina is not conspecific with Fig. 2). An exception is E. mellifera, previously classified in sect. E. barrelieri as assumed in the past (e.g., Trinajstic´, 2007). Neither Balsamis Webb and Berth. Section Helioscopia comprises annual Euphorbia segueriana and E. niciciana nor E. glareosa and E. nicaeen- and perennial herbs or shrubs with highest diversity in Europe, sis are most closely related. Consequently, they should be treated the Mediterranean, and temperate Asia. Its members are glabrous as independent species rather than as subspecies as suggested by or pubescent, have alternate, pinnately veined leaves, trans- Smith and Tutin (1968). More detailed studies with broader geo- versely ovate nectarial glands with convex outer margin and graphic sampling suggest that the evolutionary history of this clade bracts between the male flowers. Capsules are either smooth or is even more complicated, and incongruences are observed be- tuberculate, with indurated pericarp, and the seeds are mostly tween the plastid and ITS data sets (Frajman and Schönswetter, smooth, rarely punctate-rugulose or faveolate (see Fig. 2a). unpubl.). 422 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. difficult to specify their common characteristics (but see, e.g., the 1904. Type: E. myrsinites L. common characteristics of E. terracina and E. dendroides in This clade contains E. myrsinites and closely related taxa, which Fig. 2b) and several different (sub)sections might need to be recog- have also traditionally been recognised as a separate section. Its nised within this group. Further analyses, including more taxa as members are characterised by palmately veined, glaucous, equifa- well as plastid data for the Macaronesian group, are needed to clar- cial leaves, and nectarial glands with short, often dilated and lobed ify the relationships among the taxa included and to provide a solid horns. Bracts between male flowers are absent, representing a basis for a sectional revision. synapomorphy for this section. Capsules are smooth to granulate, and seeds are smooth to vermiculate-rugose. The species of sect. 4.4.8. Euphorbia sect. Esula Dumort., Fl. Belg. 87. 1827. Type: E. esula L Myrsiniteae mostly grow in dry, exposed habitats and are in this This clade contains perennial taxa with deeply sulcate capsules respect similar to most members of sect. Conicocarpae. and smooth seeds, often growing in dry grasslands. Most of them were already in the past classified in this section (see Fig. 2). Mem- 4.4.4. Euphorbia sect. Carunculares (Boiss.) Tutin in Feddes Repert. 79: bers of sect. Patellares as well as E. terracina (see above) are not 55. 1968. Type: E. serrata L. most closely related to E. esula and its allies, although they were of- In the ITS tree (Fig. 2b) E. serrata and E. calyptrata form a poly- ten classified together (Fig. 2, see also Geltman, 2007). tomy with the clade including sections Aphyllis, Esula, Oppositifoli- Relationships among the taxa are partly conflicting between ITS ae, Paralias, Patellares, and Peplus. They have both been grouped in and plastid phylogenies. Plastid data suggest that E. tshuensis from subsect. Carunculares by Boissier (1862), including species with Altai and E. valliniana, a narrow endemic from the southwestern mostly roughly serrate, palmately veined leaves, and truncate to Alps form the basal-most branches of the clade, whereas in the two-horned nectarial glands, but their monophyly is not supported ITS tree there is no support for such relationships. However, E. val- by the ITS data (Figs. 2b and 3b). Further analyses are needed to liniana and E. variabilis from the southeastern Alps as well as E. clarify the phylogenetic relationship between the two taxa and kraussiana and E. genistoides from South Africa (the latter two are their allies. Prokhanov (1949) classified E. serrata and E. calyptrata not included in the trnTF data set) have isolated positions in the in sect. Chylogala (Fourr.) Prokh., the type being E. bungei Boiss. As Bayesian tree (Fig. 2). Several taxa from this section form polyploid E. bungei is not included in our analyses, we follow the classifica- series (Fig. 2b), and some of them have been reported to hybridise, tion by Tutin. whereas hybrids have not been reported from other clades (Hegi and Beger, 1924; Chrtek and Krˇísa, 1992). Low phylogenetic resolu- Taxa of the following sections (Patellares to Peplus; 4.4.5–4.4.10) tion within the group, especially in the ITS tree (Fig. 2b), as well as are characterised by mostly alternate (not in sect. Oppositifoliae), partly incongruent positions of some taxa in ITS and trnTF trees, bifacial, pinnately veined (not in sect. Paralias) leaves, crescent, might result from reticulation and polyploidisation events, which, semilunate to bicornate nectarial glands with mostly slender together with concerted evolution, might have blurred the phylo- horns, and smooth to granulate capsules. genetic signal (Álvarez and Wendel, 2003). Euphorbia lamprocarpa from Steinmann and Porter (2002; 4.4.5. Euphorbia sect. Patellares (Prokh.) Frajman, comb. and stat. nov. GenBank number AF537545), as well as E. polychroma from Wurdack Basionym: Euphorbia subsect. Patellares Prokh. in Komarov, Fl. USSR et al. (2005; GenBank number AY794606), traditionally classified 14: 743. 1949. Type: E. amygdaloides L. within sect. Helioscopia, are included in sect. Esula in our study. This clade comprises perennial species characterised by connate Inspection of the herbarium voucher of E. lamprocarpa revealed raylet leaves and pubescent–villous indumentum, composed of rel- that it actually belongs to E. virgata s.l., whereas the herbarium atively long, multicellular hairs. Its members were included in sect. voucher of E. polychroma was not available at NCU as indicated Esula subsect. Patellares by Prokhanov (1949). We propose its treat- (Wurdack et al., 2005). The obvious misplacement of Wurdack’s ment as an independent section Patellares, considering its clear accession of ‘‘E. polychroma’’ indicates that it is certainly misidentified. morphological differentiation as well as its monophyly, clearly dis- tinct from sect. Esula as circumscribed here. All members of sect. 4.4.9. Euphorbia sect. Paralias Dumort., Fl. Belg.: 87. 1827. Type: Patellares are relatively mesophilic. E. paralias L. This clade comprises glabrous and glaucous annuals (E. segetal- 4.4.6. Euphorbia sect. Oppositifoliae (Boiss.) Baikov, Molochan Severn. is) and perennials with imbricate, isolateral, palmately veined Azii 114. 2007. Type: E. inderiensis Kar. et Kir. leaves (Figs. 2b and 4), characteristics not found in any other sec- Euphorbia turczaninowii was included in subsect. Oppositifoliae tion of the clade including section Esula and allies (Fig. 1–3). The by Boissier (1862), comprising annuals with opposite leaves dis- species constituting sect. Paralias have similar habitat preferences; tributed in Central Asia. As E. turczaninowii, the only representative most of them, with exception of E. segetalis, grow in coastal, often of the sect. Oppositifoliae in our ITS analysis, is phylogenetically saline areas. Section Paralias in our circumscription is less species- divergent from its sister sect. Aphyllis, and there are several mor- rich than traditionally circumscribed (see Fig. 2). phological differences between the two groups (Fig. 2), we treat sect. Oppositifoliae as an independent section. 4.4.10. Euphorbia sect. Peplus Lázaro, Comp. Fl. Españ. 282. 1896. Type: E. peplus L. 4.4.7. Euphorbia sect. Aphyllis Webb and Berthel., Hist. Nat. Iles It contains annual and perennial taxa with shallowly sulcate Canaries 3: 253. 1846-47. Type E. aphylla Brouss. ex Willd capsules, which are mostly two-winged on the keels (not in This clade is a heterogeneous assemblage of annual and peren- E. brachycera). The taxa included were never assumed to be closely nial species that were in the past, due to their different habit and related (see Fig. 2) and are dissimilar in habit, but winged capsules diverse morphology, classified into several (sub)sections (Fig. 2). might be synapomorphic for this clade, and were probably second- Most of the Macaronesian dendroid spurges (included only in the arily lost in E. brachycera (Figs. 2b and 4). ITS analyses) as well as E. dendroides were by Boissier (1862) clas- sified within subsect. Pachycladae, and their characteristics were 4.4.11. Euphorbia sect. Lathyris Dumort., Fl. Belg. 87. 1827. Type: E. discussed by Molero et al. (2002). The other taxa included in this lathyris L. clade were always classified in other sections (see Fig. 2); therefore This section includes a single species, E. lathyris, which is mor- their phylogenetic alliance to E. dendroides was unexpected. It is phologically unique within subg. Esula, having decussate leaf B. Frajman, P. Schönswetter / Molecular Phylogenetics and Evolution 61 (2011) 413–424 423 arrangement and spongy pericarp, characteristics not found in any Boissier, P.E., 1862. Subordo 1. Euphorbieae. In: De Candolle, A. (Ed.), Prodromus other member of the subgenus (see Fig. 2). It is an annual to bien- systematis naturalis regni vegetabilis 15. Victoris Masson et filii, Parisiis, pp. 3– 188. nial species with linear to oblong-lanceolate, pinnately veined bifa- Bruyns, P.V., Mapaya, R.J., Hedderson, T., 2006. A new subgeneric classification for cial leaves, and nectar glands with two, mostly clavate, dilated Euphorbia (Euphorbiaceae) in southern Africa based on ITS and psbA-trnH horns. It was already in the past included in its own (sub)section sequence data. Taxon 55, 397–420. Bryant, D., Moulton, V., 2004. Neighbor-net: an agglomerative method for the and our data support this. The exact phylogenetic position of construction of phylogenetic networks. Mol. Biol. Evol. 21, 255–265. 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