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Molecular phylogeny of Macaranga, Mallotus, and related genera (Euphorbiaceae s.s.): insights from plastid and nuclear DNA sequence data¹

Nationaal Herbarium Nederland, Leiden University branch, P.O. Box 9514, 2300 RA Leiden, The Netherlands; Department of Systematic and Evolutionary Botany, Universität Wien, Rennweg 14, A-1030 Vienna, Austria

Received for publication November 8, 2006. Accepted for publication July 28, 2007.

ABSTRACT

Macaranga and Mallotus (Euphorbiaceae s.s.) are two closely related, large paleo(sub)tropical genera. To investigate the phylogenetic relationships between and within them and to determine the position of related genera belonging to the subtribe Rottlerinae, we sequenced one plastid (trnL-F) and three nuclear (ITS, ncpGS, phyC) markers for species representative of these genera. The analyses demonstrated the monophyly of Macaranga and the paraphyly of Mallotus and revealed three highly supported main clades. The genera Cordemoya and Deuteromallotus and the Mallotus sections Hancea and Oliganthae form a basal Cordemoya s.l. clade. The two other clades, the Macaranga clade and the Mallotus s.s. clade (the latter with Coccoceras, Neotrewia, Octospermum, and Trewia), are sister groups. In the Macaranga clade, two basal lineages (comprising mostly sect. Pseudorottlera) and a crown group with three geographically homogenous main clades were identified. The phylogeny of the Mallotus s.s. clade is less clear because of internal conflict in all four data sets. Many of the sections and informal infrageneric groups of Macaranga and Mallotus do not appear to be monophyletic. In both the Macaranga and Mallotus s.s. clades, the African and/or Madagascan taxa are nested in Asian clades, suggesting migrations or dispersals from Asia to Africa and Madagascar.

Key Words: Afro-Asian distribution • biogeography • Cordemoya • Euphorbiaceae • Macaranga • Mallotus • molecular phylogenetics • Rottlerinae

Macaranga and Mallotus are two paleo(sub)tropical genera of shrubs, trees, and, exceptionally, woody climbers in the angiosperm family Euphorbiaceae s.s. (uniovulate Euphorbiaceae; Wurdack et al., 2005 ). These large genera (with c. 260 species in Macaranga and c. 150 in Mallotus; Radcliffe-Smith, 2001 ; Whitmore, in press ) have a wide habitat range from primary forest understorey to heavily disturbed sites and from swamp forests to montane forests. They are characteristic components of secondary forests in Southeast Asia and can be used as indicators of forest disturbance (Slik et al., 2003 ). Moreover, both genera have an array of interesting morphological features, most striking undoubtedly being the ant-housing adaptations of myrmecophytic Macaranga species (Ridley, 1910 ). To deepen the knowledge on the evolution of these important ecological and morphological traits, a robust hypothesis about the phylogenetic relationships between and within these two genera is needed. The phylogeny will also clarify the biogeography and taxonomic delimitations in Mallotus and Macaranga.

In the Euphorbiaceae classifications of Webster (1994) and Radcliffe-Smith (2001), Macaranga and Mallotus are placed in the tribe Acalypheae of the uniovulate subfamily Acalyphoideae. Mallotus is a member of the subtribe Rottlerinae, together with seven other genera (Table 1; additionally, the New World genus Avellanita was included by Radcliffe-Smith, 2001 ), whereas Macaranga is placed in the monogeneric subtribe Macaranginae. This classification implies that Macaranga and Mallotus are clearly distinct, well-separated genera. Morphologically, however, these genera are very similar. Both genera, with few species excepted, possess conspicuous, usually colorful, glandular hairs (also called glandular scales). This character is rare within Euphorbiaceae and might indicate a common origin for Macaranga and Mallotus. Furthermore, the only clear-cut difference between them is the number of locules in the anthers (two in Mallotus, three or four in Macaranga).

View this table: [in this window] [in a new window]   Table 1. Distribution and some typical morphological characters for Mallotus, other genera in the subtribe Rottlerinae, and Macaranga (Webster, 1994 ). The number of known species per genus and the number of sampled species are given in parentheses following the generic name.

The phylogenetic relationships between Macaranga, Mallotus, and related genera are poorly understood. In a molecular phylogenetic study of the Euphorbiaceae s.s. (Wurdack et al., 2005 ), Macaranga, Mallotus, and Trewia form a well-supported clade, which is sister to Blumeodendron (tribe Pycnocomeae). Further, one of the Rottlerinae genera, the Australian Rockinghamia, was shown to be unrelated to this clade. However, only limited conclusions can be drawn from this study because of the limited taxon sampling (only one or two species were sampled from each of the aforementioned genera, and no other Rottlerinae taxa were included).

Although several studies have focused on the phylogeny of the myrmecophytic Macaranga species (Blattner et al., 2001 ; Davies et al., 2001 ; Bänfer et al., 2004 ), only one study tried to clarify the relationship between Mallotus and Macaranga (Slik and Van Welzen, 2001a ). According to this morphological phylogenetic study, Mallotus is paraphyletic for two reasons. First, Mallotus sections Hancea and Oliganthae (Table 2) are separated from the rest of the genus by some of the outgroup taxa. Second, Macaranga is nested within Mallotus, being sister to Mallotus sect. Mallotus. This study is, however, hampered by a couple of shortcomings. First and foremost, the taxon sampling was limited: only three Macaranga species and none of the small genera related to Mallotus were included. Second, the resulting phylogeny was poorly supported, perhaps because of a low characters to taxa ratio (76/50) and a high number of polymorphic characters (50 of 76). Thus, a comprehensive study of the phylogeny of this interesting plant group is clearly needed before the drastic taxonomic rearrangements suggested by the morphological study (e.g., merging Macaranga and Mallotus) can be executed (as was already concluded by Slik and Van Welzen, 2001a ).

View this table: [in this window] [in a new window]   Table 2. Distribution, number of known species, and number of species sampled for the infrageneric groups of Macaranga and Mallotus (Airy Shaw, 1968 ; Whitmore, in press). The groups occurring on the western side of the Indian Ocean are underlined.

For both Macaranga and Mallotus, infrageneric classifications exist (Table 2), but they are far from satisfactory and not based on a phylogenetic framework. In their revision of Macaranga, Pax and Hoffmann (1914 , 1919 , 1931 ) divided the genus rather artificially into 36 sections. Their circumscription was criticized by Airy Shaw (1969 , 1971 ), and recently Davies suggested, based on a phylogenetic analysis, new delimitations for the sections Pachystemon and Pruinosae (Davies, 2001 ; Davies et al., 2001 ). The genus Macaranga was Whitmore's long-time research subject (e.g., 1965 , 1969 , 1980 ), but he could not finish the monograph during his lifetime. However, in a manuscript being published as a prodromus (Whitmore, in press ), a new subdivision of Macaranga is presented. Apart from three previously clearly established sections (Pachystymon, Pruinosae, and Pseudorottlera), Whitmore could not classify all species in proper sections but instead recognized 15 "natural species groups" (Gestalt groups). These preliminary groupings (for which diagnostic characters were not clearly given) have only a limited correspondence to the sections of Pax and Hoffmann.

The first sectional delimitations of Mallotus were made by Müller (1865 , 1866 ; five sections) and by Pax and Hoffmann (1914 ; 10 sections). The classification was later refined by Airy Shaw (1968) to contain eight sections. This subdivision has been used, with slight modifications, as the basis for revisions of a part of the genus (Bollendorff et al., 2000 ; Slik and Van Welzen, 2001b ; Sierra and Van Welzen, 2005 ; Sierra et al., 2005 , 2007 ; Van Welzen and Sierra, 2006 ; Van Welzen et al., 2006 ). Unfortunately, the infrageneric division is based on only a few, and sometimes dubious, characters. For example, a diverse group of opposite-leaved Mallotus species is divided into two sections only by the character of penninerved or tripli/palminerved leaves. In the morphological phylogenetic analysis (Slik and Van Welzen, 2001a ), many of the sections were indicated to be nonmonophyletic, but low levels of support prevented definitive conclusions.

The aim of this study was to reconstruct the phylogeny of Mallotus, Macaranga, and related small genera to address the following questions: (1) Are Macaranga and Mallotus monophyletic, or is Macaranga nested within Mallotus as suggested by Slik and Van Welzen (2001a) ? (2) Is the merging of Coccoceras and Deuteromallotus with Mallotus (described earlier) justified, and what is the phylogenetic position of the other small genera? (3) What are the main infrageneric clades, and how do they relate to the infrageneric groupings of Airy Shaw (1968) and Whitmore (in press )? (4) How are the species in Africa, Madagascar, and the Mascarene Islands related to those in the Asia-Pacific, and what kind of biogeographical scenario could explain this Afro-Asian distribution pattern?

To answer these questions, we have sequenced four DNA markers, from both plastid and nuclear genomes, sampling thoroughly the study genera and the infrageneric groups of Macaranga and Mallotus. Two of the markers, plastid trnL-F (consisting of trnL intron and trnL-F spacer) and nuclear rDNA ITS, have been commonly used to infer plant phylogenies at low taxonomic levels (e.g., Kathriarachchi et al., 2006 ; Samuel et al., 2006 ), whereas two other markers are relatively novel fragments of low-copy number nuclear genes. The chloroplast-expressed glutamine synthetase gene (ncpGS) plays a role in the nitrogen metabolism in chloroplasts, and it has been shown to exhibit more sequence divergence than ITS between closely related Oxalis species (Emshwiller and Doyle, 2002 ). The second low-copy number marker used in this study, a photoreceptor gene phytochromeC (phyC), has been used for the family-level phylogeny of the Phyllanthaceae, a family closely related to Euphorbiaceae s.s. (Samuel et al., 2005 ).

These four markers had various levels of sequence divergence, and their analysis provided, in most parts, a robust phylogeny illuminating the evolution of this plant group and providing a framework for taxonomic rearrangements and for further studies.

MATERIALS AND METHODS

Taxon sampling and outgroup choice
A pilot study was conducted to investigate whether all genera in the subtribe Rottlerinae (sensu Webster, 1994 ) are, in fact, closely related to Macaranga and Mallotus. Representatives of these genera were sequenced for the rbcL and/or trnL-F genes (data not shown). A maximum parsimony analysis with the large uniovulate Euphorbiaceae data set (Wurdack et al., 2005 ) showed that all these taxa, except the genus Rockinghamia, form a well-supported clade, which is sister to the genus Blumeodendron (Appendix S1, see Supplemental Data accompanying the online version of this article). Therefore, Rockinghamia was excluded from subsequent analyses, and Blumeodendron was selected as the outgroup. Additional analyses of the individual gene data sets with more distant outgroup taxa (e.g., Cleidion; data not shown) were either cumbersome because of divergent, barely alignable outgroup sequences or had results highly similar to those presented here.

Taxon names, voucher information, and GenBank accession numbers of the samples used in this study are listed in the Appendix (see also the number of species sampled per genus or infrageneric group in Tables 1 and 2). The taxon sampling includes nearly all satellite genera (except Avellanita), 57 species of Macaranga and 31 of Mallotus, covering all the species groups of Macaranga (Whitmore, in press ) and the sections of Mallotus (Airy Shaw, 1968 ). All Mallotus species from Africa and Madagascar and a considerable sample of Macaranga species from these areas were sampled. For several species, more than one specimen was sequenced to determine possible infraspecific variation.

Laboratory methods
Total DNA was extracted from leaf tissue using the DNeasy Plant Mini kit (Qiagen, Hilden, Germany). For silica-dried material, manufacturer instructions were followed. For samples from herbarium specimens, a modified protocol was used (with a prolonged lysis step with proteinase K and ß-mercaptoethanol; Wurdack et al., 2004 ). Additionally, a few samples were extracted in collaborative laboratories with various other methods. Some of the herbarium specimen extracts were further diluted (10–100x) or cleaned with PCR cleaning columns (see later in Laboratory methods) to facilitate PCR.

The marker trnL-F was amplified with primer pairs c+d (trnL intron) and e+f (trnL-F intergenic spacer; Taberlet et al., 1991 ). For the ribosomal ITS region, the primer pair ITS5+ITS4 was mostly used; additionally, ITS1 and ITS2 regions of certain degraded templates were amplified separately with primer pairs ITS5+ITS2 and ITS3+ITS4 (White et al., 1990 ). A fragment of ncpGS containing introns 7 and 8 was initially amplified with primers GScp687f and GScp856r (Emshwiller and Doyle, 1999 ). However, because these primers worked poorly with some taxa, a set of new primers was designed for the study group (Fig. 1):

View larger version (5K): [in this window] [in a new window]   Fig. 1. Primers used in PCR and sequencing of ncpGS. Published primers (Emshwiller and Doyle, 1999 ) are above, the newly designed primers below.

GSKKf1 [5'-GGCACCAATGGAGAGGTTAT-3'],

GSKKf2 [5'-GATCACATCTGGTGTGCWAG-3'],

GSKKr1 [5'-AGCTTCAATTCCCACRCTGG-3'], and

GSKKr2 [5'-YAACACCAGCYTGTTCWGTGA-3'].

Most taxa were amplified with primer pair GSKKf1+GSKKr2, but in some cases other combinations were used. The phyC fragment (part of exon 1) was primarily amplified with primer pair PHYC-F+PHYC-R (Samuel et al., 2005 ), although for a few degraded samples a newly designed forward primer PHYCiF2 [5'-GGGTTTRGTGGTYTGCYAYCA-3'] was used in combination with PHYC-R to amplify a shorter fragment.

PCR amplifications were carried out in 50-µL reactions using 0.2–2.0 µL of total DNA extract as a template. The reaction mixture also contained 1x PCR Buffer (Qiagen, Hilden, Germany), 20 pmol of each primer, 5 nmol dNTPs, 0.5 µg bovine serum albumin (BSA; Promega, Madison, Wisconsin, USA), and 1 U Taq DNA polymerase (Qiagen, Hilden, Germany). The concentration of MgCl₂ was 2.5 mM for trnL-F, 2 mM for ITS, and 1.5 mM for ncpGS and phyC. The PCR program consisted of 4 min initial denaturation at 94°C; 30–36 cycles of 30 s denaturation at 94°C, 30 s annealing at 52.5°C (48°C for phyC), and 1 min extension at 72°C; followed by a final extension of 5 min at 72°C.

PCR products were checked for length and yield by electrophoresis on 1% agarose gels and cleaned with either QIAquick PCR Cleanup (Qiagen) or Nucleospin Extract II (Macherey-Nagel, Düren, Germany) columns. The latter was also used to recover fragments of correct size from agarose gels when multiple bands were present. In cases of degraded templates yielding very weak PCR products, one of two approaches was taken. Either the products from several parallel PCR reactions were pooled in the cleaning step, or gel-excised products were used as a template in a reamplification PCR. The cleaned PCR products were sequenced either on an ABI 377 automated sequencer using the ABI BigDye Terminator chemistry for cycle sequencing (Applied Biosystems, Foster City, California, USA) and Sephadex G50 AutoSeq columns (GE Healthcare, Diegem, Belgium) for reaction cleaning, or by external service (using ABI 3730xl; Applied Biosystems).

Generally, samples were sequenced with both forward and reverse PCR primers, though additional internal ITS and ncpGS primers were used as needed. The chromatograms were inspected, and sequence contigs assembled, with Sequencher v4.1.4 (Gene Codes Corp., Ann Arbor, Michigan, USA). In this process, special attention was paid to sites with overlapping nucleotide peaks, possibly indicating infraindividual variation (polymorphisms). If an obviously overlapping signal was detected at both forward and reverse chromatograms, then the site was deemed to be putatively polymorphic between alleles or copies and was coded with IUPAC ambiguity codes.

PCR products were cloned to facilitate the sequencing of a few difficult samples and to further determine infraindividual polymorphisms in ITS and ncpGS. The pGEM-T Easy Vector System (Promega, Madison, Wisconsin, USA) was used following the instructions of the manufacturer. Bacterial cells picked from insert-containing colonies were directly used as a template for standard PCR with M13 forward and reverse primers. The resulting products were size-selected using agarose gel electrophoresis. Three clones per individual were sequenced as described earlier.

Sequence alignment and indel characters
The sequences were aligned either completely by eye using MacClade version 4.08 (Maddison and Maddison, 2001 ) and Bioedit version 7.0.5.2 (Hall, 1999 ), or with the multiple sequence alignment algorithm of ClustalW version 1.81 (Thompson et al., 1994 ) followed by extensive manual adjustments. In the alignment process, both sequence similarity and mechanisms of molecular evolution were taken into account (Kelchner, 2000 ). Specifically, the following guidelines were used: (1) Indels were assumed to be less likely than substitutions, i.e., a gap was inserted only if otherwise at least two substitutions had to be assumed. (2) The length variation in long mononucleotide repeats, and possible substitutions within, were considered to have uncertain homologies. Therefore, the variation in mononucleotide repeats of 6 bp or longer were excluded from the alignment. (3) If the gap could be clearly postulated to have resulted from an insertion or deletion of a multinucleotide tandem repeat, then this information was used to place the gap. (4) Because undetected inverted repeats can bias phylogenetic analysis (Quandt et al., 2003 ), we specifically looked for them in the alignment. (5) In the cases of overlapping gaps, the gaps were placed in a way to minimize the total number of indel events. (6) Sometimes a gap could be reasonably placed in two or more positions. If the choice could affect the phylogenetic analyses, question marks or ambiguity codes were introduced in the data matrix to account for this uncertainty while still preserving as much phylogenetic information as possible. (6) Ambiguously alignable regions with uncertain homologies were excluded.

The indel information from the alignments was incorporated into parsimony analyses with the program SeqState (Müller, 2005a ). The indel coding algorithm of SeqState (Müller, 2006 ) automates the coding of indel characters, outputting a NEXUS file containing the original data matrix followed by an extra character block comprising the indel characters. Simple indel coding (SIC; Simmons and Ochoterena, 2000 ) was used. Additionally, inverted repeats were coded as binary characters.

Phylogenetic analyses
The phylogenetic analyses were generally conducted using both maximum parsimony (MP) and Bayesian inference (BI) methods. Additionally, a maximum likelihood (ML) analysis was used in a specific case as an alternative model-based phylogeny method.

For MP analyses, PAUP* version 4.0b10 (Swofford, 2003 ) was used, treating nucleotide characters unordered and unweighted, and the polymorphic character states as uncertainties. Gaps in the alignment were treated as missing data, and the indel character block from SeqState was either included or excluded to assess the effect of indel characters. The parsimony ratchet (Nixon, 1999 ) was used to search for the most parsimonious trees. The ratchet batch files for PAUP* were generated with PRAP v.1.21 (Müller, 2004 ). In a ratchet run, each of 20 starting trees built with random addition sequence (RAS) and tree-bisection-reconnection (TBR) branch swapping underwent 50 iterations (25% of characters given double weight). This fast search strategy proved to be thorough enough for our data sets; experiments with more extensive ratchet searches and further swapping of trees found by ratchet did not result in shorter trees or changes in the strict consensus. Support for clades was assessed by bootstrap analysis (BS; Felsenstein, 1985 ) running 2000 pseudoreplicates. Because only a moderate exploration of tree space is necessary for estimating bootstrap and jackknife support values (Farris et al., 1996 ; Freudenstein et al., 2004 ; Müller, 2005b ), only a single tree, resulting from one replication of RAS+TBR, was saved per pseudoreplicate.

Bayesian inference (BI) of phylogeny with posterior probabilities (PP) as a support measure was done with MrBayes version 3.1.2 (Huelsenbeck and Ronquist, 2001 ; Ronquist and Huelsenbeck, 2003 ). The model of molecular evolution for each gene data set was selected using the Akaike information criterion (AIC) as implemented in MrModel test version 2.2 (Nylander, 2004 ); for advantages of AIC over hierarchical likelihood ratio test (hLRT), see Posada and Buckley (2004) . The selected models were: GTR+G for trnL-F, GTR+G+I for ITS (except GTR+G in the Cordemoya s.l. clade analysis; see Results), HKY+G for ncpGS (except HKY in the Macaranga clade and Cordemoya s.l. clade analyses), and HKY+G for phyC. The default priors of MrBayes were used. For each analysis, two simultaneous runs were done (starting from random trees), having three heated and one cold chain with default temperature (0.2). Markov chains were sampled every 100th generation. Analyses were run until the average standard deviation of the split frequencies approached 0.02, indicating that two runs converged onto a stationary distribution. Additionally, the plot of generation vs. log probability was inspected after the run to ensure that stationarity was reached and to determine the burn-in. Depending on the data set, 1 000 000–4 000 000 generations were run, and typically c. 10% of the samples were discarded as burn-in.

An additional maximum likelihood (ML) bootstrap analysis for the combined data set of the Mallotus s.s. clade (see Results) was conducted with PHYML v.2.4.4 (Guindon and Gascuel, 2003 ); the GTR+G+I model with four rate categories and 500 pseudoreplicates was used. Model parameters were estimated from data for the whole concatenated data set (because PHYML does not allow splitting a data set into partitions with different models).

Both MP and BI analyses were done with two different taxon-sampling strategies. First, all the taxa were analyzed together, with Blumeodendron as the outgroup. Second, each of the three main clades found in the analyses of all taxa (see Results) were analyzed individually, with one or two species selected from two other clades to serve as an outgroup (indicated in the Appendix). In both of these cases, four markers were first analyzed separately, and the results were screened for hard incongruences (i.e., incongruences with bootstrap support >70%; Hillis and Bull, 1993 ) before the combined analysis of all four data sets. Statistical tests for incongruence (e.g., incongruence length difference test; Farris et al., 1994 ) were not conducted, because these tests have been shown to be unreliable in certain conditions (e.g., Dolphin et al., 2000 ; Yoder et al., 2001 ; Darlu and Lecointre, 2002 ; see also Hipp et al., 2004 ). Moreover, because combining incongruent data sets can sometimes lead to a more robust phylogeny (Sullivan, 1996 ; Flynn and Nedbal, 1998 ; Wiens, 1998 ), we think that automatic rejection to combine them is a too strict approach. Instead, we take the view advocated by Wiens (1998) : data sets with hard incongruences can be combined, but parts of the resulting tree that are in strongly supported conflict between data sets should be regarded as questionable.

The internal conflict within each data set was inspected with the consensus network approach (Holland and Moulton, 2003 ; Holland et al., 2005 ). This tree-based method visualizes the conflict between input trees in a network. By selecting the trees from bootstrap pseudoreplicates as input trees, a consensus network provides a view to the character conflict in the data set. For this analysis, SplitsTree version 4.3 was used (Huson and Bryant, 2006 ), with threshold proportion x = 0.1.

RESULTS

Sequence characteristics
Properties of the sequence data sets of each marker are given in Table 3. For a few taxa, some of the nuclear markers could not be sequenced (see Appendix), mainly because of difficulties in amplifying low copy number nuclear genes from degraded samples. These taxa with missing data were nevertheless included in the combined phylogenetic analyses. Generally, the forward and reverse sequencing reactions fully covered the sequence contigs. In this respect, ncpGS was more problematic. In several cases, the chromatogram quality dropped drastically after mononucleotide repeats; consequently, parts of the ncpGS contigs were based on a single direction only. In these cases, the chromatograms were closely inspected, and the sequencing reaction repeated if required to obtain an unambiguous result.

Sequence alignment and the indel characters
The alignments are available online as supplemental data in Appendices S2–S5. Insertion of gaps was required to align all noncoding regions; moreover, two gaps (3 and 12 bp long) were needed to align the phyC exon. The other coding regions (the exons of ncpGS, and 5.8S of ITS) were gap free. The most extensive length variation was observed in ncpGS: a number of long and overlapping gaps were needed in intron 8. Because of these indel events, the ncpGS sequences of the Cordemoya s.l. and Macaranga clades (see the phylogenetic results described later) were much shorter than the sequences of the Mallotus s.s. clade (c. 300 bp instead of c. 600 bp).

The inclusion of indel characters into the MP analyses had only limited impact on the phylogenetic results. Within the three main clades (see the phylogenetic results described later), indel characters had no or very little effect in the Cordemoya s.l. and Mallotus s.s. clades, but in the Macaranga clade they provided additional resolution and support. Here we report only the cladograms based on analyses including the indel characters, and, where necessary, we mention the differences with analyses where the indels were omitted.

Infra-specific and -individual polymorphism
Two or three separate specimens (collected from different parts of the distribution of the species, if possible) were sequenced for eight species to assess the infra-specific variation. The acquired sequences were either identical or highly similar, and the specimens were always placed together in the phylogenetic analyses (result not shown). For the subsequent analyses only one of the specimens was chosen to represent the species.

Polymorphic sites with overlapping nucleotide peaks were detected with direct sequencing in all nuclear data sets, but their number was generally low (Table 3), and visual inspection of the alignments revealed no clear additive patterns possibly indicating hybridization (e.g., Sang et al., 1995 ). Two ITS sequences with a relatively high number of these putatively polymorphic sites (Mallotus griffithianus and M. lackeyi) were cloned. The clone sequences confirmed the presence of either all (M. griffithianus, 10 out of 10) or some (M. lackeyi, two out of five) of the putative polymorphisms (data not shown). Additionally, one 2 bp indel polymorphism was found in M. griffithianus. Several additional differences between clones were also observed; some of them do not appear to be Taq errors and could be traced back to weak, previously unnoticed overlapping peaks in the chromatograms. In a phylogenetic analysis of ITS data (result not shown), the clone sequences were placed near the corresponding direct sequence. Moreover, using the direct sequence or any of the clones resulted in the same phylogenetic position for the specimen in question.

Two ncpGS samples were also cloned to confirm infra-individual polymorphisms, and, especially, to investigate whether sequencing problems related to mononucleotide repeats (described earlier) were caused by alleles with a difference in the number of repeated nucleotides. However, the chromatograms of all the clone sequences suffered from the same deteriorated signal after the repeats as the direct sequences. Thus, this phenomenon is likely due to technical problems in the sequencing reactions and not to infra-individual polymorphisms.

Analysis with all taxa and the major relationships
Most of the single-marker analyses, as well as the combined analysis of all four markers, revealed the same three highly supported main clades: (1) a Cordemoya s.l. clade, consisting of the genera Cordemoya and Deuteromallotus, and the Mallotus sections Hancea and Oliganthae; (2) a Mallotus s.s. clade with the remaining Mallotus species and the genera Coccoceras, Neotrewia, Octospermum, and Trewia; (3) a Macaranga clade with all sampled Macaranga species.

The relationships between and support for these clades are summarized in Fig. 2 (for detailed trees, see the online Appendices S6–S9). TrnL-F, phyC, and BI analysis of ITS support the sister group relationship of the Macaranga and Mallotus s.s. clades, placing the Cordemoya clade in a basal position. In contrast, in the MP analysis of ITS, the Macaranga clade is highly nested inside the Mallotus s.s. clade, and, in particular, sister to a clade consisting of Mallotus sect. Mallotus, Mallotus discolor, and Octospermum pleiogynum. Analysis of ncpGS also gave deviating results: the three main clades are present in the MP analysis, but the Macaranga and Cordemoya s.l. clades are now sister groups. Moreover, BI analysis of ncpGS fails to separate the members of the Macaranga and Cordemoya clades.

View larger version (13K): [in this window] [in a new window]   Fig. 2. Summary of the phylogenetic analyses with all sampled taxa, showing the relationships of the major clades. (A–D) Single-marker analyses. (E) Combined analysis of all four markers. (F) Bayesian phylogram from the combined analysis. MP, maximum parsimony; BI, Bayesian inference. MP bootstrap values are above the branches, BI posterior probabilities below. The outgroup (Blumeodendron) is not shown.

Cordemoya s.l. clade
All single-marker analyses (not shown) resulted in similar trees without hard incongruences. The combined analysis (Fig. 3) also have the same pattern of two highly supported subclades: one with Cordemoya integrifolia and all three Deuteromallotus species, and the other with the Mallotus sections Hancea and Oliganthae.

View larger version (12K): [in this window] [in a new window]   Fig. 3. Phylogenetic relationships inferred from the individual analysis of Cordemoya s.l. clade (see Fig. 2) with the combined data set of four markers. A Bayesian majority consensus tree is shown with parsimony bootstrap values above the branches and posterior probabilities below. A hyphen (-) indicates that the node does not exist in parsimony strict consensus. Infrageneric group (see Table 2) indicated with three letter abbreviation. Species occurring at western side of Indian Ocean (Africa, Madagascar, Mascarenes) are indicated with stars.

View larger version (19K): [in this window] [in a new window]   Fig. 4. Phylogenetic relationships inferred from the individual analysis of Macaranga clade (see Fig. 2) with the combined data set of four markers. A Bayesian majority consensus tree is shown with parsimony bootstrap values above the branches, posterior probabilities below. A hyphen (-) indicates that the node does not exist in the parsimony strict consensus. Infrageneric group (see Table 2) indicated with three letter abbreviation (shown in boldface if the particular group is nonmonophyletic). Species occurring at western side of Indian Ocean (Africa, Madagascar, Mascarenes) are indicated with stars.

Mallotus s.s. clade
The ITS data set provided the most resolved tree, whereas trees based on the three other markers have roughly the same lesser resolution. On the other hand, the number of well supported clades (BS 70) is almost equal in all single-marker analyses, including ITS. All data sets resemble each other also in the distribution of the supported clades: they are predominantly small, and the relationships between them are not resolved and/or supported, resulting in a large basal polytomy.

There is one hard incongruence between the data sets: in the ITS tree M. barbatus groups with M. paniculatus (BS 77), whereas in the ncpGS tree it forms a clade with M. macrostachyus and M. tetracoccus (BS 99) with M. paniculatus sister to this clade. Furthermore, two incongruences almost reach the cut-off level of BS 70. First, phyC groups M. repandus as sister to sect. Mallotus (BS 69), whereas ncpGS places M. repandus with M. philippensis (BS 98). Second, ncpGS groups M. resinosus with M. decipiens (BS 100), but ITS places it with M. leucocalyx (BS 68; M. decipiens being sister to the clade of these two taxa).

The combined analysis of the four markers for the Mallotus s.s. clade is shown in Fig. 5. Trees obtained by BI and MP analyses are congruent (except for one difference in the clade consisting of M. leucocalyx, M. resinosus, and M. decipiens), the BI tree being more resolved. The basal nodes of the MP tree are essentially not supported, whereas BI analysis gives support (often strong) for several additional nodes. ML bootstrap analysis (not shown) resulted in a topology very similar to the BI and MP analyses, but no support (BS < 50) was given to the nodes supported only by BI.

View larger version (14K): [in this window] [in a new window]   Fig. 5. Phylogenetic relationships inferred from the individual analysis of Mallotus s.s. clade (see Fig. 2) with the combined data set of four markers. A Bayesian majority consensus tree is shown with parsimony bootstrap values above the branches, posterior probabilities below. A hyphen (-) indicates that the node does not exist in the parsimony strict consensus. Infrageneric group (see Table 2) indicated with three letter abbreviation (shown in boldface if the particular group is nonmonophyletic); eHA, a group of species excluded from sect. Hancea (Slik and van Welzen, 2001b ). Species occuring at western side of Indian Ocean (Africa, Madagascar, Mascarenes) are indicated with stars.

Phylogenetic analysis methods and support values
Although unweighted Maximum Parsimony (MP) and model-based Bayesian Inference (BI) are fundamentally different methods, analyzing our data sets with them resulted to a large extent in similar topologies. Also the support for clades was measured in distinctly different ways: bootstrap analysis for MP and posterior probabilities for BI. It has become clear that these two indices are not directly comparable, and that posterior probabilities are generally higher than bootstrap values (e.g., Rannala and Yang, 1996 ). This trend can be observed in our results as well. Moreover, some clades without bootstrap support received high posterior probabilities, especially in the Mallotus s.s. clade (discussed later in detail). Because recent studies have shown that posterior probabilities can overestimate the support or even give high support for incorrect nodes (Suzuki et al., 2002 ; Alfaro et al., 2003 ; Douady et al., 2003 ; Simmons et al., 2004 ), we regard these clades as dubious. However, we also recognize that the difference in these two kinds of support values can also arise from the general dissimilarities of MP and BI as optimality criteria for phylogeny reconstruction.

Major relationships and the monophyly of Macaranga and Mallotus
The analyses of all sequenced taxa, including a representative sample of the diversity in Macaranga and Mallotus, show that Macaranga is nested in the subtribe Rottlerinae, and, therefore, there is no basis for Webster's (1994) decision to place it into a separate subtribe Macaranginae. Furthermore, three well-supported main clades are revealed, allowing the monophyly of Macaranga and Mallotus to be assessed (Fig. 2).

First, all markers agree on the monophyly of Macaranga, as suggested by earlier studies with limited taxon sampling (Blattner et al., 2001 ; Slik and Van Welzen, 2001a ). The 3- or 4-locular anthers of Macaranga are thus a good synapomorphy for the genus, and were uniquely derived from 2-locular anthers present in the other clades and outgroup. As an exception, one Macaranga species, M. heudelotii Baill. (not sampled), is reported to have 2-locular anthers (Whitmore, in press ). This species possesses spiny branches and branched staminate inflorescences and, therefore, would morphologically fit well in a deeply nested position with other African Macaranga species (clade C2; see discussion on the Macaranga clade and Fig. 4). We thus regard the 2-locular condition in M. heudelotii as a reversal from the 3/4-locular state.

On the other hand, our results clearly show that Mallotus, as currently delimited, is not a monophyletic genus. All markers support the paraphyly of Mallotus, caused by the placement of a few Mallotus taxa away from the main Mallotus clade (= Mallotus s.s. clade) and forming a separate clade with Cordemoya and Deuteromallotus (= Cordemoya s.l. clade). These Mallotus segregates, namely the Asian sections Hancea and Oliganthae, were already separated from the rest of Mallotus in the morphological phylogeny (Slik and Van Welzen, 2001a ). The species assemblage of the Cordemoya s.l. clade has not been suggested before, although Müller (1866) placed Cordemoya integrifolia, Deuteromallotus acuminatus, and Mallotus penangensis (sect. Hancea) together in his Mallotus sect. Cordemoya.

The genus Deuteromallotus was originally considered to differ from Mallotus by characters in pistillate flowers (style/stigma very short and style scarcely papillose) (Pax and Hoffmann, 1914 ). Later, McPherson (1995) demonstrated that the fragile stigmas of Deuteromallotus break easily, and when the flowers are intact they do not differ from those of Mallotus. We confirm this observation, but disagree with his suggestion to merge Deuteromallotus with Mallotus because in our results it falls into the Cordemoya s.l. clade as well.

The Cordemoya s.l. clade, with the taxon composition described in the previous paragraphs, is also supported by morphological characters. Most importantly, the conspicuous, spherical to disclike glandular hairs, typical for the Macaranga and Mallotus s.s. clades, are missing in the members of the Cordemoya s.l. clade. The latter have capitate glandular hairs and/or peltate-stellate hairs instead. Moreover, the pollen of the Cordemoya s.l. clade has areolate ornamentation instead of the perforate/microreticulate ornamentation of the Mallotus s.s. clade (Sierra et al., 2006 ).

In addition to Cordemoya and Deuteromallotus discussed earlier, the results presented here clarify the relationship between Mallotus and the other small Rottlerinae genera, revealing second cause for the paraphyly of Mallotus: Neotrewia, Octospermum, and Trewia are part of a well-supported Mallotus s.s. clade. Also, the inclusion of Coccoceras into Mallotus (Airy Shaw, 1963 ; Bollendorff et al., 2000 ) is confirmed here (for the further discussion about these genera, see the following section about Mallotus s.s. phylogeny). Rockinghamia, a genus already considered to be distant to Mallotus in an earlier study (Wurdack et al., 2005 ), was confirmed to be not closely related. However, the position of the unsampled genus Avellanita, placed in the Rottlerinae by Radcliffe-Smith (2001) , remains to be investigated. This genus was placed incertae sedis by Webster (1994) , and we regard its having a close relationship with the taxa studied here as dubious because of its discordant distribution (endemic to Chile) and inflorescence structure (bisexual cymes rather than the typical, mostly unisexual, spikes, racemes, or panicles). Preliminary molecular phylogenetic results indicate that Avellanita is far removed in the Acalyphoideae from our study clade (K. Wurdack, Smithsonian Institution, Washington, personal communication).

Both plastid (trnL-F) and nuclear (phyC) data strongly support the monophyly of Mallotus s.s. and its sister group relationship with the Macaranga clade, contradicting the nested placement of Macaranga shown by the analysis of morphological data (Slik and Van Welzen, 2001a ). The same result, with moderate support for monophyly of Mallotus s.s. (PP 0.94) is obtained from the BI analysis of ITS. On the other hand, the result of the MP analysis of ITS, with Macaranga deeply embedded in Mallotus s.s. and sister to a clade containing Mallotus sect. Mallotus (for a detailed tree, see Appendix S7 in Supplemental Data with the online version of this article), clearly resembles the phylogeny inferred from the morphology.

There are, however, reasons to believe that the result of the MP analysis of ITS does not reflect the underlying organismal phylogeny. The nested position of Macaranga does not have bootstrap support, and the consensus network analysis of the ITS bootstrap trees (not shown) revealed a relatively strong alternative split supporting the monophyly of Mallotus s.s. (BS 16, whereas the split placing Macaranga nested in Mallotus s.s. had BS 24). In other words, although not visible in the strict consensus, ITS data has characters supporting Mallotus s.s. clade, even in the MP framework. Furthermore, the ITS data set is highly variable, and the plot of transition vs. transversion distances has some signs of saturation. MP analysis could therefore have failed to detect the obscured signal supporting the separation of Mallotus s.s. and Macaranga, whereas the BI, based on molecular evolutionary models, which accounts for multiple hits and different rates for substitution classes (e.g., Swofford et al., 1996 ), resulted in the same relationship as revealed by most of the other data. We choose therefore two monophyletic sister clades, Mallotus s.s. and Macaranga, as our phylogenetic result.

The analyses of ncpGS also strongly support the monophyly of Mallotus s.s. On the other hand, the results of the ncpGS analyses deviate from those of other markers, because the MP analysis of ncpGS data places Macaranga and Cordemoya s.l. clades together, and the BI fails even to separate the members of these two clades. These deviating results are, however, weakly supported, and, because of the long gaps required to align these taxa with the Mallotus s.s. clade, there is only a limited number of characters available to infer the relationships between the major clades. A deviating, but unsupported, result could thus have arisen by chance. Moreover, the gaps in the Macaranga and Cordemoya s.l. clades, although occurring in the same area in the ncpGS intron 8, are not homologous and provide no evidence for a sister-group relationship between these clades.

The phylogeny of the Cordemoya s.l. clade
Both single-marker and combined analyses of the Cordemoya s.l. clade reveal a strongly supported geographical signal (Fig. 3): one of the subclades comprises only taxa from Madagascar and the Mascarene Islands (genera Deuteromallotus and Cordemoya, respectively), whereas the other consists of the purely Asian Mallotus sections Oliganthae and Hancea. Futhermore, the monophyly of sect. Hancea as circumscribed by Slik and Van Welzen (2001b) is strongly supported (see also the following discussion on the Mallotus s.s. clade). The morphology of this clade and taxonomic rearrangements are further discussed in a separate paper (Sierra et al., 2006 ).

The phylogeny of the Macaranga clade
Combining the four Macaranga data sets, which have no hard incongruences, resulted in a more resolved and more highly supported phylogeny than any of the single-marker analyses (Fig. 4). Previous studies on the Macaranga phylogeny (Blattner et al., 2001 ; Davies et al., 2001 ; Bänfer et al., 2004 ) included mainly myrmecophytic species and their west Malesian relatives. Therefore, this study, with samples from all of Whitmore's (in press ) Macaranga groups, provides the first comprehensive phylogeny of the genus. In our results (Fig. 4), half of the 18 infrageneric groups recognized by Whitmore proved to be, although sometimes with low support, nonmonophyletic. Nevertheless, taxon sampling is still limited (several large Macaranga groups are represented by one or a few species only), and the tree is only partially supported; caution is thus necessary when interpreting the results. In the following discussion, the information about the Macaranga morphology is based on Whitmore (in press) and personal observations, unless indicated otherwise.

Basal clades B1 and B2
The analysis revealed two relatively small basal lineages (Fig. 4: clades B1 and B2), which are separated with strong support from a large crown group. These two basal clades consist mainly of species belonging to Macaranga sect. Pseudorottlera, a section suggested to be transitional between Mallotus and Macaranga (Zollinger, 1856 ; Airy Shaw, 1965 ). Species falling into clades B1 and B2 are all shrubs or small trees that grow in primary forest and that usually have small, penninerved leaves, and 2-locular fruits. Furthermore, their staminate inflorescences are unbranched and bear small bracteoles without disc-shaped glands (nectaries), whereas staminate inflorescences in the rest of Macaranga are variously branched (often with more than two axis orders, but exceptionally unbranched in very few species) and either have disc-shaped glands or not. In Mallotus, staminate inflorescences are either unbranched or scantily branched and have small bracteoles that always lack disc-shaped glands.

Clade B1, sister to the rest of Macaranga, brings two Australian species, M. subdentata (sect. Pseudorottlera) and M. inamoena (Dioica group) together with M. alchorneoides (Coriacea group, the only New Caledonian species sampled); this grouping has never been suggested before. All three species are frequently monoecious, a condition additionally present only in most of the other New Caledonian species and M. glaberrima (Hassk.) Airy Shaw (sect. Pseudorottlera, distributed from Java to New Guinea; not sampled). Macaranga inamoena was placed in the Dioica group by Whitmore (in press ), but with its unbranched staminate inflorescences it fits better in the B1 clade (staminate inflorescences are generally branched in the Dioica group s.s.). The next clade, B2, is sister to the Macaranga crown group (clades C1–C3) and consists of the remaining sampled Pseudorottlera species.

The composition of the basal clades B1 and B2 agrees with the results from previous molecular phylogenetic studies (Blattner et al., 2001 ; Davies et al., 2001 ), which placed sect. Pseudorottlera as sister to the rest of Macaranga (other members of clades B1 and B2 were not sampled in those studies). In contrast, the morphological analysis placed a pioneer species M. tanarius at the base of the Macaranga clade (Slik and Van Welzen, 2001a ). This result, together with the embedded position of Macaranga in a clade of pioneer Mallotus species, led to a conclusion that Macaranga originated in open vegetation and that primary forest understorey species (e.g., sect. Pseudorottlera) evolved from pioneer ancestors (Slik and Van Welzen, 2001a ). According to our results, the Macaranga ancestor could have had either ecology, depending on the results in the sister clade Mallotus s.s., which is unfortunately poorly resolved.

The crown group, clades C1–C3
The Macaranga crown group is a well-supported clade containing the majority of the species and most of the morphological diversity of the genus. It consists of three subclades (C1–C3 in Fig. 4) with varying support and one ambiguously placed species, M. trichocarpa (also not placed in any of the Whitmore's groups). The relationships among the clades C1–C3 are still ambiguous: BI strongly supports a clade of C1+C2, whereas MP either unites C2+C3 (indel characters included) or C1+C2 (indel characters excluded); neither of the MP groupings is supported by bootstrap. Each of these clades, especially C2 and C3, presents a high level of morphological diversity, and no morphological synapomorphies are known for them at the moment. However, examination of the crown group clade reveals a clear geographical structure: the species from the three main centers of diversity of the genus, i.e., west Malesia, Africa + Madagascar, and New Guinea, roughly correspond with the clades C1, C2, and C3, respectively.

The Macaranga clade C1 is well supported and comprises all taxa from the sections Pachystemon and Pruinosa, and all taxa from the Bicolor, Conifera, Javanica and Winkleri species groups, with the exception of M. siamensis (sect. Pruinosa), which is placed in clade C2. All these groups have a west-Malesian-centered distribution, with some outlier species mainly in Indochina, Sulawesi, and the Philippines. This clade contains all myrmecophytic Macaranga species; their phylogenetic relationships and the evolution of myrmecophytism have been studied in detail elsewhere (Blattner et al., 2001 ; Davies et al., 2001 ; Bänfer et al., 2004 ). In our analyses, this clade is rather poorly resolved, perhaps partly due to the hybridization between myrmecophytic species revealed in a phylogeographic analysis of chloroplast haplotype data (Bänfer et al., 2006 ). However, our results demonstrate the close relationship of the Bicolor and Conifera groups with the myrmecophytic sections Pachystemon and Pruinosa. These two groups, together with the Javanica group, should, therefore, be thoroughly sampled for future studies of myrmecophytic Macaranga.

Clade C2 unites the mainly continental Asian Denticulata group, one aberrant species from the sect. Pruinosae (M. siamensis), and all species sampled from Africa, Madagascar, and the Mascarenes. Macaranga siamensis has often been confused with M. gigantea (because of their enormous, similarly shaped leaves), and, although differing in several characters, was tentatively placed with it in sect. Pruinosae (Davies, 2001 ). It, however, differs from other members of the sect. Pruinosae in having prominent extrafloral nectaries on the apical part of leaves, disc-shaped glands on the staminate bracteoles, and globose seeds (in the sect. Pruinosae, extrafloral leaf nectaries are not prominent, disc-shaped glands are absent, and seeds are lenticular). Considering the overall morphology and habit, the placement of M. siamensis among the Denticulata group is rather surprising, but they do share roughly the same distribution and globose-shaped seeds. Moreover, some Denticulata species also have staminate bracteoles with disc-shaped glands.

Most Asian species of clade C2 form a grade leading to a moderately supported clade comprising Asian M. indica and all species from the western side of the Indian Ocean. In the MP analysis, the latter forms an unsupported clade, but in BI M. mauritiana (from Mauritius) groups together with M. indica. Nevertheless, and although denser sampling of African and Madagascan species might enhance the picture, these results demonstrate the phylogenetic affinity of all Macaranga species occurring in the western side of the Indian Ocean and suggest a possible single origin of them.

The type species of Macaranga, M. mauritiana, with hollow stems, unique capitulate staminate inflorescences, and bizarre fusiform fruits, was placed in a group of its own (Mauritiana) and was even discussed as belonging to a separate genus (Whitmore, in press ). Our results show that this species is clearly related to the Denticulata group (with which it shares the general leaf shape) and African and Madagascan Macaranga.

African Macaranga species are a diverse group of 26 species with a wide array of growth forms (including lianas) and other morphological adaptations (e.g., ant-housing stipules of M. saccifera). Also, many species have a spiny trunk and branches. Pax and Hoffmann (1914) classified the African species into five sections (in one case even together with Asian species), whereas Whitmore left them ungrouped. In our analysis, all nine sampled African species (belonging to three different sections of Pax and Hoffmann), form one monophyletic but poorly supported group. Therefore, our data suggest that all African Macaranga species originated from a single, common ancestor. Also, all species endemic to Madagascar (five of 10 sampled) form a single clade, a result supporting Whitmore's decision to unite them in the Oblongifolia group (classified in four sections by Pax and Hoffmann).

C3 is a well-supported clade with all taxa sampled from the groups Angustifolia, Brunneofloccosa, Dioica, Gracilis, Longistipulata, Mappa, and Tanarius (except M. inamoena of the Dioica group, which belongs to clade B1). Together, these groups comprise almost 120 species and display huge morphological variation, from montane species with delicate leaves (Gracilis group) to large-leaved species (Mappa group). Most of these groups are clearly New Guinea centered, with a few species occuring in the neighboring areas, such as the Moluccas, Sulawesi, the Philippines, Australia, and the west Pacific Islands. The Mappa group (only one species sampled) has a markedly west Pacific distribution, with some species reaching Micronesia and Polynesia. Only one species from C3 clade, the widespread M. tanarius, occurs also in west Malesia and continental Asia.

That members of both the Dioica group and sect. Pseudorottlera have fruits subtended by leafy bracts led Whitmore (1980 , in press ) to suggest a close affinity between them. The present study indicates, however, that the Dioica group, as a member of clade C3 (except the misplaced M. inamoena, see the discussion on basal clades earlier), is phylogenetically distant to sect. Pseudorottlera (clades B1 and B2).

Our results group the Brunneofloccosa and Gracilis groups together; species in both groups are restricted to montane forests (except two Brunneofloccosa species). Furthermore, taxa from the Dioica, Longifolia, and Tanarius groups form a well-supported clade. However, none of these groups appears to be monophyletic. A study with denser taxon sampling is needed to clarify the phylogeny of this clade.

The phylogeny of the Mallotus s.s. clade
Single-marker analyses of the Mallotus s.s. clade produced largely polytomous trees, and, in contrast to the effects of combining data in the Macaranga clade, the combined Mallotus s.s. analyses yielded only a limited amount of additional supported clades (Fig. 5), especially in the MP analysis. The topologies of the MP and BI trees are largely the same, and both analyses gave strong support to small terminal clades. On the other hand, these analyses differ greatly in the support given to the basal and inner nodes: many of them are highly supported (PP 0.95–1.00) by BI but do not receive any BS support in the MP analysis.

Apart from its general tendency to overestimate support, BI is especially prone to give high confidence to very short internodes (Alfaro et al., 2003 ). Although it is not obvious what should be considered as a short internode, the Mallotus s.s. internodes supported only by PP are on average clearly shorter than those supported by both PP and BS (result not shown). Further evidence that BI overestimated the support for these nodes comes from the ML bootstrap analysis conducted for this data set. The ML bootstrap results in high support for nodes supported by both MP and BI, but the nodes supported only by BI receive ML bootstrap values of less than 50. In PHYML, different models cannot be used for different partitions, and, therefore, the results of BI and ML analyses might not be directly comparable. However, this result strengthens the hypothesis that the high support of BI for the basal nodes unsupported by MP is not because of general methodological differences between unweighted MP and model-based BI, but because of the tendency of BI to overestimate support in some circumstances. Therefore, we regard the backbone of combined Mallotus s.s. phylogeny to be essentially unresolved.

The failure of all four gene regions, each with different properties (e.g., plastid vs. nuclear genomes, exons vs. introns, and different levels of variation), to reliably resolve the Mallotus s.s. phylogeny is puzzling. The sequence divergence is in an acceptable range: the divergence in the Mallotus s.s. data sets is similar or higher than in the Macaranga clade, but the transition vs. transversion distance plots (result not shown) revealed no signs of saturation for the Mallotus s.s. clade.

Additional insights into data sets were gained with consensus networks produced from MP bootstrap pseudoreplicate trees. The consensus networks from all single-marker Mallotus s.s. data sets have a similar structure: small groups (corresponding to the highly supported terminal clades) are connected to a central reticulation, indicating that data sets have internal conflicts (i.e., homoplasy) in the relationship between these groups. Moreover, studying the incompatible splits causing the central reticulations revealed no common pattern among the four data sets, i.e., the conflicting splits involve different taxa in different data sets.

The Mallotus s.s. analyses also revealed incongruences between single-marker data sets: one incongruence is hard (i.e., BS > 70) and two are almost hard (BS 68–69). We do not consider them to contribute significantly to the lack of resolution and support observed in the combined analysis, because excluding the taxa causing the incongruences did not improve the results. Also, the incongruences are unlikely to cause the basal polytomy, because most of them are localized, i.e., they occur between closely related species.

The present study is a first molecular phylogenetic analysis of the genus Mallotus. Even though all analyses resulted in poorly resolved phylogenetic trees, several conclusions can be drawn. Apart from the placement of certain Mallotus species in the Cordemoya s.l. clade (discussed earlier), our results confirm the inclusion of the genus Coccoceras with Mallotus sect. Polyadenii (Bollendorff et al., 2000 ) and demonstrate the close relationship of Neotrewia, Octospermum, and Trewia with Mallotus. All four genera are placed in the Mallotus s.s. clade, and all of them form strongly supported clades with morphologically similar Mallotus species. The indehiscent or tardily dehiscent fruits are probably independently derived in each of these genera from the typical dehiscent capsules of Mallotus (two unsampled Mallotus species, M. blumeanus and M. sphaerocarpus, also have indehiscent fruits but with a thick, fleshy layer surrounding the seeds). A similar phenomenon can be observed in several other euphorb groups (Esser, 2003 ; Wurdack et al., 2005 ). In the following paragraphs we discuss Neotrewia, Octospermum, and Trewia briefly; more detailed morphological descriptions and the taxonomic rearrangements are provided in a separate paper (Kulju et al., 2007 ).

The Malesian genus Neotrewia (one sp.) is characterized by unilocular (or rarely bilocular) indehiscent fruits. This contrasts with the mainly dehiscent, and typically 3-locular (sometimes 2–5-locular) fruits of Mallotus (however, unilocular fruits are common in Macaranga). Because no other morphological differences from Mallotus exist, it is no surprise to see this genus deeply embedded in the Mallotus s.s. clade and as sister to a group of quite similar species.

New Guinean Octospermum (one sp.) with 7–9-locular and indehiscent fruits groups with M. discolor (Australia). This result is supported by morphology as well: M. discolor together with M. chromocarpus Airy Shaw and M. nesophilus Müll.Arg. (both not sampled) form a small group of New Guinean and Australian species, which shares several characters with Octospermum: stipules absent, anther connectives broadened, and fruits indehiscent. These Mallotus species have traditionally been placed in sect. Philippinenses (alternate leaves and unarmed fruits), but in considering only Malesian taxa, Sierra et al. (2005) noticed the morphological similarity of M. chromocarpus with Octospermum pleiogynum and excluded the former from sect. Philippinenses. This result is supported by our data, although the exact relationship of Octospermum and allies with this section needs further investigation.

Trewia nudiflora, the type species of Asian genus Trewia (two spp.), differs from Mallotus primarily in its indehiscent, hard, somewhat fleshy, and often thick-walled fruits, which have no match in Mallotus and are possibly an adaptation to megafaunal dispersal (Dinerstein and Wemmer, 1988 ). Here we see a strongly supported sister group relationship of T. nudiflora with M. khasianus, a species whose distribution partially overlaps with Trewia (both occurring from India to Thailand). Although M. khasianus has the dehiscent, thin-walled capsules typical of Mallotus, these two species share an often deciduous habit and very long staminate inflorescences.

Neotrewia and Octospermum are clearly embedded in the Mallotus s.s. clade, and because both N. cumingii and O. pleiogynum were originally described as Mallotus species, they can readily be merged with it again. The case of Trewia is less straightforward because the clade of T. nudiflora and M. khasianus is placed as the sister to the rest of Mallotus s.s. clade. This raises the possibility of transferring M. khasianus to Trewia instead of merging the two genera (Trewia L. is the older generic name and has priority over Mallotus!). However, this position of the Trewia-M. khasianus clade does not appear in any of the individual gene analyses (whether analyzed by MP or BI) and is only supported by the combined BI analysis. More research is thus needed to clarify the issue.

Our Mallotus s.s. phylogeny allows only a limited assessment of the monophyly of the Mallotus sections (as defined by Airy Shaw, 1968 ). Sections Mallotus, Polyadenii, and Stylanthus seem to be monophyletic, although this might change with further sampling. On the other hand, section Philippinenses is polyphyletic, even after the removal of M. discolor (see previous discussion under Octospermum), although the polyphyly is not supported by the MP bootstrap.

Most of the Mallotus species belong to two sections with truly opposite leaves, Axenfeldia and Rottleropsis. These sections differ from each other by having either a penninerved or tripli/palminerved leaf venation, respectively. Our analyses with limited taxon sampling suggest that the venation character is homoplastic and also that these two sections together do not form a clade. Members of Mallotus sect. Hancea (Cordemoya s.l. clade) also have opposite leaves, but in this case one leaf of a pair is stipuliform. Slik and Van Welzen (2001b) and Van Welzen et al. (2006) excluded five species from this section on the basis that one leaf of a pair is reduced but not stipuliform (one species excepted) and/or other characters, suggesting instead an affinity with sections Axenfeldia and Rottleropsis. As two former Hancea species sampled in this study fall into the Mallotus s.s. clade, their separation from the sect. Hancea is confirmed here, although their relationship with the aforementioned sections is still unclear.

Afro-Asian biogeography
Both Macaranga and Mallotus s.s. have disjunct distributions across the Indian Ocean basin. These patterns could be explained by vicariance caused by the Gondwanan breakup only if these genera date from the Cretaceous (the last major separation of Gondwanan elements, between Madagascar and Seychelles–Indian block, happened c. 95–84 Ma; McLoughlin, 2001 ). We consider this unlikely because the Acalyphoideae s.l. clade (the clade in which Macaranga, Mallotus, and related genera are nested, see Wurdack et al., 2005 ) has been estimated to have a crown group age of c. 70 Ma (Davis et al., 2005 ); the Macaranga and Mallotus s.s. clades are probably considerably younger (the inferred dates are, however, minimum ages based on fossil calibration points, and therefore an older, even Gondwanan, age for these clades cannot be ruled out). Moreover, instead of a basal split between the two geographical areas, the African and Madagascan taxa are in both cases clearly nested inside Asian (and Australasian) clades (Figs. 4 and 5). Therefore, Macaranga and Mallotus s.s. clades should already have largely diversified in Gondwana before the breakup to make the Gondwanan vicariance hypothesis possible.

The observed pattern of African and Madagascan taxa being nested in Asian clades is, therefore, more compatible with dispersal and/or migration from Asia to Africa and Madagascar. Three different scenarios can be hypothesized for these events: (1) a direct and perhaps relatively recent long-distance dispersal, (2) an Eocene–Oligocene dispersal from India to Madagascar through "Lemurian stepping stones" in the western Indian Ocean (Schatz, 1996 ), or (3) an overland migration from Asia through SW Asia and NE Africa before the Miocene aridification of the climate and subsequent disappearance of (sub)tropical forests in these areas (Zohary, 1973 ; Raven and Axelrod, 1974 ). Several leaf fossils from the Eocene onwards have been assigned to Macaranga and Mallotus (e.g., Akhmetiev and Vikulin, 1995 ; Horiuchi and Takimoto, 2001 ), but their identification is based on the leaf shape and venation pattern and can therefore be questioned. However, a leaf fossil that can be confidently placed in the Macaranga-Mallotus clade was recently found in an Oligocenic stratum in NW Ethiopia (27.36 Ma; J. L. Garcia Massini, Southern Methodist University, personal communication). This fossil shows two typical features for these genera: extrafloral nectaries and globose to disc-shaped glandular hairs. Although Mallotus cannot be ruled out as an identification, the leaf shape and long petioles strongly suggest that it belongs to Macaranga (e.g., similar to extant East African M. kilimandscharica Pax). This fossil suggests that African Macaranga are old enough to have reached the continent through the "Lemurian stepping stones" or by overland migration. A carefully conducted molecular dating analysis of Macaranga, Mallotus, and relatives with multiple calibration points and smoothing methods might shed more light on this issue.

Although the exact scenario of dispersal or migration in Macaranga and Mallotus s.s. is unclear, the direction, from Asia to Africa and Madagascar, can be deduced from the phylogeny. Similar results have been found in biogeographical studies in rodents (Jansa et al., 1999 ) and in some Melastomataceae genera (Renner, 2004 ), but many of the Afro-Asian biogeographical studies, both with plants and animals, have demonstrated an opposite direction of dispersal (Dayanandan et al., 1999 ; Raxworthy et al., 2002 ; Austin et al., 2004 ; Alejandro et al., 2005 ; Yuan et al., 2005 ). More studies are thus needed to find out whether one direction has been prevailing in dispersal between Asia and Africa and Madagascar; several other paleo- or pantropical euphorb genera, like Acalypha, Claoxylon, Cleidion, and Croton, could work as feasible study systems (for initial results in Croton, see Berry et al., 2005 ).

Another remarkable result of our study is that the only two African species in the Mallotus s.s. clade, Mallotus oppositifolius and M. subulatus (the widespread former species occurs in Madagascar as well), are clearly not each other's closest relatives (Fig. 5; a result also supported by morphology; Sierra et al., 2007 ). This indicates two separate introductions to Africa, a result contrasting with that of the Macaranga clade, where all the 14 sampled African and Madagascan species (of 36 total) form a distinct clade (although in the Baysian analysis Indian Macaranga indica is included in this clade; see Fig. 4 and online Appendix S9).

Conclusions
In this study we used DNA sequence data to investigate the phylogeny of Macaranga, Mallotus, and related genera. The results clarified the relationships among these genera and the question of the monophyly of Macaranga and Mallotus. Macaranga is a monophyletic genus, whereas Mallotus, as currently delimited, is distinctly paraphyletic. A new monophyletic Mallotus s.s. can be obtained by excluding sections Hancea and Oliganthae, which group together with the genera Cordemoya and Deuteromallotus in a separate basal clade, and by including the genera Coccoceras, Neotrewia, Octospermum, and (probably) Trewia. These taxonomic rearrangements, together with morphological treatments, are published in separate papers (Sierra et al., 2006 ; Kulju et al., 2007 ). Insights into the infrageneric phylogeny of Macaranga were also gained, and five mostly well-supported main clades were identified. On the other hand, the phylogeny of the Mallotus s.s. clade is still poorly known; additional studies of this group with new data sets and denser taxon sampling might help to resolve the issue. Biogeographically, the results of this study suggest that migrations or dispersals from Asia to Africa and Madagascar have occurred in both the Macaranga and Mallotus s.s. clades.

APPENDIX

Taxon— GenBank accessions: trnL-F, ITS, ncpGS, phyC; Origin; Voucher (Herbarium).

Blumeodendron calophyllum Airy Shaw—DQ899180, —, DQ901956, DQ767726; Indonesia, East Kalimantan, ITCI-concession; Slik 2826 (L). Blumeodendron kurzii J.J.Sm.—DQ899181, DQ866525, DQ901957, DQ767727; Indonesia, Java, Bogor Botanic Garden IX.C.144; Gravendeel et al. 521 (L).

Coccoceras muticum Müll.Arg.—DQ899183, DQ866527, DQ901959, DQ767729; Indonesia, East Kalimantan, Labanan; Slik M1234 (L). Coccoceras muticum Müll.Arg.—DQ899182, DQ866526, DQ901958, DQ767728; Indonesia, East Kalimantan, ITCI-concession; Slik M901 (L). Cordemoya integrifolia (Willd.) Baill.—DQ899184, DQ866528, DQ901960, DQ767730; Reunion, Mare Longue; Coode 4958 (L). Cordemoya integrifolia (Willd.) Baill.—DQ899185, DQ866529, DQ901961, —; Mauritius, Piton de Milieu; Lorence 2231 (K).

Deuteromallotus baillonianus (Baill.) Pax & K.Hoffm.—DQ899186, DQ866530, DQ901962, DQ767731; Madagascar, Toamasina Prov., Soanierana Ivongo; Ralimanana et al. 387 (K). Deuteromallotus capuronii Leandri—DQ899187, DQ866531, DQ901963, DQ767732; Madagascar, Fianarantsoa; Rabenantoandro et al. 739 (MO). "Deuteromallotus" spinulosus^c (= Mallotus spinulosus McPherson)—DQ899188, DQ866532, DQ901964, DQ767733; Madagascar, Fianarantsoa; Rabenantoandro & McPherson 681 (MO).

Macaranga albescens Perry—DQ899189, DQ866533, DQ901965, DQ767734; Papua New Guinea, Chimbu valley; Sterly 80–350 (L). Macaranga alchorneoides Pax & Lingelsheim—DQ899190, DQ866534, DQ901966, DQ767735; New Caledonia, Province du Nord; McPherson & Lowry 18526 (MO). Macaranga aleuritoides^a F.Muell.—DQ899191, DQ866535, DQ901967, DQ767736; Papua New Guinea, Madang; Weiblen 2049 (MIN). Macaranga alnifolia Baker—DQ899192, DQ866536, DQ901968, DQ767737; Madagascar, Toliara Prov.; Hoffmann et al. 191 (K). Macaranga angustifolia K.Schum. & Lauterb.—DQ899193, DQ866537, DQ901969, DQ767738; Papua New Guinea, Morobe Prov., near Bubia; Takeuchi & Ama 15542 (L). Macaranga auriculata (Merr.) Airy Shaw—DQ899194, DQ866538, —, DQ767739; Indonesia, East Kalimantan, ITCI-concession; Slik M958 (L). Macaranga barteri Müll.Arg.—DQ899195, DQ866539, DQ901970, DQ767740; Ghana, Brong-Ahafo; Jongkind & Nieuwenhuis 1604 (WAG). Macaranga bicolor Müll.Arg.—DQ899196, DQ866540, DQ901971, DQ767741; Philippines, Luzon, Los Baños, Mt. Makiling; Fernando 1736 (L). Macaranga bifoveata J.J.Sm.—DQ899197, —, DQ901972, —; Papua New Guinea, Madang Prov., Ohu; Novotny & Molem EUP258 (L). Macaranga cf. brachytricha Airy Shaw—DQ899198, DQ866541, DQ901973, —; Papua New Guinea, Madang Prov., Wannang; Weiblen 1713 (L). Macaranga clavata Warb.—DQ899200, DQ866543, DQ901975, DQ767743; Papua New Guinea, East Sepik Province, April River; Stancik 5122 (LAE). Macaranga conifera (Reichb.f. & Zoll.) Müll.Arg.—DQ899201, DQ866544, DQ901976, DQ767744; Indonesia, East Kalimantan, Bukit Bangkirai; Slik M628 (L). Macaranga densiflora^a Warb.—DQ899202, DQ866545, DQ901977, DQ767745; Papua New Guinea, Madang; Weiblen 2022 (MIN). Macaranga denticulata (Blume) Müll.Arg.—DQ899203, DQ866546, DQ901978, DQ767746; Thailand, Eastern Floristic Distr., Nakhon Ratchasima Prov., Khao Yai National Park; van Welzen 2003-20 (L). Macaranga diepenhorstii (Miq.) Müll.Arg.—DQ899204, DQ866547, DQ901979, DQ767747; Thailand, Peninsular Floristic Region, Thung Khai; Chamchumroon 2017 (L). Macaranga domatiosa Airy Shaw—DQ899205, DQ866548, DQ901980, DQ767748; Papua New Guinea, Morobo Prov., Wau-area, Mt. Kaindi; van Valkenburg 281 (L). Macaranga ducis^a Whitmore—DQ899206, DQ866549, DQ901981, DQ767749; Papua New Guinea, Madang; Weiblen 2025 (MIN). Macaranga echinocarpa Baker—DQ899207, DQ866550, DQ901982, DQ767750; Madagascar, Toamasina; Miller et al. 8765 (MO). Macaranga gabunica Prain—DQ899208, DQ866551, DQ901983, DQ767751; Gabon, Nyanga, chantier SFN, Igotchi; van Valkenburg et al. 2611 (WAG). Macaranga gigantea (Reichb.f.& Zoll.) Müll.Arg.—DQ899209, DQ866552, DQ901984, DQ767752; Indonesia, East Kalimantan, Sungai Wain; Slik M91 (L). Macaranga grallata McPherson—DQ899210, DQ866553, DQ901985, DQ767753; Madagascar, Toamasina Prov., Soanierana Ivongo; Ralimanana et al. 408 (K). Macaranga grandifolia (Blanco) Merr.—DQ899211, DQ866554, DQ901986, DQ767754; Philippines, Luzon, Los Baños, Mt. Makiling; Fernando 1737 (L). Macaranga heterophylla (Müll.Arg.) Müll.Arg.—DQ899212, DQ866555, DQ901987, DQ767755; Liberia, Grand Cape Mount; Jongkind et al. 6135 (WAG). Macaranga heynei I.M.Johnson—DQ899214, DQ866556, DQ901989, DQ767756; Malaysia, W.Malaysia, Salangor/Pahaus, Genting Highlands; Moog 98-011 (KAS). Macaranga heynei I.M.Johnson—DQ899213, —, DQ901988, —; Malaysia, W-Malaysia, road to Tanah Rata; Moog 01-032 (L). Macaranga cf. hispida Müll.Arg.—DQ899199, DQ866542, DQ901974, DQ767742; Papua New Guinea, East Sepik, near Yuat River; Weiblen 1831 (L). Macaranga hullettii King ex Hook.f.—DQ899215, DQ866557, DQ901990, DQ767757; Indonesia, East Kalimantan, Sungai Wain; Slik M132 (L). Macaranga hurifolia Beille—DQ899216, DQ866558, DQ901991, DQ767758; Liberia, Sino, Sapo NP; Jongkind et al. 5549 (WAG). Macaranga inamoena^b F.Muell. ex Benth.—DQ899217, DQ866559, DQ901992, DQ767759; Australia, Queensland, Cook Distr.; Forster 29763 (BRI, L). Macaranga indica Wight—DQ899218, DQ866560, DQ901993, DQ767760; India, Dindigul Distr., Kodaikanal, Palni Hilss; Steward & Balcar RHT 55221 (L, RHT). Macaranga induta Perry—DQ899219, DQ866561, DQ901994, DQ767761; Papua New Guinea, Madang Prov., nr. Kaironk; Weiblen et al. 1064 (L). Macaranga involucrata (Roxb.) Baill.—DQ899220, DQ866562, DQ901995, DQ767762; Australia, Queensland, Cook Distr.; Forster PIF29768 (BRI, L). Macaranga klaineana Pierre ex Prain—DQ899221, DQ866563, DQ901996, DQ767763; Gabon, Estuaire; Breteler et al. 14289 (WAG). Macaranga lamellata Whitmore—DQ899222, DQ866564, DQ901997, DQ767764; Indonesia, East Kalimantan, ITCI-concession; Slik M1060 (L). Macaranga lowii King ex Hook.f. var. kostermansii Airy Shaw—DQ899223, DQ866565, DQ901998, DQ767765; Indonesia, East Kalimantan, Bukit Bangkirai; Slik M208 (L). Macaranga lowii King ex Hook.f. var. lowii—DQ899224, DQ866566, DQ901999, DQ767766; Indonesia, East Kalimantan, Sungai Wain; Slik M56 (L). Macaranga mauritiana Bojer ex Müll.Arg.—DQ899225, DQ866567, DQ902000, —; Mauritius, Mt. Cocotte; Lorence 2349 (K). Macaranga monandra Müll.Arg.—DQ899226, DQ866568, DQ902001, DQ767767; Gabon, Nyanga, Concession SFN; van Valkenburg et al. 2531 (WAG). Macaranga novo-guineensis^a J.J.Sm.—DQ899227, DQ866569, DQ902002, DQ767768; Papua New Guinea, Madang; Weiblen 1803 (MIN). Macaranga oblongifolia Baill.—DQ899228, DQ866570, DQ902003, DQ767769; Madagascar, Fianarantsoa; McPherson & Rabenantoandro 18350 (MO). Macaranga obovata Baill.—DQ899229, DQ866571, DQ902004, DQ767770; Madagascar, Fianarantsoa; McPherson & Rabenantoandro 18279 (MO). Macaranga pachyphylla Müll.Arg.—DQ899230, DQ866572, DQ902005, DQ767771; Thailand, Peninsular Floristic Region, Thung Khai; Chamchumroon 2016 (L). Macaranga pearsonii Merr.—DQ899231, DQ866573, DQ902006, DQ767772; Indonesia, East Kalimantan, Sungai Wain; Slik M104 (L). Macaranga poggei Pax—DQ899232, DQ866574, DQ902007, DQ767773; Gabon, Woleu-Ntem, Tchimbele; Tabak & Feijen 10 (WAG). Macaranga praestans Airy Shaw—DQ899233, DQ866575, DQ902008, DQ767774; Brunai, Belait; Challen et al. 31 (K). Macaranga puncticulata Gage—DQ899234, DQ866576, DQ902009, DQ767775; Indonesia, East Kalimantan, Labanan; Slik M1179 (L). Macaranga quadriglandulosa^a Warb.—DQ899235, DQ866577, DQ902010, DQ767776; Papua New Guinea, Madang; Weiblen 1853 (MIN). Macaranga repando-dentata Airy Shaw—DQ899236, DQ866578, DQ902011, DQ767777; Indonesia, East Kalimantan, ITCI-concession; Slik M994 (L). Macaranga rhizinoides (Blume) Müll.Arg.—DQ899237, DQ866579, DQ902012, DQ767778; Indonesia, Java, Cibodas Botanic Gardens VII.C.64–64a; Gravendeel et al. 676 (L). Macaranga saccifera Pax—DQ899238, DQ866580, DQ902013, —; Gabon, Ogooue-Ivindo; Wieringa et al. 3565 (WAG). Macaranga schweinfurthii Pax—DQ899239, DQ866581, DQ902014, DQ767779; Gabon, Ogooue-Lolo; Wieringa et al. 4088 (WAG). Macaranga siamensis S.J.Davies—DQ899240, DQ866582, DQ902015, DQ767780; Thailand, Eastern Floristic Distr., Nakhon Ratchasima Prov., Khao Yai National Park; van Welzen 2003–16 (L). Macaranga subdentata Benth.—DQ899241, DQ866583, DQ902016, DQ767781; Australia, Queensland, near Wyvuri Swamp; Forster et al. 24032 (L). Macaranga tanarius (L.) Müll.Arg.—DQ899242, DQ866584, DQ902017, DQ767783; Indonesia, East Kalimantan, Bukit Bangkirai; Slik M705 (L). Macaranga tanarius (L.) Müll.Arg.—DQ899243, DQ866585, DQ902018, DQ767782; Australia, Queensland, Port Curtis Distr.; Forster 29660 (BRI, L). Macaranga tessellata Gage—DQ899244, DQ866586, DQ902019, —; Indonesia, Papua, Mt. Jaya, Mimika Regency; Utterridge 329 (L). Macaranga trichocarpa^b (Reichb.f. & Zoll.) Müll.Arg.—DQ899245, DQ866587, DQ902020, DQ767784; Indonesia, East Kalimantan, Bukit Bangkirai; Slik M398 (L). Macaranga triloba (Thunb.) Müll.Arg.—DQ899246, DQ866588, DQ902021, DQ767785; Indonesia, Java, Jawa Barat, Halimun National Park; Gravendeel et al. 619 (L). Macaranga umbrosa S.J.Davies—DQ899247, DQ866589, DQ902022, DQ767786; Brunai, Tutong, Tasek Merimbun Heritage Park; Challen et al. 41 (K). Macaranga winkleri Pax & K.Hoffm.—DQ899248, DQ866590, DQ902023, DQ767787; Indonesia, East Kalimantan, Bukit Bangkirai; Slik M678 (L). Mallotus barbatus Müll.Arg.—DQ899249, DQ866591, DQ902024, DQ767788; Leiden Botanic Garden, acc. 920695; Kulju 90 (L). Mallotus brachythyrsus Merr.—DQ899250, DQ866592, DQ902025, DQ767789; Indonesia, East Kalimantan, ITCI-concession; Slik M900 (L). Mallotus caudatus Merr.—DQ899251, DQ866593, DQ902026, DQ767790; Indonesia, East Kalimantan, Labanan; Slik M1243 (L). Mallotus claoxyloides^b (F.Muell.) Müll.Arg.—DQ899252, DQ866594, DQ902027, DQ767791; Australia, Queensland, Port Curtis Distr.; Forster 29663 (BRI, L). Mallotus connatus M. Aparicio—DQ899279, DQ866620, DQ902050, DQ767816; Indonesia, Java, Bogor Botanic Gardens, IX.C.; Gravendeel et al. 522 (L). Mallotus decipiens Müll.Arg.—DQ899253, DQ866595, DQ902028, DQ767792; Thailand, Southwestern, Prachuap Khiri Khan, Kaeng Kra Chan National Park; Middleton et al. 1104 (L). Mallotus decipiens Müll.Arg.—DQ899254, DQ866596, DQ902029, DQ767793; Thailand, Southwestern, Prachuap Khiri Khan, Kaeng Kra Chan National Park; Middleton et al. 1065 (L). Mallotus discolor F.Muell. ex Benth.—DQ899255, DQ866597, DQ902030, DQ767794; Australia, Queensland, Port Curtis Distr.; Forster 29659 (BRI, L). Mallotus eucaustus Airy Shaw—DQ899256, DQ866598, DQ902031, DQ767795; Indonesia, East Kalimantan, Labanan; Slik M1085 (L). Mallotus ficifolius (Baill.) Pax & K.Hoffm.—DQ899257, DQ866599, DQ902032, DQ767796; Australia, Queensland, Cook Distr.; Forster PIF29782 (BRI, L). Mallotus glomerulatus Welzen—DQ899258, —, —, —; Thailand, Northeastern, Nakhon Phanom Prov., Phu Langka National Park; Koonkhanthod et al. 517 (L). Mallotus griffithianus^b (Müll.Arg.) Hook.f.—DQ899259, DQ866600, DQ902033, DQ767797; Indonesia, East Kalimantan, Labanan; Slik M1076 (L). Mallotus khasianus Hook.f.—DQ899260, DQ866601, DQ902034, DQ767798; Thailand, Northern, Nan Prov., Doi Phu Ka National Park; Kessler PK3276 (L). Mallotus lackeyi Elmer—DQ899261, DQ866602, DQ902035, DQ767799; Indonesia, East Kalimantan, ITCI-concession; Slik M912 (L). Mallotus leucocalyx Müll.Arg.—DQ899262, DQ866603, —, DQ767800; Thailand, Surat Thani Prov., Phanom Distr., Khlongphanom National Park; Middleton et al. 2122 (L). Mallotus macrostachyus (Miq.) Müll.Arg.—DQ899263, DQ866604, DQ902036, DQ767801; Indonesia, East Kalimantan, Bukit Bangkirai; Slik M262 (L). Mallotus miquelianus (Scheff.) Boerl.—DQ899264, DQ866605, DQ902037, DQ767802; Indonesia, East Kalimantan, ITCI-concession; Slik M879 (L). Mallotus oppositifolius (Geisel.) Müll.Arg.—DQ899265, DQ866606, DQ902038, DQ767803; Gabon, Ngounié, Sindara; Wieringa et al. 4384 (WAG). Mallotus pallidus (Airy Shaw) Airy Shaw—DQ899266, DQ866607, DQ902039, DQ767804; Thailand, Southwestern, Prachuap Khiri Khan, Khao Sam Roi Yot National Park; Middleton et al. 1136 (L). Mallotus paniculatus^b (Lam.) Müll.Arg.—DQ899267, DQ866608, DQ902040, DQ767806; Indonesia, East Kalimantan, Sungai Wain; Slik M144 (L). Mallotus paniculatus (Lam.) Müll.Arg.—DQ899268, DQ866609, DQ902041, DQ767805; Australia, Queensland, Cook Distr.; Forster 29762 (BRI, L). Mallotus peltatus (Geisel.) Müll.Arg.—DQ899269, DQ866610, DQ902042, DQ767807; Indonesia, East Kalimantan, ITCI-concession; Slik M896 (L). Mallotus penangensis Müll.Arg.—DQ899270, DQ866611, DQ902043, DQ767808; Indonesia, East Kalimantan, Gunung Meratus; Slik M845 (L). Mallotus philippensis (Lam.) Müll.Arg.—DQ899272, DQ866613, DQ902045, DQ767809; Australia, Queensland, Port Curtis Distr.; Forster 29664 (BRI, L). Mallotus philippensis (Lam.) Müll.Arg.—DQ899271, DQ866612, DQ902044, DQ767810; Indonesia, Java, Bogor Botanic Gardens, IX.C.23; Gravendeel 504 (L). Mallotus philippensis (Lam.) Müll.Arg.—DQ899273, DQ866614, —, DQ767811; Sri Lanka, Matale Distr., Illukkumbura; Kathriarachchi et al. 64 (K, WU). Mallotus pierrei (Gagnep.) Airy Shaw—DQ899274, DQ866615, DQ902046, —; Thailand, Southwestern, Prachuap Khiri Khan, Huay Yang National Park; Middleton et al. 1320 (L). Mallotus polyadenos F.Muell.—DQ899275, DQ866616, DQ902047, DQ767812; Australia, Queensland, Port Curtis Distr.; Forster 29780 (BRI, L). Mallotus repandus (Rottler) Müll.Arg.—DQ899276, DQ866617, DQ902048, DQ767813; Indonesia, Java, Bogor Botanic Gardens XV.C.20–20a; Gravendeel et al. 515 (L). Mallotus resinosus (Blanco) Merr.—DQ899277, DQ866618, DQ902049, DQ767814; Sri Lanka, Kurunegala Distr., Weuda; Kathriarachchi et al. 67 (K, WU). Mallotus rhamnifolius (Willd.) Müll.Arg.—DQ899278, DQ866619, —, DQ767815; Sri Lanka, Ratnapura Distr., Puwakgahawala; Kathriarachchi et al. 38 (K, WU). Mallotus subpeltatus (Blume) Müll.Arg.—DQ899280, DQ866621, DQ902051, DQ767817; Thailand, Peninsular, Krabi, Khao Phanom Bencha National Park; Middleton et al. 494 (L). Mallotus subulatus Müll.Arg.—DQ899281, DQ866622, —, —; Cameroon, South West Prov., Fako, Buea; Wheatley 16 (K). Mallotus tetracoccus Kurz—DQ899282, DQ866623, DQ902052, DQ767818; Sri Lanka, Matale Distr., Knuckles; Kathriarachchi et al. 2 (K, WU). Mallotus thorellii Gagnep.—DQ899283, DQ866624, DQ902053, DQ767819; Cambodia, Kampong Speu Prov., Chabarmon Distr.; Huq et al. 10865 (L).

Neotrewia cumingii (Müll.Arg.) Pax & K.Hoffm.—DQ899284, DQ866625, DQ902054, DQ767820; Philippines, Luzon, Los Baños, Mt. Makiling; Fernando 1735 (L).

Octospermum pleiogynum (Pax & K.Hoffm.) Airy Shaw—DQ899285, DQ866626, DQ902055, DQ767821; Indonesia, Irian Jaya, Bird's Head Peninsula; Polak NT11610 (L).

Trewia nudiflora L.—DQ899286, DQ866627, DQ902056, DQ767823; Thailand, Central Floristic District, Saraburi Prov., Phu Khae Botanical Garden; van Welzen 2003–5 (L). Trewia nudiflora L.—DQ899287, DQ866628, DQ902057, DQ767822; India, Tiruchi Dist., Srirangam; Perianayagam RHT 74579 (L, RHT).

FOOTNOTES

¹ The authors thank the following persons and institutes for plant material: F. Blattner, G. Challen, V. Chamchumroon, K. Chayamarit, S. Davies, H. J. Esser, E. Fernando, B. Fiala, P. Forster, B. Gravendeel, P. Hoffmann, H. Kathriarachchi, P. Keßler, P. Phonsena, M. Polak, K. M. Mathew, G. McPherson, D. Middleton, F. Slik, W. Takeuchi, G. Weiblen, K. Weising, and J. Wieringa; K, MO, WAG, Hortus Botanicus Leiden, Nationale Plantentuin van België; and K. Wurdack for sharing the uniovulate euphorb trnL-F/rbcL data set and the Royal Botanic Gardens, Kew for sharing the late Tim Whitmore's unpublished Prodromus of Macaranga manuscript. Additional help and/or valuable comments on the manuscript were provided by P. Baas, F. Bakker, P. Berry, C. Bödeker, D. Co, M. Eurlings, B. Fiala, P. Hoffmann, J. van Konijnenburg-van Cittert, M. Vorontsova, J. Zuccarello, and one anonymous reviewer. The fieldwork was supported by the Finnish Cultural Foundation.

⁴ Author for correspondence (kulju{at}nhn.leidenuniv.nl )

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