Molecular Phylogenetics and Evolution 54 (2010) 553–560
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Molecular Phylogenetics and Evolution
journal homepage: www.elsevier.com/locate/ympev
A three-genome phylogeny of Momordica (Cucurbitaceae) suggests seven returns from dioecy to monoecy and recent long-distance dispersal to Asia
Hanno Schaefer *, Susanne S. Renner
Systematic Botany and Mycology, University of Munich (LMU), Menzinger-Str. 67, D-80638 Munich, Germany article info abstract
Article history: The bitter gourd genus Momordica comprises 47 species in Africa and 12 in Asia and Australia. All have Received 17 May 2009 unisexual flowers, and of the African species, 24 are dioecious, 23 monoecious, while all Asian species are Revised 9 August 2009 dioecious. Maximum likelihood analyses of 6257 aligned nucleotides of plastid, mitochondrial and Accepted 10 August 2009 nuclear DNA obtained for 122 accessions of Momordica and seven outgroups show that Momordica is Available online 15 August 2009 monophyletic and consists of 11 well-supported clades. Monoecy evolved from dioecy seven times inde- pendently, always in Africa and mostly in savanna species with low population densities. Leaky dioecy, Keywords: with occasional fruit-producing males, occurs in two African species and might be the first step in an evo- Ancestral state reconstruction lutionary transition towards monoecy. Dated biogeographic analyses suggest that Momordica originated Dioecy Leafy in tropical Africa and that the Asian species are the result of one long-distance dispersal event about Molecular clock 19 million years ago. The pantropical vegetable Momordica charantia is of African, not Asian origin as Monoecy had previously been suggested. Sexual system transitions Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction sha and Vasudeva, 2001; H.S., pers. obs.), and self-incompatibility is unknown in Cucurbitaceae. Details of flowering biology are In the gourd family (Cucurbitaceae), flowers are mostly unisex- known only for the bitter gourd, M. charantia, which is an impor- ual, and about 50% of the 900–1000 species are monoecious, the tant crop species in parts of Asia and Africa. In this species, the first remainder dioecious (Schaefer and Renner, unpublished). Details flowers appear 45–55 days after sowing, and under long-day con- of sexual system evolution in the family, however, are poorly ditions male flowers bloom 2 weeks before the first female flow- known. Zhang et al. (2006) reconstructed bisexual flowers as ers; the female to male flower ratio is ca. 1 in 25, and flowering ancestral in the order Cucurbitales (seven families) and dioecy as lasts ca. 6 months (Palada and Chang, 2003). Sex expression and having evolved in the common ancestor of the four core cucurbita- inheritance have been studied in M. dioica, which is used as a veg- lean families Begoniaceae, Cucurbitaceae, Datiscaceae, and Tetram- etable in India (Hossain et al., 1996). Crossing experiments that elaceae. Dioecy was inferred as ancestral for the Cucurbitaceae, and used artificially induced monoecious plants indicated that sex in monoecy in the family would then be the result of repeated sexual M. dioica is controlled by a single factor, with heterozygous males system shifts. The only evolutionary analysis to date focused on the and homozygous recessive females. small cucurbit genus Bryonia with about ten species, where either In this study, we use field and herbarium data, molecular mark- two shifts from dioecy to monoecy or four shifts from monoecy to ers from three genomes, ancestral state reconstruction, and molec- dioecy occurred, depending on the assumed ancestral state (Volz ular dating to infer how often, when, and in which direction and Renner, 2008). transitions between monoecy and dioecy have occurred in Momor- Momordica comprises 59 species growing in rainforest, decidu- dica and if they are related to habitat, life form, or pollinators. ous forest, bushland, savannas, or grassland. Most species are perennial climbers, two species are small shrubs and two species 2. Materials and methods are annuals. Momordica flowers measure 1–10 cm in diameter and are short-lived. About a third of the species are monoecious, 2.1. Taxon sampling the remaining dioecious. Three of the monoecious species have been shown to be self-compatible (Palada and Chang, 2003; Loke- Online Supplement Table 1 provides a complete list of the 58 species of Momordica included in this study (out of 59 accepted species in the genus), with authors, geographic origin, sexual sys- * Corresponding author. Present address: Imperial College London, Silwood Park Campus, Buckhurst Road, Ascot SL5 7PY, Berkshire, UK. tem, habitat, life form, pollinator, fruit type, chromosome number, E-mail address: [email protected] (H. Schaefer). and GenBank Accession Numbers for all sequences. We included
1055-7903/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2009.08.006 554 H. Schaefer, S.S. Renner / Molecular Phylogenetics and Evolution 54 (2010) 553–560 six as yet undescribed species and one recently described African the separate plastid, nuclear, and mitochondrial data partitions species (Schaefer, 2009), a full taxonomic revision of the genus will produced congruent phylogenetic estimates (not shown), with all be published elsewhere. To detect intra-specific variation, we se- areas of discordance being restricted to branches with low support quenced 2–6 accessions for 80% of the species; for 11 rare species (i.e., bootstrap proportions <60%). Given this overall congruence, only one accession was available. Our analysis includes 605 se- the three data partitions were concatenated, with specific model quences, of which 593 were generated for this study. In addition, parameters estimated for each partition. To detect if analyses had the first author studied several thousand herbarium specimens in become trapped in local optima, we conducted ten separate ML 21 European, African, American and Chinese institutions (A, B, searches. The tree with the best likelihood score was then selected BM, BR, CAS, DSM, E, EA, FI, FT, GH, K, KUN, L, M, MO, P, UPS, W, as the optimal tree. Non-parametric bootstrapping as implemented WAG and WU, herbarium acronyms follow Index Herbariorum at in the ‘‘fast bootstrap” algorithm of RAxML used 500 replicates. http://sciweb.nybg.org/science2/IndexHerbariorum.asp), and 19 Bayesian inference used the GTR + G model (with the default of the 59 species in their natural habitat in Australia, Tanzania, four rate categories) plus a proportion of invariable sites and relied Nigeria, Sierra Leone, and China. Ten species were cultivated at on MrBayes (Huelsenbeck and Ronquist, 2001). We again analyzed the botanical garden Munich. Two species could not be included: the combined dataset with three partitions (plastid, nuclear, mito- M. macrosperma (Cogn.) Chiov., which is known only from the leaf- chondrial), allowing partition models to vary by unlinking gamma less type at FT and was not available for DNA extraction, and M. shapes, transition matrices, and proportions of invariable sites. sahyadrica Kattuk. & V. T. Antony, a recently described entity from Markov chain Monte Carlo (MCMC) runs started from independent India, which based on morphological characters seems to be a hy- random trees, were repeated twice, and extended for 1 million brid (H.S., unpubl.). Following the Cucurbitaceae phylogeny of generations, with trees sampled every 100th generation. We used Schaefer et al. (2009), we included two distant outgroups, Bol- the default priors in MrBayes, namely a flat Dirichlet prior for the bostemma paniculatum and Gerrardanthus grandiflorus, and the relative nucleotide frequencies and rate parameters, a discrete uni- close relatives Baijiania borneensis, B. yunnanensis, Thladiantha cor- form prior for topologies, and an exponential distribution (mean difolia, T. nudiflora, and T. pustulata. 1.0) for the gamma-shape parameter and branch lengths. Conver- gence was assessed by checking that the standard deviations of 2.2. DNA isolation, amplification, sequencing, and alignment split frequencies were <0.01; that the log probabilities of the data given the parameter values fluctuated within narrow limits; that Total genomic DNA was isolated from silica-dried leaves and the convergence diagnostic (the potential scale reduction factor gi- herbarium specimens using the NucleoSpin plant kit (Macherey- ven by MrBayes) approached one; and by examining the plot pro- Nagel, Düren, Germany). DNA amplification by polymerase chain vided by MrBayes of the generation number versus the log reaction (PCR), fragment purification and cycle sequencing fol- probability of the data. Trees saved prior to convergence were dis- lowed standard protocols. We amplified six markers from the three carded as burn-in (1000 trees) and a consensus tree was con- genomes (i) one coding and three non-coding chloroplast regions: structed from the remaining trees. the plastid maturase K (matK) gene, the trnL intron and adjacent trnL-F spacer, the rpl20–rps12 intergenic spacer between the ribo- 2.4. Analysis of sexual system evolution somal protein genes S12 and L20, and the trnH–psbA spacer. Prim- ers used to amplify the first three regions are given in Schaefer To analyse sexual system evolution, we used a reduced dataset et al. (2009). The trnH–psbA spacer was amplified with primers of 65 species with one accession per species (58 Momordica and trnH and psbA given in Volz and Renner (2009). (ii) From the nu- seven outgroup species). We mapped the evolution of sexual sys- cleus, we amplified the second intron of the Leafy gene (referred tems onto the phylogeny by defining three unordered states, (0) to herein simply as LFY) using the primers LFY CucF 5-TCT TCC unknown, (1) monoecious, and (2) dioecious. Information on ACC TST ATG ARC AGT GTC GTG AAT-3 and LFY CucR 5-CGA AAT sexual systems came from personal observation in the natural CAC AAA AGY TAT TGS GYA KTY CA-3 (developed by HS). (iii) habitat and greenhouse, from the study of herbarium specimens, The mitochondrial region used in this study is the nad1 intron floras, and monographs (online Supplement Table 1). To infer (NADH dehydrogenase subunit 1, exon B-C), which was amplified ancestral states, we used parsimony and maximum likelihood as using the nad1/B-C universal primers from Demesure et al. (1995). implemented in Mesquite ver. 2.6 (Maddison and Maddison, Five randomly chosen samples of the nuclear and mitochondrial 2009; http://mesquiteproject.org/mesquite/mesquite.html). All products were cloned as described in Volz and Renner (2008) to as- analyses were carried out on the preferred highest likelihood tree, sess within-plant sequence divergence. For all markers, sequencing with branch lengths set to the maximum likelihood values ob- primers were the same as for amplification. Both strands were se- tained under the GTR + G model (above). Likelihood analyses in quenced and used to generate consensus sequences in Sequencher Mesquite used the Markov k-state one-parameter model, which (ver. 4.1.2, GeneCodes Corp.). Sequence alignment was done man- is a generalization of the Jukes–Cantor model (Lewis, 2001) and ually in MacClade ver. 4.0.6 (Maddison and Maddison, 2003). The assumes a single rate for all transitions between character states. alignments have been deposited in TreeBASE (www.treebase.org). We let Mesquite estimate the transition parameters of the model, based on the tip trait states in the 65-taxon tree and its branch 2.3. Phylogenetic analyses lengths.
Maximum likelihood (ML) tree searches and ML bootstrapping 2.5. Molecular clock analyses were performed using RAxML ver. 7.0.4 (Stamatakis et al., 2008; available at http://phylobench.vital-it.ch/raxml-bb/). RAxML To transform the likelihood tree inferred from the combined searches relied on the general time-reversible (GTR) model with dataset into a time-calibrated phylogeny, we used Bayesian clock among-site rate heterogeneity modelled by a gamma distribution estimations with an uncorrelated rates model (Drummond et al., with 25 rate categories (all model parameters were estimated over 2006). The 65-species alignment was first imported in BEAUti ver. the duration of specified runs). Although RAxML can incorporate a 1.4.8 (part of the BEAST package; Drummond and Rambaut, 2007) further parameter for the proportion of invariant sites, we chose to generate BEAST input files. We used the GTR + G model with six not to use it because the 25 gamma rate categories sufficiently ac- rate categories. Based on the results of a family-wide analysis with count for rate heterogeneity (Stamatakis et al., 2008). Analyses of a four-plastid-marker matrix and four fossil-calibration points H. Schaefer, S.S. Renner / Molecular Phylogenetics and Evolution 54 (2010) 553–560 555
Fig. 1. Maximum likelihood tree for Momordica obtained from combined plastid, nuclear and mitochondrial sequence data (6257 aligned nucleotides). Likelihood bootstrap values are indicated above branches, Bayesian posterior probability values below branches, and the geographic origin of each accession follows species names.
(Schaefer et al., 2009), we used the following five constraints (all graphic regions as an unordered multi-state character using the with a normal prior distribution): Momordica stem node following six regions as character states (i) East and Southeast Asia 46 ± 3 Ma, Momordica crown node 31 ± 5 Ma, Thladianthinae including New Guinea and tropical Australia, (ii) West Asia, (iii) (Thladiantha and Baijiania) crown node 45 ± 4 Ma, Baijiania crown East and Northeast Africa, (iv) Central Africa, (v) West Africa, and node 32 ± 6 Ma, and Thladiantha crown node 14 ± 5 Ma. The (vi) Southern Africa. For the widespread species M. balsamina, M. performance of the BEAST runs was checked using Tracer ver. 1.4 charantia, and M. foetida, we used an additional state ‘‘all Africa.” (Rambaut and Drummond, 2007). Following convergence, the Recent human-mediated range expansions of M. balsamina and resulting trees were combined using TreeAnnotator ver. 1.4.8 (part M. charantia into the New World tropics, Asia, and the Pacific is- of the BEAST package), with a burn-in of 2000 trees. Final trees were lands were ignored. Momordica cymbalaria (syn. M. tuberosa)is checked and edited in FigTree ver. 1.2.2 (Rambaut, 2006-2009). known from North and East Africa to India and Pakistan but likely was introduced to Asia as a vegetable and medicinal plant (Lokesha 2.6. Biogeographical analyses and Vasudeva, 2001); it was therefore coded as East and Northeast Africa only. We then used maximum likelihood as implemented in To estimate the biogeographical history of Momordica, we used Mesquite to reconstruct shifts between biogeographic regions. This ancestral area reconstruction (Ronquist, 1994). We estimated geo- method incorporates branch-length information, which is why we 556 H. Schaefer, S.S. Renner / Molecular Phylogenetics and Evolution 54 (2010) 553–560 employed it as an alternative to dispersal-vicariance analysis field, female plants are much less common than male plants (ca. (DIVA; Ronquist, 1997). 1:50). In the two predominantly dioecious species M. friesiorum and M. pterocarpa, three herbarium specimens of 150 and 4 of ca. 500 were monoecious (i.e., they were fruit-producing males). 3. Results
3.1. Morphological analyses and field observations 3.2. Phylogenetic analyses
Of the 59 described and new Momordica species (58 of them in- The final plastid sequence matrix consisted of 1217 aligned cluded in Fig. 1), 34 are dioecious and 22 monoecious. For three of nucleotides of the matK gene, 1177 nucleotides of the trnL and the new species it remains unclear whether they are monoecious trnL-F region, 869 nucleotides of the rpl20–rps12 intron, and 620 or dioecious because the available collections are too incomplete. nucleotides of the trnH–psbA spacer. Plastid sequences were ob- Based on field observations and herbarium specimens, all species tained for all included species and accessions. The nuclear dataset of the closely related genera Indofevillea, Thladiantha, Baijiania, Tel- consisted of 976 aligned nucleotides of LFY, which were obtained fairia, and Cogniauxia are dioecious. In monoecious Momordica, the for 49 accessions representing 36 species. The mitochondrial ma- number of female flowers is much lower than that of male flowers, trix consisted of 1659 aligned nucleotides of nad1, which were ob- ranging from about 1:10 to 1:25. In dioecious species studied in the tained for 31 samples belonging to 31 species.
M. camerounensis M. jeffreyana 7. M. spec. nov. HS538 M. enneaphylla M. peteri M. rostrata M. trifoliolata M. cardiospermoides M. littorea 6. M. dissecta M. gilgiana M. cissoides M. trifolia M. clarkeana M. cochinchinensis M. sphaeroidea M. macrophylla M. denticulata M. suringarii M. laotica M. denudata M. subangulata M. renigera M. dioica M. repens 5. M. corymbifera M. pterocarpa M. nuda M. spec. nov. HS590 M. spec. nov. HS438 M. friesiorum M. anigosantha M. spinosa M. glabra M. spec. nov. HS81 M. multiflora M. spec. nov. HS78 4. M. parvifolia M. silvatica M. angustisepala M. henriquesii M. boivinii M. humilis M. kirkii M. cymbalaria 3. M. spec. nov. HS436 M. sessilifolia M. leiocarpa M. foetida M. angolensis M. charantia 2. M. welwitschii M. balsamina M. involucrata M. obtusisepala M. mossambica M. cabrae M. calantha 1. Baijiania borneensis Baijiania yunnanensis Thladiantha nudiflora Thladiantha pustulata Thladiantha cordifolia Gerrardanthus grandiflorus Bolbostemma paniculatum
Fig. 2. The evolution of monoecy and dioecy in Momordica as inferred on the preferred maximum likelihood tree (character states: dioecious – white, monoecious – black, unclear – grey); numbers on the right indicate sexual system shifts. H. Schaefer, S.S. Renner / Molecular Phylogenetics and Evolution 54 (2010) 553–560 557
Maximum likelihood analyses of the individual datasets re- 3.3. Sexual system evolution vealed no topological contradictions with significant bootstrap support (Section 2), and the four partitions were therefore com- Parsimony and maximum likelihood produced identical infer- bined into a matrix with 6257 aligned nucleotides. The topology ences about changes between monoecy and dioecy in Momordica. of the resulting maximum likelihood tree (Fig. 1) is identical to We therefore show only the reconstruction obtained under likeli- the Bayesian consensus tree (not shown) in all statistically sup- hood (Fig. 2). The ancestral state in Momordica most likely is ported nodes and shows that Momordica is monophyletic (with dioecy. Monoecy then evolved independently in seven species or 100% bootstrap support as well as Bayesian posterior probability) groups of species (1) M. calantha, a perennial liana of secondary and that the genus can be divided into 11 clades that mostly corre- rainforests in Northern Tanzania and Kenya; (2) the M. charantia spond to the morphological clades proposed by Jeffrey and de clade, three savanna species of southern and southwestern Africa Wilde (2006). Multiple accessions of the same morphological spe- and two weed species common along forest margins and on dis- cies clustered together, but in M. cissoides, M. charantia, and M. turbed ground throughout tropical Africa; (3) the sister species enneaphylla, large genetic differences among accessions (long M. corymbifera and M. repens, tuberous perennials from southern branches) suggest additional cryptic species. African savannas; (4) the Raphanocarpus group of six perennials
M. camerounensis M. jeffreyana M. spec. nov. HS538 M. enneaphylla M. peteri M. rostrata M. trifoliolata M. cardiospermoides M. littorea M. dissecta M. gilgiana M. cissoides M. trifolia M. clarkeana M. cochinchinensis M. sphaeroidea M. macrophylla M. denticulata M. suringarii M. laotica M. denudata M. subangulata M. renigera M. dioica M. repens M. corymbifera M. pterocarpa M. nuda M. spec. nov. HS590 M. spec. nov. HS438 M. friesiorum M. anigosantha M. spinosa M. glabra M. spec. nov. HS81 M. multiflora M. spec. nov. HS78 M. parvifolia M. silvatica M. angustisepala M. henriquesii M. boivinii M. humilis M. kirkii M. cymbalaria M. spec. nov. HS436 M. sessilifolia M. leiocarpa M. foetida M. angolensis M. charantia M. welwitschii M. balsamina M. involucrata M. obtusisepala M. mossambica M. cabrae M. calantha Baijiania borneensis Baijiania yunnanensis Thladiantha nudiflora Thladiantha pustulata Thladiantha cordifolia Gerrardanthus grandiflorus Bolbostemma paniculatum East and Southeast Asia East and Northeast Africa West Asia Central Africa West Africa Southern Africa all Africa
Fig. 3. The historical biogeography of Momordica inferred on the preferred maximum likelihood tree under maximum likelihood optimisation (character states as indicated in the legend). 558 H. Schaefer, S.S. Renner / Molecular Phylogenetics and Evolution 54 (2010) 553–560 with dry fruits, mostly from savannas or dessert habitats of Eastern Northeast Africa” and ‘‘West Africa.” Of the 11 monophyletic or sub-Saharan Africa; (5) the M. multiflora clade, six species from groups within Momordica, four are reconstructed as East and tropical forest in Central Africa; for two of them the sexual system Northeast African with >60% likelihood, two with >50%. The vege- is unknown; (6) M. cardiospermoides, a perennial savanna species table species M. charantia and M. balsamina are both reconstructed of East and Southern Africa; (7) M. jeffreyana, a perennial liana of as originating in Africa. the West African rainforests. 3.5. Molecular clock analyses 3.4. Biogeography Divergence time estimates for Momordica are shown in Fig. 4. Reconstructions of the ancestral area (Fig. 3) and habitat (not The oldest of the 11 clades within the genus (labelled in Fig. 1)is shown) yielded humid forests in East and Southeast Asia as the the Dimorphochlamys group (29 (32–14) Ma) with four tall peren- most likely states for the Momordica stem node. The area occupied nial liana species in the East and West African Tropics and Mozam- by the crown group node is reconstructed as most likely East and bique. The Asian M. cochinchinensis group is estimated to be ca. 19 Northeast Africa. The genus subsequently diversified in ‘‘East and (24–14) My old. Age estimates for the nodes inferred to have shifted
M. denticulata -3,87 M. macrophylla M. suringarii M. cochinchinensis M. sphaeroidea -0,37 M. denudata -1,0 -2,0 M. subangulata M. laotica -3,27 M. renigera M. dioica M. trifolia M. clarkeana M. cissoides M. gilgiana M. nuda M. friesiorum M. spec. nov. HS590 M. spec. nov. HS438 M. pterocarpa M. anigosantha -2,02 M. repens M. corymbifera -2,33 M. spec. nov. HS78 M. multiflora spec. nov. HS81 -5,18 M. M. silvatica M. parvifolia M. glabra M. angustisepala M. henriquesii M. spinosa M. spec. nov. HS538 -2,32 M. cardiospermoides M. littorea M. trifoliolata -1,01 M. rostrata M. dissecta M. peteri M. enneaphylla M. camerounensis M. jeffreyana M. cymbalaria M. spec. nov. HS436 M. kirkii M. humilis M. boivinii -2,14 M. sessilifolia M. involucrata M. balsamina M. welwitschii M. angolensis M. charantia M. leiocarpa M. foetida M. cabrae M. calantha M. mossambica M. obtusisepala Thladiantha nudiflora Thladiantha pustulata Thladiantha cordifolia Baijiania yunnanensis Baijiania borneensis Gerrardanthus grandiflorus Bolbostemma paniculatum
Ma
Fig. 4. Chronogram for 58 species of Momordica and outgroups obtained under a model of uncorrelated rate change using five calibration points (see text for details). Node heights are the median ages, 95% highest posterior density intervals are indicated by grey bars at the nodes. H. Schaefer, S.S. Renner / Molecular Phylogenetics and Evolution 54 (2010) 553–560 559 from dioecy to monoecy range from 34 to 2 Ma. The South East males. These consequences must depend on the quantity and qual- Asian and Australian M. sphaeroidea is about 3.9 (4.9–1.5) My old. ity of the seeds produced by ‘‘leaky” males relative to co-occurring females. Without data on these aspects, one cannot predict whether a particular leaky-dioecious species is on its way back to monoecy 4. Discussion or not. Nevertheless it is clear that the spread of bisexual mutants must be at the start of any transition from dioecy to monoecy. The inferred seven transitions from dioecy to monoecy in The biogeographic history of Momordica reconstructed in this Momordica illustrate the flexibility of Cucurbitaceae sexual sys- study fits well into the scenario developed for the history of the tems. The genus also provides a so far unique example of a consis- cucurbit family in Schaefer et al. (2009) if a tropical Asian origin tent trend away from dioecy and towards monoecy. In animals, of the Momordica stem lineage is assumed (so far no fossil evidence with their more determined body plans, this reversal may be available). Having spread into Northern and Eastern Africa, inde- impossible (Bull and Charnov, 1985), but in plants such reversals pendent Momordica lineages expanded their range into Central have now been reported from several other genera, such as Fuchsia and West Africa, and Southern Africa. About 19 Ma, one lineage (Onagraceae; Sytsma et al., 1991), Sagittaria (Alismataceae; Dorken then dispersed back to Asia with one species, M. sphaeroidea, finally et al., 2002), Mercurialis (Euphorbiaceae; Obbard et al., 2006), Bryo- reaching Australia less than 4 Ma. The important vegetable species nia (Volz and Renner, 2008), and Iphigenia/Wurmbea (Colchicaceae; M. charantia and the less commonly grown vegetable M. balsamina Case et al., 2008). In Iphigenia (nested in the genus Wurmbea) and both belong to a South African clade and are therefore most likely Fuchsia, monoecy appears to be associated with long-distance dis- of African origin and not Asian as suggested by Marr et al. (2004). persal, in agreement with Baker’s law (1967) that a single plant can only start a new population if it is self-fertile and produces her- Acknowledgments maphroditic flowers or unisexual flowers of both sexes. In Momor- dica, reversals to monoecy are not associated with long-distance The authors thank B. Duyfjes, C. Jeffrey, M. Thulin, W. de Wilde, dispersal events, and the Asian lineage, which is the result of the and J. Wieringa for material and helpful comments, and the cura- longest dispersal event in the genus, is entirely dioecious. It seems tors of A, B, BM, BR, CAS, DSM, E, EA, FI, FT, GH, K, KUN, L, M, more likely that the transitions to monoecy in Momordica are re- MO, P, UPS, W, WAG and WU for hospitality and/or loan of mate- lated to plant–pollinator relationships and how these relationships rial. Financial support for the project came from the German Sci- are moulded by population density, which in turn relates to habi- ence Foundation (DFG) Grant No. RE603/3-1. tat. The pollinators of all but three species of Momordica are spec- ialised oil bees of the genus Ctenoplectra (Schaefer and Renner, Appendix A. Supplementary data 2008; online Supplement Table 1). Male flowers in Momordica (ex- cept those of M. balsamina, M. charantia, and M. dioica) produce Supplementary data associated with this article can be found, in nectar, pollen and fatty oil, while female flowers produce fatty oils the online version, at doi:10.1016/j.ympev.2009.08.006. only (Vogel, 1990; H.S., unpubl.). Male flowers are borne singly or in few-flowered umbels in leaf axils throughout the flowering sea- References son. Female flowers are produced towards the end of the season and always singly. Moreover, the amount of oil in female flowers Baker, H.G., 1967. Support for Baker’s Law – as a rule. Evolution 21, 853–856. tends to be lower than that offered in male flowers (Vogel, 1990, Baker, H.G., Cox, P.A., 1984. Further thoughts on dioecism and islands. Ann. Mo. Bot. p. 75), and in some species, female flowers are entirely rewardless. Gard. 71, 244–253. Bull, J.J., Charnov, E.L., 1985. On irreversible evolution. Evolution 39 (5), 1149–1155. 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