American Journal of Botany 91(5): 767±777. 2004.

ON THE ORIGIN OF THE FIG: PHYLOGENETIC RELATIONSHIPS OF FROM NDHF SEQUENCES1

SHANNON L. DATWYLER AND GEORGE D. WEIBLEN2

Department of Biology, University of Minnesota, St. Paul, Minnesota 55108 USA

The majority of species in the mulberry family (Moraceae) are ®gs (), marked by a specialized in¯orescence (syconium) and an obligate mutualism with pollinating ®g wasps. Because of the unique morphology of the syconium, it has been dif®cult to investigate the evolutionary position of the ®g. We sequenced the gene ndhF to examine relationships in Moraceae and to elucidate shifts in reproductive traits. The reclassi®cation of tribes is warranted, and the limits of , Moreae, and are revised to re¯ect evolutionary relationships. The results point to ancestral dioecy in Moraceae and multiple origins of monoecy, androdioecy, and gynodioecy. Ancestral wind pollination gave way to insect pollination at least twice. Strong support for the sister-group relationship of a revised Castilleae with Ficus suggests that entomophily and involucral bracts encircling the ¯owers preceded the evolution of the syconium. Bracts surround ¯owers in Castilleae only during early development, but in Ficus the involucre and the receptacle enclose the fruit as well. Molecular dating suggests that ®g pollination is at least 80±90 million years old. The diversity of Ficus relative to its is a likely consequence of ancient specialization and cospeciation with pollinating ®g wasps.

Key words: breeding systems; Ficus; molecular dating; Moraceae; phylogenetic classi®cation; pollination.

The 37 genera of Moraceae have a striking array of in¯o- taxon sampling in these studies was not suf®cient to resolve rescence forms, pollination syndromes, and breeding systems the sister group to the ®gs. Investigating the ®g origin requires (Fig. 1). Most of the 1100 species are ®gs (Ficus), known for a detailed phylogenetic hypothesis that has been lacking for their unique in¯orescence and obligate pollination mutualism the family until now. with ®g wasps (Agaonidae). The ®g in¯orescence (syconium) Molecular studies have demonstrated that Moraceae are part is an urn-shaped receptacle lined with unisexual ¯owers and of the Rosidae and are closely allied to the Urticalean , enclosed at the apex by involucral bracts (ostiole). Pollinating including Cannabaceae, Celtidaceae, , Cecropi- ®g wasps negotiate these tightly arranged bracts to access the aceae, and Ulmaceae (Zavada and Kim, 1996; Wiegrefe et al., interior of the syconium where some of the ¯owers are galled 1998; Sytsma et al., 2002). The Urticalean rosids differ from and others develop into fruits. Ultimately, mutualism centers most other rosids in the presence of solitary ovules, lactifers, on the exchange of pollination services for the rearing of pol- cystoliths, paired in¯orescences in leaf axils, and unisexual linator offspring. The remarkable speci®city and complexity ¯owers. Urticaceae plus Cecropiaceae are sister to Moraceae, of the ®g/pollinator interaction is widely appreciated and has distinguished from the latter in having lactifers only in the often served as a model for the study of coevolution (reviewed bark, clear latex, and orthotropous ovules (Sytsma et al., in Weiblen, 2002; Cook and Rasplus, 2003; Jousselin et al., 2002). 2003). However, the origin of the ®g in¯orescence has re- Moraceae are characterized by milky latex in all parenchy- mained a mystery because its unusual morphology is not easily matous tissue, unisexual ¯owers, anatropous ovules, and ag- related to other Moraceae in¯orescences (Fig. 1). Different gregated drupes or achenes. Growth forms include trees, evolutionary pathways to the syconium have been suggested shrubs, hemiepiphytes, climbers, and herbs. Flowers are re- based on the diversity of in¯orescences in the family. Corner duced and, when present, the perianth is four- or ®ve-merous, (1978) speculated that the ®g evolved from an urn-shaped re- tepaloid, and often membranous. Filaments are either in¯exed ceptacle resembling or Sparattosyce (Fig. 1), while in bud or straight. In¯exed , often referred to as ``ur- Berg (1989) hypothesized a cymose ancestor. Recent molec- ticaceous,'' are associated with a pistillode against which the ular studies suggested a close relationship with anthers are appressed in bud. These stamens, springing out- (Herre et al., 1996) or (Sytsma et al., 2002). However, ward at anthesis to release their pollen, are indicative of wind 1 Manuscript received 7 August 2003; revision accepted 8 January 2004. pollination (Corner, 1962; Berg, 2001). Straight ®laments are For assistance, advice, and specimens the authors thank C. Berg, V. Borla, often but not always associated with pollination involving in- G. Bush, J. Chave, W. Clement, J. Feldcamp, S. Goodwin, D. Huebert, M. sects. The perianths of carpellate ¯owers are often connate or Janda, E. Jousselin, T. Jaffre, S. Mathews, V. Novotny, D. Middleton, P. Nu- adnate to the receptacle (Berg, 2001), a condition hypothesized nez, S. Mori, C. Niezgoda, A. Small, W. Takeuchi, E. Wood, and N. Zerega. to protect ¯owers against phytophagous insects (Berg, 1989, We also acknowledge the US National Science Foundation, US Drug Enforce- ment Administration, National Research Institute of Papua New Guinea, For- 1990). est Research Institute of Papua New Guinea, Parataxonomist Training Center, Moraceae have been divided into ®ve tribes (Table 1; Roh- Organization for Tropical Studies, Centre National de la Recherche Scienti- wer, 1993). Ficeae are monotypic with a pantropical distribu- ®que, National Museum of Natural History, Smithsonian Tropical Research tion and ϳ750 species. can be either monoecious with Institute, Field Museum of Natural History, Harvard University Herbaria, Na- bisexual in¯orescences or gynodioecious but functionally di- tionaal Herbarium Nederland (Leiden), and the Supercomputing Institute at the University of Minnesota. This research was funded by NSF grant DEB oecious (Weiblen, 2000). Artocarpeae are represented by 12 0128833. genera and 87 species, including the economically important 2 E-mail: [email protected]. (jackfruit, breadfruit). Species are either monoe- 767 768 AMERICAN JOURNAL OF BOTANY [Vol. 91

Fig. 1. In¯orescences and infructescences of Moraceae. (A) bi¯ora; solitary carpellate ¯owers. (B) af®nis, racemose infructescence. (C) brasilensis infructescences. (D) Artocarpus heterophyllus, carpellate (right) and staminate (left) in¯orescences. (E) Ficus dammaropsis Diels, cross- section through syconium showing the involucral bracts (ostiole). (F) hildebrantii, bisexual in¯orescence with marginal bracts. (G) rubes- cens, bisexual in¯orescences. (H) guianensis, staminate in¯orescences with involucral bracts surrounding developing ¯owers. (I) krukovii developmental series from carpellate in¯orescence (top left) to infructescence (bottom right). (J) Sparattosyce dioica; carpellate in¯orescence (bottom) and infructescence (top). (K) Antiaropsis decipiens; carpellate in¯orescence with involucral bracts surrounding carpels. (L) Antiaropsis decipiens; staminate in¯o- rescence, top view. (M) Antiaropsis decipiens, infructescence developmental series. Scale bars are 1 cm unless noted. May 2004] DATWYLER AND WEIBLENÐMORACEAE PHYLOGENY 769

TABLE 1. Classi®cation, species richness, and distribution of Moraceae according to Rohwer (1993).

Tribe Species Distribution Artocarpeae R.Br. Antiaropsis K.Schum. 1 New Guinea Artocarpus J.R.Forst. & G.Forst. ϳ50 Asia & Indo-Paci®c Aubl. 1 Neotropics H.Karst. 4 Neotropics Clarisia Ruiz & Pav. 3 Neotropics Hullettia King ex Hook.f. 2 SE Asia Baill. 3 Indo-Paci®c Poulsenia Eggers 1 Neotropics Prainea King 4 Indo-Paci®c Sorocea St. Hil. 14 Neotropics Sparattosyce Bur. 1 New Caledonia Decne. ex TreÂcul 3 Afrotropics Castilleae C.C.Berg Lesch. 1 Paleotropics Castilla Sesse in Cerv. 3 Neotropics TreÂcul 7 Neotropics Aubl. 5 Neotropics Engl. 1 Afrotropics Naucleopsis Miq. ϳ20 Neotropics Perebea Aubl. 9 Neotropics TreÂcul ϳ9 Neotropics Dorstenieae Gaudich. Bosqueiopsis Wildem. & Th.Dur. 1 Afrotropics Brosimum Sw. 13 Neotropics Dorstenia L. ϳ105 Afrotropics & Neotropics Helianthostylis Baill. 2 Neotropics Baill. 2 Afrotropics Thouars 1 Afrotropics Trymatococcus Poepp. & Endl. 3 Neotropics Utsetela Pellegr. 1 Afrotropics Ficeae Gaudich. Ficus L. ϳ750 Pantropical Moreae Gaudich. Bleekrodea Blume 3 Madagascar & SE Asia L'HeÂr. ex Vent. 8 Madagascar & SE Asia Gaudich. 3 Asia, Australia & Madagascar Maclura Nutt. 11 Pantropics & North America Milicia Sim 2 Afrotropics L. ϳ12 Cosmopolitan Lour. ϳ25 SE Asia & Africa P.Browne 9 Neotropics & SE Asia cious or dioecious, with unisexual in¯orescences of variable relatively simple racemes, spikes, or globose heads. Most spe- architecture including racemes, spikes, capitula, globes, discs, cies have urticaceous stamens, apparently related to anemo- and solitary ¯owers (Jarrett, 1959; Berg, 1988). Berg (1988, phily. However, there is a great deal of variation in vegetative 2001) recognized that the Artocarpeae lack the homogeneity and ¯oral morphology, and generic delimitation is somewhat of other Moraceae tribes and suggested that it might be sub- uncertain (Berg, 2001). Up to 40 generic names have been divided into three subtribes on account of the morphological recognized at one time or another in this group (Berg, 2001). variation. Several genera have been enlarged in an attempt to devise a Castilleae include eight genera (Table 1) distributed more satisfactory and cohesive classi®cation (Corner, 1962, throughout the neotropics and to a limited extent in tropical 1975; Berg, 1982, 1986, 1988, 2001). It has been suggested Africa. The tribe has unisexual, discoid to urceolate recepta- that taxonomic uncertainty within Moreae stems from the re- cles with involucrate bracts in most species, the presence of tention of plesiomorphic features (Berg, 1989, 2001). septate wood ®bers, and self-pruning branches (Berg, 1972, We examined Moraceae phylogenetic relationships, repro- 1977a, 2001). Species are monoecious, dioecious, or andro- ductive character evolution, and the origin of the ®g pollina- dioecious (Berg, 1972, 1977a, 2001). tion mutualism. We employed sequences from the chloroplast The eight African and neotropical genera of Dorstenieae are gene ndhF because it has proven useful for examining ordinal dominated by ϳ100 species of Dorstenia. The tribe is de®ned and familial relationships among plants (Olmstead et al., 2001; by discoid or globose bisexual in¯orescences with the carpel- Sytsma et al., 2002). Moraceae chloroplast DNA phylogeny late ¯owers often embedded within the receptacle, peltate in- provided insights into (1) tribal relationships, including the ter¯oral bracts, and uncinate hairs in many species. Growth sister group to the ®gs, (2) ancestral changes in breeding sys- form varies from arborescent in most genera to herbaceous or tems and pollination syndromes, (3) ancestral traits associated suffructescent in Dorstenia (Berg and Hijman, 1999). Al- with the origin of the syconium, and (4) molecular divergence though most species are monoecious, dioecy and androdioecy time estimates for the origin of ®g pollination. also occur (Berg, 1988). The eight genera of Moreae include approximately 70 spe- MATERIALS AND METHODS cies. Species are mostly dioecious, although a few species are Eighteen outgroups in four families and 83 taxa representing 33 genera of monoecious with bisexual in¯orescences. In¯orescences are Moraceae were sampled (see Supplemental Data accompanying the online 770 AMERICAN JOURNAL OF BOTANY [Vol. 91 version of this article). Although 89% of Moraceae genera were sampled, of Ficeae ϩ Castilleae. The log likelihood difference of the empirical data Bosquieopsis, Hullettia, Scyphosyce, and Treculia were not included because yielded the probability of obtaining this clade in error. fresh material was unavailable and DNA extraction from herbarium material was unsuccessful. DNA extraction was performed using the Qiagen DNeasy Character evolutionÐThe following four reproductive traits were scored plant extraction kit (Valencia, California, USA) from 10±15 mg of silica-gel for each taxon based on information from the taxonomic literature, herbarium preserved leaf fragments or herbarium specimens. Polymerase chain reaction specimens, and observations in the ®eld. Pollination was scored as (0) ane- (PCR) ampli®cation of a 2100 bp fragment was performed in two reactions mophilous or (1) entomophilous. Taxa with ``urticaceous'' stamens were of ϳ1200 bp each using primers from Olmstead and Sweere (1994) including scored for wind pollination because no species with this morphology are ndhF8f, ndhF972, ndhF1318r, and ndhF2110r. Ampli®cation conditions were known to be insect pollinated. Carpellate in¯orescences were scored as (0) as follows: ϳ20 ng genomic DNA, 1ϫ TaKaRa Ex Taq buffer (2 mmol/L lacking an involucre of bracts around the carpels or (1) with an involucre of

MgCl2), 160 nmol/L each primer, 0.2 mmol/L each dNTP, 1.25 unit TaKaRa bracts surrounding carpels. Breeding system was scored as (0) monoecious, Ex Taq DNA polymerase (Otsu, Shiga, Japan). Thermal cycling was per- (1) dioecious, (2) androdioecious, or (3) gynodioecious. Monoecious, andro- formed in 25 cycles of 94ЊC for 30 s, 48ЊC for 60 s, 68ЊC for 90 s, and a dioecious, and gynodioecious plants were further scored for (0) unisexual ®nal extension at 72ЊC for 7 min. The PCR products were cleaned using the in¯orescences or (1) bisexual in¯orescences. Ancestral state reconstruction QIAquick or MinElute PCR puri®cation spin columns (Qiagen, Valencia, Cal- was performed on the maximum likelihood tree using MacClade version 3.0 ifornia, USA). Clean PCR products were quanti®ed using Hoechst 33258 ¯uo- (Maddison and Maddison, 2000). rescent dye (Acros Organics, Morris Plains, New Jersey, USA) in a Turner Correlated change in pollination syndrome, breeding system, and the pres- Quantech ¯uorometer (Barnstead-Thermolyne, Dubuque, Iowa, USA). Ten- ence of an involucre of bracts around the carpels on a randomly resolved ML microliter sequencing reactions were performed with Big Dye sequencing re- tree was examined using the concentrated changes test (Maddison, 1990). agents and protocols (versions 2, 3; Applied Biosystems, Foster City, Cali- Correlated evolution was also examined in a likelihood framework using Dis- fornia, USA) and data were collected using an ABI 377 Automated DNA crete (Pagel, 1994, 1997). Omnibus tests evaluated the hypothesis of corre- sequencer (Applied Biosystems). Between seven and nine sequencing primers lated evolution in two characters by examining the ®t of an independent model were used for each taxon, including the aforementioned primers, ndhF972r, of character evolution to a dependent model. Likelihood tests were conducted ndhF1318, and ndhF1603r. In addition, we designed two primers for Urti- on a pruned data set of only species for which pollination syndrome is known cales, ndhF84f (5Ј-TCT TCG CCG TAT AGT GGG TTT TTC C-3Ј), and (63 of 102 taxa) because pollination syndromes are unknown for many spe- ndhF713r (5Ј-ATC RGG TAA CCA TAC ATG AAG RGG-3Ј). Sequences cies. Branch lengths were calculated using likelihood parameters estimated on were edited using Sequencher version 3.0 (Gene Codes, Ann Arbor, Michigan, the ML tree. USA). Sequence alignment was approximated with ClustalX (Thompson et al., 1997), followed by manual alignment to maintain open reading frames Molecular datingÐA likelihood ratio (LR) test compared the likelihood of for the entire portion of the gene. the data with and without the assumption of a molecular clock (Felsenstein, ϭ Ϫ ␹2 1988), where LR 2 (lnLclock lnLno clock) was assumed to be distributed Parsimony analysesÐTree searches were performed using PAUP* version with the degrees of freedom equal to n taxa minus two. Because rates of 4.0b10 (Swofford, 2002) with the tree-bisection-reconnection (TBR) branch- evolution varied greatly across Moraceae, we performed dating procedures swapping algorithm and 10 000 random addition sequence replicates. Max- using a penalized likelihood approach (Sanderson, 2002) as implemented in trees was set to increase without limit. Support was assessed with 1000 boot- the program r8s version 1.6 (Sanderson, 2003). Penalized likelihood is a sem- strap replicates (10 addition sequence replicates per bootstrap replicate) and iparametric method that allows substitution rates to vary among lineages ac- maxtrees set at 10 000. The decay index (Bremer, 1988, 1994) was assessed cording to a smoothing parameter. The optimal smoothing parameter was cho- using TreeRot version 2 (Sorenson, 1999) with 20 random addition sequences sen by means of a data-driven cross validation procedure in which taxa were per replicate and maxtrees set at 10 000. All clade support and tree length sequentially pruned from the tree. The r8s program calculated the parameter ␹2 calculations were conducted with uninformative characters excluded. estimate that best predicted the removed data (i.e., minimized the error). Cross-validation and initial dating were performed with the age of the root Likelihood analysesÐModeltest (Posada and Crandall, 1998) was used to node ®xed at one. Nodes in the resulting ultrametric tree were constrained identify the best ®tting model of sequence evolution with the fewest additional with the minimum ages of Moraceae fossils including Ficus achenes aged at parameters. Tree-bisection-reconnection branch swapping was performed un- 60 mya, Morus leaves dated at 40 mya, and Moraceae fruits dated at 90 mya der the likelihood criterion using PAUP* 4.0b10 on a starting tree generated (Collinson, 1989). The root node of the tree was also set to a maximum age by neighbor joining with the parameters obtained from Modeltest. After the of 135 mya based on the oldest known angiosperms fossil (Magallon et al., search located the tree with the highest likelihood, model parameters were 1999). estimated for that tree. These parameters set a new round of branch swapping on the maximum likelihood (ML) tree. Branch swapping and parameter es- RESULTS timation were iterated until analyses converged on the same likelihood score The aligned data set included 2089 base pairs (bp) of which and model parameters. 652 positions were parsimony-informative. Parsimony recov- ered 11 057 trees of 2003 steps (CI ϭ 0.518; RI ϭ 0.844). Parametric bootstrappingÐParticular phylogenetic hypotheses were also There was strong support for a Moraceae clade in the bootstrap evaluated by parametric bootstrapping (Huelsenbeck and Hillis, 1996). We consensus trees (Fig. 2). Cecropiaceae plus Urticaceae clade focused on support for the monophyly of Ficeae plus Castilleae. Parsimony was strongly supported, but the Cecropiaceae were paraphy- searches were performed in which Ficeae plus Castilleae were constrained to letic due to the position of Poikilospermum. Moreae comprised be non-monophyletic. Likelihood parameters and branch lengths were esti- mated from one of the parsimony trees resulting from the reverse constraint a paraphyletic grade, with a lineage including Morus, Milicia, search. The tree postulating the non-monophyly of Ficeae plus Castilleae and Streblus, and Trophis that was sister to Artocarpeae, while the its likelihood parameters were used to simulate 100 replicate data sets using remaining Moreae were more closely related to Dorstenieae. SEQ-GEN (Rambaut and Grassly, 1997). Maximum parsimony trees were The sister-group relationship of the Artocarpeae plus Moreae inferred for each simulated data set, and ϪlnL scores given the aforemen- sensu stricto had relatively weak support (59% bootstrap). Tro- tioned parameters were compared between an MP tree and the tree used to phis and Streblus were polyphyletic, with some species more simulate the data. The log-likelihood difference of the constrained (null) and closely related to Artocarpeae and others more closely related unconstrained (best) topologies provided a distribution of the test statistic to Dorstenieae. The position of Maclura and Streblus smithii under the null hypothesis that systematic error accounted for the monophyly was not resolved. May 2004] DATWYLER AND WEIBLENÐMORACEAE PHYLOGENY 771

Fig. 2. Strict consensus of 11 057 most parsimonious trees. Numbers above branches refer to bootstrap percentage/decay index. Classi®cation follows Rohwer (1993) with outgroup families and ingroup tribes indicated in brackets. Taxa in boldface type indicate genera for which tribal placement is revised. 772 AMERICAN JOURNAL OF BOTANY [Vol. 91

Artocarpeae were polyphyletic. A core group included Ar- tween bracts and insect pollination was signi®cant according tocarpus, Prainea, Parartocarpus, Clarisia, and Batocarpus, to the omnibus test (LR ϭ 17.0; df ϭ 4; P Ͻ 0.05). while Sorocea and Bagassa grouped with the Moreae sensu The concentrated changes test indicated that changes to stricto. Three genera previously attributed to Artocarpeae monoecy were not signi®cantly concentrated in wind-pollinat- (Sparattosyce, Antiaropsis, Poulsenia) were embedded in a ed lineages (P ϭ 0.11), when breeding system was considered paraphyletic Castilleae. With the inclusion of these genera in to be dependent on pollination syndrome. Likewise, changes a broadened Castilleae, the tribe was sister to Ficus with 88% to wind pollination were not signi®cantly concentrated in mon- bootstrap support (Fig. 2). Parametric bootstrapping indicated oecious lineages (P ϭ 1.00). The omnibus test indicated an that the probability of recovering the Castilleae plus Ficus overall association between breeding system and pollination clade in error was less than 0.01. Not surprisingly, Ficus was syndrome (LR ϭ 14.66; df ϭ 4; P Ͻ 0.05). However, models monophyletic with 100% bootstrap support. restricting the probability of change in pollination syndrome Modeltest identi®ed a model with equal substitution rates to be dependent on the state of the breeding system (LR ϭ for transitions and unequal rates for transversions and a pa- 0.4; df ϭ 1), and vice versa (LR ϭ 0.6; df ϭ 1), were not rameter for heterogeneity in rates of substitution across sites signi®cantly more likely than the independent model. (TVM ϩ⌫; Posada and Crandall, 1998) as the most appro- priate model of sequence evolution with the fewest additional Molecular datingÐEven with a parameter-rich model of parameters. After three iterations, the analysis converged on nucleotide substitution, the data strongly rejected the assump- the same likelihood tree and parameter estimates. Branch tion of a molecular clock (LR ϭ 709.42; df ϭ 100; P Ͻ swapping yielded two equally likely trees with a score of ϪlnL 0.001). Therefore, a penalized likelihood analysis assuming 17 928.474. The rate matrix for the tree shown in Fig. 3 was nonclock-like evolution was performed. Cross-validation anal- 1.69381, 2.02679, 0.39974, 1.17943, 2.06549, with ␣ϭ ysis estimated the optimal smoothing parameter at 1.12. Based 0.688340. Base frequencies were A ϭ 0.303690, C ϭ on a maximum age constraint of 135 mya for the root of Ur- 0.142979, G ϭ 0.159156, and T ϭ 0.394175. The topology ticalean rosids and minimum age constraints for Ficus (60 of the two trees differed only with respect to the position of mya), Morus (40 mya), and Moraceae (90 mya), the minimum the Bleekrodea madagascariense plus Streblus elongatus divergence for the stem lineage of Ficeae plus Castilleae was clade. ϳ83 mya. These estimates indicated that Moraceae diverged at least 99.4 mya while the Cecropiaceae plus Urticaceae di- Character evolutionÐEqually weighted ancestral state re- verged at least 98 mya, which is consistent with fossil Urti- construction indicated that dioecy is ancestral in Moraceae, caceae fruits dated at 90 mya (Collinson, 1989). with between two and four shifts to monoecy (Fig. 4). Within the Dorstenieae, Helianthostylis represented a single origin of DISCUSSION androdioecy. Ficus showed at least one origin of gynodioecy. In Ficus and Dorstenieae, monoecious species have bisexual Phylogenetic analyses based on ndhF sequence data support in¯orescences, whereas in Artocarpeae and Castilleae, mon- the monophyly of Moraceae with the exclusion of Cecropi- oecious species have unisexual in¯orescences. aceae. Moraceae ndhF sequences also con®rm that ®g polli- Wind pollination was likely the ancestral condition of Urt- nation evolved only once, as suggested by other chloroplast icales (Fig. 4). Although the pollination biology of many Mor- genes and mitochondrial DNA from ®g pollinators (Herre et aceae is not known, there appear to have been at least two al., 1996; Machado et al., 2001). Previous studies based on independent origins of entomophily, in the paleotropical Ar- limited sampling of Moraceae suggested that some members tocarpeae and in the Castilleae plus Ficus clade. Many genera of Castilleae are close relatives of the ®gs (Herre et al., 1996; have morphologies suggestive of insect pollination, but obser- Sytsma et al., 2002). We demonstrate for the ®rst time that the vations of ¯oral visitors are lacking, particularly in Dorsten- entire Castilleae plus some Artocarpeae are sister to Ficus, and ieae and Sorocea. Ancestral state reconstruction suggests that we propose taxonomic changes in accord with these ®ndings. insect pollination preceded the divergence of Castilleae and Ficus (Fig. 4). Taxonomic implicationsÐWe present a new classi®cation An involucre of bracts surrounding the developing carpels of Moraceae to re¯ect the evolutionary relationships of the also occurs in Castilleae and Ficus (Fig. 4). The involucre tribes (Appendix; see Supplemental Data accompanying the encircles the developing ¯owers in Castilleae, and the bracts online version of this article). Minor revisions in the placement are pushed apart as the receptacle expands. By contrast, the of particular genera result in four monophyletic tribes. Moreae involucre in Ficus forms an ostiole that limits access to the remain paraphyletic in our classi®cation pending more detailed ¯owers, traps most pollinators inside of the syconium, and sampling of the group, which encompasses extreme morpho- seals the infructescence as well. Outside of the Castilleae plus logical heterogeneity and is recognized on the basis of ple- Ficus clade, involucral bracts surrounding the carpels are pre- siomorphic features of Urticalean rosids, including in¯exed sent only in Trophis caucana (Pittier) C.C.Berg. A few Dor- stamens and wind pollination (Sytsma et al., 2002). Our results stenieae and Artocarpeae have small bracts associated with the point to a Moreae sensu stricto (Figs. 3±4) but further study receptacle but these do not enclose the ¯owers at any stage of of Moreae genera is warranted. Trophis and Streblus are es- development. pecially problematic and have been divided into many mono- The evolution of involucral bracts was not signi®cantly con- typic or oligotypic genera by various authors. These genera centrated in insect-pollinated lineages (P ϭ 0.50), despite the display tremendous variation in growth form, breeding system, perfect correlation of entomophily and bracts in the Castilleae and in¯orescence morphology. Berg (1988) broadly circum- plus Ficeae clade. The concentrated changes test is sensitive scribed these genera based on the presence of free tepals in to limited numbers of evolutionary events, in this case, a single Streblus vs. connate tepals in Trophis. Streblus is further di- origin of involucral bracts. By contrast, the correlation be- vided into ®ve sections, two of which are monotypic. We sam- May 2004] DATWYLER AND WEIBLENÐMORACEAE PHYLOGENY 773

Fig. 3. One of two trees equaling ϪlnL 17 928.48. The revised tribal classi®cation of Moraceae is indicated in brackets. The arrow indicates the difference between the two equally likely trees. The tree not shown places Bleekrodea madagascariense plus Streblus elongatus as the sister group to Dorstenieae. 774 AMERICAN JOURNAL OF BOTANY [Vol. 91

Fig. 4. Breeding system (left) and pollination syndrome (right) mapped onto the ML tree shown in Fig. 3. For breeding system, closed circles and open circles indicate bisexual in¯orescences and unisexual in¯orescences, respectively, for monoecious, androdioecious, and gynodioecious species. On the pollination syndrome reconstruction, closed circles indicate the presence of involucral bracts surrounding the carpels at anthesis. May 2004] DATWYLER AND WEIBLENÐMORACEAE PHYLOGENY 775 pled three representatives of sect. Paratrophis and found that ily (Fig. 4). Functional dioecy in Ficus, resulting from the S. pendulinus and S. glaber are sister, while the position of S. interaction of gynodioecious in¯orescences with pollinating smithii remains uncertain. In addition, only sect. Sloetia was seed eaters, appears to have evolved at least twice with several represented in our analysis by S. elongatus, which is sister to reversals to monoecy (Weiblen, 2000; Jousselin et al., 2003). Bleekrodea. Even with limited sampling, our results suggest Androdioecy, a very rare breeding system in angiosperms the disintegration of the genus. (Renner and Ricklefs, 1995), has been documented in Castilla Trophis is divided into six sections, four of which are mono- (Castilleae) and Helianthostylis (Dorstenieae), indicating two typic. Trophis racemosa (sect. Trophis) and T. involucrata origins within Moraceae. Androdioecy and gynodioecy might (sect. Echinocarpa) are a part of the Moreae sensu stricto but be more common in the family but incomplete collections T. scandens (sect. Malaisia) is more closely related to Brous- make this assessment dif®cult. sonetia and Dorstenieae. As with Streblus, the non-monophyly of this genus is not surprising given its morphological com- Pollination syndrome evolutionÐAn apparently plesiom- plexity, and further investigation is warranted. orphic condition in Moreae is the presence of stamens that are Artocarpeae as circumscribed by Rohwer (1993) is poly- in¯exed in bud, generally associated with a pistillode. As the phyletic, and we recognize a more restricted Artocarpeae by ®lament elongates, the anthers are oriented along the pistillode excluding ®ve genera from the tribe. Antiaropsis, Poulsenia, in bud such that the stamens spring back explosively and re- and Sparattosyce are transferred to Castilleae, and Bagassa lease pollen into the air as the ¯ower opens. This adaptation and Sorocea are transferred to Moreae sensu stricto (Appen- to wind pollination is common in the Urticaceae. Some mem- dix). This delimitation is strongly supported by ndhF (Fig. 2) bers of Moreae have straight ®laments in bud, including Ba- and nuclear ribosomal DNA (G. D. Weiblen, unpublished data) gassa, Sorocea, and Maclura sect. Cudrania, suggesting one and is not contradicted by morphology (Fig. 4). All genera in or two losses of ``urticaceous stamens'' in the Moreae sensu the revised Artocarpeae have a reduction in number lato. Bagassa has pendant, staminate in¯orescences that are (four stamens) and have large seeds that lack endosperm. Al- often associated with anemophily, but Sorocea has no obvious though Hullettia and Treculia were not sampled, we include features of either wind- or insect-pollination. Pluricellular tri- these genera in the revised Artocarpeae based on morphology. chomes on the carpellate in¯orescences exude a nutritious sub- There is moderate support for the Dorstenieae based on strate that can serve as a medium for fungal mycelium. This ndhF sequences although the position of Trilepisium remains exudate and the associated fungus may serve as a reward for uncertain (Fig. 2). African Scyphosyce and Bosqueiopsis were pollinators (Berg, 2001). unavailable, and the inclusion of these genera might in¯uence Many species of Dorstenieae are hypothesized to be insect relationships in the tribe. We expect, however, that they are pollinated based on ¯oral structure and scents, and beetles closely allied with Dorstenieae based on morphology. Many have been observed visiting some African species (Berg and closely allied species of Moreae and Dorstenieae are monoe- Hijman, 1999). However, seed set in the absence of pollinators cious with bisexual in¯orescences. Dorstenieae are distin- has been recorded in Dorstenia (Berg and Hijman, 1999). In guished by the presence of one to several carpellate ¯owers Trilepisium, staminate in¯orescences with a strong odor are embedded in the receptacle and surrounded by staminate ¯ow- known to attract beetles that may breed in the in¯orescences ers. (Berg, 1977b). Among the neotropical Dorstenieae, Brosimum Sequence data support the inclusion of Sparattosyce, Antia- alicastrum has been reported to produce clouds of pollen from ropsis, and Poulsenia in a broadened Castilleae, characterized staminate in¯orescences suggestive of wind pollination (Berg, by self-pruning branches (Berg, 1977a) and unisexual in¯o- 2001). However, visitation by ``small diverse insects'' has also rescences with involucral bracts. Interestingly, these three gen- been reported (Bawa et al., 1985; Kress and Beach, 1994), and era were the only members of the Artocarpeae sensu Rohwer other authors suggest that in¯orescences of Brosimum are (1993) with involucrate in¯orescences. Berg (1990) hypothe- adapted for insect pollination (Croat, 1978; Berg, 1990). sized that both involucral and inter¯oral bracts represent ad- In Artocarpus integer, pollination appears to be mediated aptations preventing insects from feeding on the developing by gall midges feeding on fungal parasites of staminate in¯o- carpels and stamens. The early development of the involucre rescences (Sakai et al., 2000). Gall midges feed on an exudate prior to ¯oral differentiation is consistent with a protective from the mycelium of the fungus and breed in the mycelium. role, although ¯owers are not completely enclosed at receptiv- Insects may be attracted to the in¯orescences by a strong, ity or anthesis in many Castilleae. These bracts are free in sweet smell that is emitted at night (Sakai et al., 2000). In the most species but are fused along most of their length in Spar- process of feeding and breeding on staminate in¯orescences, attosyce, resembling an urn-shaped receptacle. insects pick up sticky pollen grains and occasionally transport them to carpellate in¯orescences. Gall midges have been ob- Breeding system evolutionÐContrary to the traditional served at low frequency on carpellate in¯orescences, possibly view that dioecy evolves from monoecy (Renner and Ricklefs, attracted by the ¯oral fragrance that is emitted. However, the 1995; Weiblen et al., 2000), our results suggest that dioecy is fungus on which midges feed is not found on carpellate in¯o- the ancestral condition in Moraceae and that monoecy has rescences, and short visits by gall midges to these in¯ores- evolved independently between two and four times in the fam- cences suggest pollination by deceit. Fungal growth may be ily. However, if gains of dioecy are assigned twice the cost of limited by the structure of the Artocarpus in¯orescence. The losses, then monoecy is reconstructed as the ancestral condi- perianths of adjacent ¯owers fuse to form an exterior surface tion under parsimony. Monoecious lineages are neatly divided through which the stigmas protrude through a small opening into those with unisexual in¯orescences (Artocarpeae and Cas- at the apex. This structure may limit entry of phytophagous tilleae) and those with bisexual in¯orescences (Dorstenieae insects to the delicate tissue of the gynoecium (Berg, 1990). and Ficeae). Based on these differences in in¯orescence ar- We hypothesize that, in contrast to the paleotropical Artocar- chitecture, monoecy may have evolved four times in the fam- peae, the pendulous, catkin-like staminate in¯orescences of the 776 AMERICAN JOURNAL OF BOTANY [Vol. 91 neotropical genera are adapted to wind pollination. On the oth- the origin of the stem lineage. It is noteworthy that Machado er hand, some authors allude to wind pollination in Artocarpus et al. (2001) based their divergence time estimates on an anal- and nothing is known of syndromes in closely allied Parar- ysis in which taxa with nonclock-like substitution rates were tocarpus and Prainea (Jarrett, 1959; Berg, 2001). Field ob- discarded from a group that demonstrates a great deal of rate servations are needed to test these predictions based on phy- heterogeneity. Furthermore, the analysis was calibrated with a logeny. fossil ®g wasp dated at 18 mya. Dates based on clock-like Berg (1990) hypothesized that the involucral bracts of Cas- substitution rates are most accurate near the calibration point tilleae may be an adaptation to prevent phytophagous insects and tend to overestimate the timing of ancient events (Arbo- from feeding on the ¯owers. Pollination syndrome is unknown gast et al., 2002). Although Ficus appears to have a Cretaceous for about half of the genera of Castilleae, but thrips pollination origin, the crown radiation of Ficus may have occurred more has been reported in Castilla, Antiaropsis, Naucleopsis, Per- recently than suggested by Machado et al. (2001). Molecular ebea, and Poulsenia (Sakai, 2001; G. D. Weiblen, unpublished biogeographic studies that integrate knowledge of ®gs, polli- data). The same species of thrips, Frankliniella diversa (Thri- nators, and their relatives are the next logical step in this line pidae; Thysanoptera), has been observed visiting Castilla and of investigation. Poulsenia (Sakai, 2001). Thrips pollination appears to be as- sociated with insects breeding in staminate in¯orescences. Pol- ConclusionsÐThe origin of the ®g has not ®gured in the len serves as the primary food source for both nymphs and extensive literature on coevolutionary complexity of the ®g/ adults. Females also visit carpellate in¯orescences, but for pollinator interaction (reviewed in Cook and Rasplus, 2003; shorter periods of time. Similar to the situation in Artocarpus, Weiblen, 2002). Our revised classi®cation of the Moraceae thrips may be attracted to carpellate ¯owers by a ¯oral scent based on ndhF sequences provides a phylogenetic framework that mimics the staminate in¯orescences (Sakai, 2001). Further for understanding the evolution of ®g pollination. The sister investigation of the generality of thrips pollination in the Cas- relationship of Castilleae to Ficus provides molecular corrob- tilleae is needed, but our results indicate that traits associated oration of the view that insect pollination and involucral bracts with pollination in the Castilleae are critical to understanding encircling the ¯owers in a Cretaceous ancestor were likely the evolution of ®g pollination. associated with the origin of the remarkable pollination mu- tualism. However, other aspects of the ®g origin remain a mys- Origin of the ®gÐThe sister relationship of thrips-pollinat- tery. How did the extreme protogyny evolve that established ed Castilleae and Ficus suggests that insect pollination was a the synchrony of ®g and pollinator life cycles? When and feature shared by the common ancestor of these lineages. Max- where did the Agaonidae become ®g specialists? It is hoped imum likelihood analysis of correlated evolution further de- that developmental studies and molecular biogeography may tected a signi®cant association between entomophily and the provide further insights into these questions. presence of involucral bracts that encircle the ¯oral primordia in Ficus and Castilleae (Berg, 1972; Verkerke, 1989). During ®g development, the receptacle expands to accommodate the LITERATURE CITED ¯owers, while the tightly appressed bracts form the ostiole. The involucral bracts of many Castilleae encircle the carpels ARBOGAST, B. S., S. V. EDWARDS,J.WAKELY,P.BEERLI, AND J. B. SLOW- INSKI. 2002. Estimating divergence times from molecular data on phy- and form a ring through which the receptive stigmas protrude. logenetic and population genetic timescales. Annual Review of Ecology Even partial enclosure of the carpels could provide protection and Systematics 33: 707±740. from phytophagous insects and play a role in pollinator spec- BAWA, K. S., S. H. BULLOCK,D.R.PERRY,R.E.COLVILLE, AND M. H. i®city (Berg, 1990). The staminate in¯orescences of Castilleae GRAYUM. 1985. Reproductive biology of tropical lowland rain forest provide breeding sites for pollen-feeding thrips, which are de- trees. II. Pollination systems. American Journal of Botany 72: 346±356. ceived into visiting the carpellate in¯orescences by means of BERG, C. C. 1972. Olmedieae Brosimeae (Moraceae). Hafner, New York, New York, USA. ¯oral scent (Sakai, 2001). In contrast, ostiolar bracts that com- BERG, C. C. 1977a. The Castilleae, a tribe of the Moraceae, renamed and pletely seal the ¯owers (and the fruit) inside the receptacle rede®ned due to the exclusion of the type genus Olmedia from the Ol- serve to trap ®g pollinators. We interpret the precondition of medieae. Acta Botanica Neerlandica 26: 73±82. the syconium as a partially closed, involucrate in¯orescence BERG, C. C. 1977b. Revisions of African Moraceae (excluding Dorstenia, similar to that of the modern Castilleae. The complete enclo- Ficus, Musanga, and Myrianthus). Bulletin du Jardin botanique de sure of the ®g by the ostiole was likely associated with pol- l'Etat, Bruxelles 47: 267±407. BERG, C. C. 1982. The reinstatement of the genus Milicia Sim (Moraceae). lination by specialized, parasitic Hymenoptera and led to the Bulletin du Jardin botanique de l'Etat, Bruxelles 52: 225±229. origin of the obligate mutualism. At present, we can only spec- BERG, C. C. 1986. 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