doi 10.1098/rspb.2000.1418 Phylogenetic relationships, historical biogeography and character evolution of g-pollinating wasps Carlos A. Machado1*, Emmanuelle Jousselin2, Finn Kjellberg2, Stephen G. Compton3 and Edward Allen Herre1 1SmithsonianTropical Research Institute, Apartado 2072, Balboa, Republic of Panama 2CNRS-CEFE, 1919 Route de Mende, 34293 Montpellier Ce¨ dex 5, France 3Centre for Ecology and Evolution, School of Biology, University of Leeds, Leeds LS2 9JT, UK Nucleotide sequences from the cytochrome oxidase I (COI) gene were used to reconstruct phylogenetic relationships among 15 genera of ¢g-pollinating wasps. We present evidence supporting broad-level co- cladogenesis with respect to most but not all of the corresponding groups of ¢gs. Using fossil evidence for calibrating a molecular clock for these data, we estimated the origin of the ¢g^wasp mutualism to have occurred ca. 90 million years ago. The estimated divergence times among the pollinator genera and their current geographical distributions corresponded well with several features of the break-up of the southern continents during the Late Cretaceous period. We then explored the evolutionary trajectories of two char- acteristics that hold profound consequences for both partners in the mutualism: the breeding system of the host (monoecious or dioecious) and pollination behaviour of the wasp (passive or active). The ¢g^ wasp mutualism exhibits extraordinarily long-term evolutionary stability despite clearly identi¢able con£icts of interest between the interactors, which are re£ected by the very distinct variations found on the basic mutualistic theme. Keywords: ¢g wasp ; pollination; biogeography; coevolution; Gondwana; mutualism species, some individuals produce only seed-bearing fruit 1. INTRODUCTION and are functionally female, while others produce only The interaction between ¢gs (Ficus: Moraceae) and ¢g- pollen and pollen-carrying wasp progeny and are func- pollinating wasps (Agaonidae, Chalcidoidea) represents tionally male (Janzen 1979; Wiebes 1979; Kjellberg et al. perhaps the most specialized case of obligate pollination 1987; Patel & Hossaert-McKey 2000). mutualism known (Corner 1952, 1988; Ramirez 1970; The di¡erent breeding systems impose profoundly Janzen 1979; Wiebes 1979; Herre et al. 1996; Herre 1999). di¡erent reproductive consequences on both the host ¢g Host ¢g species are generally pollinated by species- and the pollinator wasp. In the monoecious case, indivi- speci¢c pollinator wasp species. With ca. 750 described ¢g dual female foundresses fertilize the £owers using the species showing a pan-tropical distribution and a variety pollen from their natal tree, thereby realizing male ¢tness of growth habits, both ¢g and wasp species are remark- for their own natal ¢g. Yet they then reproduce at the cost ably diverse. Moreover, even within this diversity, of some of those potential seeds, in£icting costs in both multiple variants on the basic themes of the interaction natal and receptive trees (Herre 1989, 1999; West & exist. This variation profoundly a¡ects the nature of the Herre 1994; Herre & West 1997). In the dioecious case, costs and bene¢ts that each member derives from the sexual functions in the trees are separated. Here, if the mutualism. foundresses enter a `female’ in£orescence, they realize Approximately half of all Ficus species are functionally ¢tness for their natal tree by pollinating £owers that will monoecious with individual in£orescences performing develop seeds, but do not reproduce themselves. Alterna- both female (seed production and dispersal) and male tively, if individual foundresses enter a `male’ in£or- (pollen production and dispersal) functions. In these escence, they are able to reproduce themselves, yet will systems, mated, pollen-bearing, female ¢g wasps (foun- produce no seeds with the pollen of their natal tree dresses) enter the enclosed ¢g in£orescences (syconia), (Wiebes 1979; Kjellberg et al. 1987; Grafen & Godfray pollinate the uniovulate £owers inside, lay eggs in some of 1991; Anstett et al. 1997; Patel & Hossaert-McKey 2000). them and die. Their o¡spring develop by consuming the Furthermore, both active and passive pollination occur contents of one potential seed each, emerge later and across di¡erent species of wasps. These di¡erent pollina- mate. The female o¡spring then gather pollen from male tion syndromes are associated with distinctive morpho- £owers within the syconia and £y o¡ in order to attempt logical adaptations in both the wasp and the ¢g. In to ¢nd a receptive ¢g tree and begin the cycle anew species with active pollination, the wasps possess specia- (Corner 1952, 1988; Galil & Eisikowitch 1968; Ramirez lized structures for carrying pollen in the external part of 1970; Herre 1989, 1999). The remaining Ficus species are the thorax and the front legs (Ramirez 1969) and show gynodioecious, but functionally dioecious. In these distinctive behaviours for collecting and depositing pollen (Frank 1984). The male £owers in actively pollinated ¢gs *Author and address for correspondence: Department of Genetics, are relatively small and less numerous (Galil & Meiri Rutgers University, Nelson Biological Laboratories, 604 Allison Road, 1981). In contrast, wasps that passively pollinate their Piscataway, NJ 08854, USA ([email protected]). hosts lack or present a signi¢cant reduction in the size of Proc. R. Soc. Lond. B (2001) 268, 685^694 685 © 2001 The Royal Society Received 14 July 2000 Accepted 27 November 2000 686 C. A. Machado and others Fig-pollinating wasp evolution the specialized structures found in active pollinators and incorporated three sections of Ficus (Sycocarpus, Neomorphe and the wasps show no pollination behaviour (Galil & Adenosperma) into the subgenus Sycomorus. Here we follow their Neeman 1977). Passively pollinated ¢gs have relatively classi¢cation with the modi¢cation that the palaeotropical higher ratios of anthers to female £owers, produce much section Oreosycea is incorporated into the subgenus Urostigma as more pollen per syconium than actively pollinated ¢gs suggested by molecular evidence (Herre et al. 1996). and their mature anthers tend to dehisce naturally, There are 20 recognized genera of ¢g-pollinating wasps, all thereby facilitating the passive `collection’ of pollen by belonging to the family Agaonidae sensu Rasplus (Rasplus et al. their pollinators (pollen adheres to various parts of the 1998) within the superfamily Chalcidoidea (Wiebes 1982, 1994; body surface) (Ramirez 1969; Galil & Eisikowitch 1971; Boucek 1988; Berg & Wiebes 1992). With the exception of wasps Galil & Neeman 1977; Galil & Meiri 1981; Ramirez & from the genera Ceratosolen, Platyscapa and Wiebesia, each genus is Malavasi 1997). restricted to a single subgenus and section of ¢g. The observation that related species of wasps generally pollinate related species of ¢gs has led to the proposal of (b) DNA methods strict-sense coevolution between the two groups (Ramirez Genomic DNA was extracted from 32 individual wasps repre- 1974; Wiebes 1979, 1982; Berg & Wiebes 1992). However, senting 15 out of the 20 genera of pollinating wasps (table 1) existing classi¢cations of ¢gs and their pollinators are using Chelex 100 (Walsh et al. 1991). The ¢ve genera not based on morphological characters that are often inti- included in this study (Agaon, Allotriozoon, Deilagaon, Nigeriella mately involved in the interactions between the two and Paragaon) are all associated with African ¢gs of the mono- mutualists (e.g. the breeding system of the ¢gs and the ecious subgenus Urostigma. Sequences of 816 nucleotides were characters involved in the pollination behaviour of the collected from the 3’-end of the mitochondrial COI gene (posi- wasps). Therefore, the apparent congruence observed in tions 2191^3007 of the Drosophila yakuba mitochondrial genome) their current classi¢cations might simply re£ect reciprocal (Clary & Wolstenholme 1985) using conserved insect poly- adaptations leading to convergent evolution (e.g. Van merase chain reaction primers and standard manual and auto- Noort & Compton 1996). Fortunately, molecular data can mated sequencing protocols (Simon et al. 1994; Machado et al. provide independent characters for reconstructing phylo- 1996; Machado 1998). Sequences have been deposited in genies and rigorously testing evolutionary hypotheses GenBank (accession numbers AF302052^AF30205 6 and concerning ¢gs and their pollinators. For example, AY014964^AY014995). molecular studies of ¢gs and wasps have been conducted at both ¢ne (between species within a pollinator genus (c) Phylogenetic analyses and their associated hosts) and broad (across genera of All analyses were carried out with version 4.0b1 of PAUP* wasps and their hosts) taxonomic scales and the data (Swo¡ord 1998). Four species from two di¡erent subfamilies of appear consistent with strict-sense coevolution (Herre et non-pollinating ¢g wasps (Sycophaginae, Sycoryctinae) were al. 1996). However, the sampling of taxa in those studies used as outgroups (table 1). Phylogenies were reconstructed was limited and support for many of the proposed rela- using the maximum-likelihood (ML) optimality criterion. The tionships was weak. Recent studies have increased the most appropriate nucleotide substitution model for explaining number of taxa sampled, but have focused on dioecious the process of nucleotide substitution in the data was chosen by ¢gs and their pollinators (Weiblen 1999, 2000, 2001). comparing three models that consider unequal