Keywords

1 Evolution of Pollination by Seed Parasites

The mutualisms between plants and their seed-parasitic pollinators, such as the fig–fig wasp, yucca–yucca moth, and leafflower–leafflower moth mutualisms, provide textbook examples of specialized pollination systems (Weiblen 2002; Cook and Rasplus 2003; Pellmyr 2003; Kato et al. 2003). Remarkably, in all three systems, the pollinator insects actively collect and transport pollen between flowers in order to ensure food for their seed-feeding larvae. Reciprocal adaptation by plants to restrict floral access by other visitors resulted in extreme mutual dependence between plants and insects. Consequently, these mutualisms served as principal model systems for the studies of coevolution and mutualism.

Despite a wealth of documented examples of specialized pollination systems in angiosperms, however, pollination by obligate seed parasites is rare. This is because seed parasitism inflicts a heavy cost on plants, whereas abundant copollinator s swamp the mutualistic effect of pollination by seed parasite s (Thompson and Pellmyr 1992; Thompson and Cunningham 2002). In fact, exclusion of pollinators has not occurred in plants that were more recently found as being pollinated by seed parasites, including Lithophragma plants pollinated by Greya moths (Thompson and Pellmyr 1992), senita cactus pollinated by senita moth s (Fleming and Holland 1998), and Silene plants pollinated by Hadena and Perizoma moths (Kephart et al. 2006). Nevertheless, there are other plant–seed parasite associations that have evolved into reciprocal specialization (globeflower –globeflower fly and Rheum nobile –fungus gnat mutualisms; Jaeger and Després 1998; Song et al. 2014). Understanding of how and under what circumstances obligate pollination mutualism s evolve would thus benefit from exploring the origin of active pollination and mutualism in the Phyllantheae–Epicephala association.

In this chapter, we describe the pollination systems and associations with Epicephala of 26 Phyllantheae species studied during 2002–2007 in Southeast Asia , New Caledonia , Australia , Madagascar , Guinea , and North America (Table 6.1). Based on this information, we explore the origin of the Phyllantheae–Epicephala mutualism using robust molecular phylogenies for 46 species of Phyllantheae and associated Epicephala moths. Although the present analysis focuses on only a small proportion of the global diversity of Phyllantheae, the sampled species cover the entire range of taxonomic diversity within the tribe (Hoffmann et al. 2006; Kathriarachchi et al. 2006), allowing an overview of broad coevolutionary history of the Phyllantheae–Epicephala association. Overall, the results reveal an unexpectedly complex origin of the Phyllantheae–Epicephala pollination mutualism and provide important general insights into how a combination of evolutionary innovation and partner shifts shapes the evolutionary dynamics of mutualism in coevolving species interactions.

Table 6.1 List of species studied
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2 Diversity of Pollination Systems in Phyllantheae

As detailed in Chaps. 3 and 4, there are five Phyllantheae lineages that are obligately pollinated by host-specific Epicephala moths (Glochidion; Breynia; and New Caledonian Phyllanthus; Phyllanthus section Anisonema; and an unclassified group of Phyllanthus endemic to Madagascar). The remaining species are pollinated by diurnal insects that visit flowers for nectar and pollen, and do not have associations with pollinating Epicephala. However, Flueggea suffruticosa is parasitized by Conopomorpha flueggella, and three herbaceous Phyllanthus species are parasitized by seed-parasitic Epicephala species that do not pollinate the flowers (Chap. 5).

The plants that are not pollinated by Epicephala employ a variety of insects as pollinators. Species of Flueggea have the broadest range of flower visitors, including bee s, flies , beetle s, and butterflies , which all probably contribute to pollination. Although observation is limited, dipteran insects appear to be important pollinators of many other Phyllantheae, such as Phyllanthus flexuosus , P. oligospermus (both subgenus Kirganelia), P. liukiuensis (subgenus Eriococcus), P. roseus (subgenus Phyllanthodendron), Breynia retusa , and Sauropus quadrangularis . Notably, most herbaceous Phyllanthus, which are phylogenetically spread across the entire Phyllantheae phylogeny (Chap. 4), are pollinated by ant s that visit flowers for nectar . In a controlled experiment where only ants were allowed to visit flowers of P. lepidocarpus grown in cages, plants regularly attained full fruit set (Fig. 6.1), whereas they produced no fruits when insects were fully excluded. Because P. lepidocarpus is self-compatible , ants are probably sufficient to pollinate this species fully in wild conditions as well.

Fig. 6.1
figure 1

Ant pollination of Phyllanthus lepidocarpus. (a) An ant, Paratrechina flavipes, consuming nectar on male flower of P. lepidocarpus. (b) P. lepidocarpus pollen attached to the head of Tetramorium sp. (c) Result of selective exclusion experiment in Phyllanthus lepidocarpus. Fruit set of the caged treatment is significantly lower than the fruit sets of other three treatments (Kruskal–Wallis test, χ2 = 40.01, df = 3, P < 0.001). Numbers inside bars are sample sizes. Error bars are too small to be seen

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Whether a plant species is pollinated by Epicephala is most clearly reflected in style morphology. In species pollinated by the moths, styles are reduced and fused to form a narrow apical cavity into which moths insert the proboscis to deposit pollen. By contrast, species diurnally pollinated by various nectar-seeking insects usually have bifid styles that are spread horizontally, which facilitates passive pollen receipt from insect bodies (Fig. 6.2). Overall, species with different pollination syndrome s have nonoverlapping degrees of style spreading (Fig. 6.3); thus, pollination systems can be reliably assigned to plant species for which sufficient ecological data are not available.

Fig. 6.2
figure 2

Floral morphology of Phyllantheae plants with Epicephala (a–h) and non-Epicephala (i–p) pollinators. (a, e) Glochidion acuminatum. (b, f) Breynia vitis-idaea. (c, g) Phyllanthus reticulatus. (d, h) Phyllanthus marojejiensis. (i, m) Flueggea suffruticosa. (j, n) Phyllanthus flexuosus. The arrowheads indicate female flowers. (k, o) Breynia retusa. (l, p) Sauropus quadrangularis. For each species, male flowers are shown above female flowers (Reproduced from Kawakita 2010)

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Fig. 6.3
figure 3

Distribution of style spreading in Phyllantheae, quantified as the ratio of apical to basal style width. Species pollinated by Epicephala (green) have reduced styles that are medially fused, whereas non-Epicephala-pollinated species (blue) have horizontally spread, bifid styles. Filled and empty boxes indicate species with and without associations with Epicephala, respectively. Ecological data were not available for species with asterisks, but because species with different pollination syndromes had nonoverlapping distributions of style spreading, their pollination modes could be assigned reliably. Female flowers are drawn for Phyllanthus marojejiensis, Glochidion acuminatum, Sauropus brevipes, and Flueggea suffruticosa (from left to right). Error bars, ±1 SE (Reproduced from Kawakita and Kato 2009)

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3 Phylogeny of Phyllantheae and Epicephala

To investigate the origin of the Phyllantheae–Epicephala mutualism, pollination systems were mapped onto the molecular phylogeny of 46 Phyllantheae species. The phylogeny is based on the combined chloroplast matK, ndhF, atpB, and nuclear PHYC gene dataset for 92 species of Phyllanthaceae including the above 46 Phyllantheae species. Maximum parsimony, likelihood, and Bayesian analyses all produced a highly resolved and well-supported phylogeny for Phyllantheae (Fig. 6.4). Similarly, the phylogeny of 26 Epicephala species associated with the above Phyllantheae species were reconstructed based on the combined mitochondrial COI, nuclear ArgK, EF-1α, Wg, and the 18S rDNA gene dataset. This produced a well-resolved phylogeny, although the phylogenetic placement of Conopomorpha flueggella with respect to Epicephala remained ambiguous (Fig. 6.5).

Fig. 6.4
figure 4

Bayesian majority consensus cladogram of 46 Phyllantheae species based on sequences of combined plastid matK, ndhF, atpB, and nuclear PHYC genes. Numbers indicate maximum parsimony and likelihood bootstrap values, and Bayesian posterior probability (from top to bottom; shown only when >50). Asterisks indicate maximal nodal support (100 for all three measures)

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Fig. 6.5
figure 5

Bayesian majority consensus cladogram of 25 Epicephala species and related Conopomorpha flueggella based on sequences of combined mitochondrial COI and nuclear ArgK, EF-1α, Wg, and 18S rDNA genes. Numbers indicate parsimony and likelihood bootstrap values, and Bayesian posterior probability (from top to bottom; shown only when >50). Asterisks indicate maximal nodal support (100 for all three measures). Associated host taxonomic groups are given

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These phylogenies provide important insights into the origin of mutualism and active pollination. First, Phyllantheae species pollinated by Epicephala are not monophyletic, indicating that there have been multiple shifts in pollination systems. Reconstruction of ancestral character state s for the pollination system along the Phyllantheae phylogeny suggest that there are five independent origin s of the obligate pollination mutualism in Phyllantheae, with a single reversal to non-Epicephala pollination in Breynia retusa (Fig. 6.6). The pollinator Epicephala species are also nonmonophyletic, and ancestral character state reconstruction indicated a likely single origin of pollination behavior with a single event of secondary loss (Fig. 6.6). Major clades of Epicephala generally have specific associations with well-defined taxonomic groups of Phyllantheae, but relationships at higher levels were largely incongruent, indicating that host shifts have occurred repeatedly (Fig. 6.6).

Fig. 6.6
figure 6

Origin of the Phyllantheae–Epicephala obligate pollination mutualism. Chronograms for Phyllantheae plants (left) and associated Epicephala moths (right). Pie charts indicate the probabilities of Epicephala and non-Epicephala pollination systems (Phyllantheae) or the presence/absence of active pollination behavior (Epicephala) occurring at ancestral nodes. Asterisks indicate significant difference in likelihoods. Mutualism is represented in green, and associations with major plant and moth clades are indicated

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The above analysis of ancestral character state reconstruction indicates that Epicephala-pollinated Phyllantheae plants evolved multiple times independently. However, because the taxon sampling was limited to 46 species amid the global diversity of Phyllantheae (>1200 species), results of ancestral state reconstruction might change with the addition of more taxa. Therefore, divergence time s for the Phyllantheae and Epicephala phylogenies were estimated to test whether the multiple origins hypothesis is in fact the preferred scenario. If the age of the most recent common ancestor of moth-pollinated plants is contemporary to that of Epicephala, a single origin of the mutualism followed by multiple losses would still be a viable hypothesis. Alternatively, evolution of pollinating behavior postdating initial host divergence would provide strong support for the multiple origins hypothesis.

A major obstacle when estimating divergence times is the scarcity of fossil s, which is also the case for Phyllantheae and Epicephala. Nevertheless, there are several fossils of Phyllantheae and plants in other tribes of Phyllanthaceae that can be used to provide minimum age constraints on Phyllanthaceae phylogeny. The fossils used are Bischofia-type pollen from Bartonian , Middle Eocene (37.2 mya); Actephila-type pollen from Late Eocene (33.9 mya); Phyllanthus-type pollen from Early Eocene (48.6 mya) (Gruas-Cavagnetto and Köhler 1992); and Glochidion leaf impression s from Middle Miocene (11.6 mya; Prasad 1994; Antal and Prasad 1996). The root node (i.e., the node splitting Phyllanthaceae and Picrodendraceae) was assumed to be no older than 108 mya, which is the oldest estimate of the corresponding node in a study of Malpighiales radiation (Davis et al. 2005). Because attribution of some of the Phyllanthaceae fossils may still need refinement (Gruas-Cavagnetto and Köhler 1992), caution may be necessary when taking the precise dates resulting from this analysis. Because gracillariid moths are extremely scarce in the fossil record (Lopez-Vaamonde et al. 2006), Epicephala divergence times were obtained assuming a molecular clock of the COI gene. Only the COI clock was used because it is generally conserved across arthropod taxa (Gaunt and Miles 2002), has been widely used for dating in insects (Kandul et al. 2004; Quek et al. 2007; Ueda et al. 2008), and clusters at approximately 1.5% myr−1 in several arthropod groups (Farrell 2001; Quek et al. 2004; Sota and Hayashi 2007).

The analysis of divergence times indicates that the most recent common ancestor of Epicephala-pollinated plants occurred 41.0 mya (95% credibility interval, 39.3–48.3 mya; Fig. 6.6). In contrast, estimated ages of the split between Conopomorpha flueggella and Epicephala clustered within a timeframe between 20 and 30 mya. These estimates for the age of active pollination postdates initial host divergence by roughly 10–20 myr (Fig. 6.6), which is consistent with delayed radiation of Epicephala and hence multiple origins of the obligate pollination mutualism in Phyllantheae. Although the estimate of the timing of Epicephala divergence depends largely on the accuracy of the COI molecular clock, the assumed 1.5% myr−1 is among the slowest of known rates for the arthropod COI gene (1.3–2.3% myr−1; Brower 1994; Quek et al. 2004), and using higher rates would only give younger estimates for the age of the Epicephala root node; thus, the method employed is conservative with respect to providing young ages.

4 Origin of Active Pollination and Mutualism

The above phylogenetic analyses and divergence time estimations allowed a general overview of the evolutionary history of the Phyllantheae–Epicephala association. Because the taxon sampling was limited to 20% of the global diversity of Phyllantheae at the section level (Kathriarachchi et al. 2006) and less than 5% at the species level, the entire picture of the evolutionary history of Epicephala pollination in Phyllantheae is probably much more complex than as depicted here. However, inclusion of other lineages would likely only strengthen the conclusion of repeated independent evolution because these plants generally have bifid, horizontally spread styles that are characteristic of non-Epicephala-pollinated plants (Fig. 6.2). Exceptions are the New World Phyllanthus subgenus Xylophylla, which consists of approximately 60 species having reduced columnar styles (Webster 1958) and section Microglochidion, which consists of approximately 10 species occurring on the tepuis of the Guiana Highlands (Chap. 5). Field observation and examination of herbarium specimens indicate that they are also associated with seed-feeding Epicephala. It is thus tempting to clarify the pollination systems of these plants and phylogenetic positions of associated Epicephala, as they may represent additional origins of Epicephala moth pollination in Phyllantheae.

Our finding that the obligate pollination mutualism arose repeatedly in Phyllantheae is in stark contrast with the situations in the fig–fig wasp and yucca–yucca moth mutualisms. Coevolutionary analyses in the fig and yucca systems indicate that these associations arose only once in each partner lineage 40–60 mya (Pellmyr and Leebens-Mack 1999; Rønsted et al. 2005). An exception is Hesperoyucca whipplei , which is phylogenetically distant from the rest of the yuccas and independently established the mutualism with a yucca moth (Bogler et al. 1995; Pellmyr et al. 2007; Smith et al. 2008a). In the Phyllantheae–Epicephala system, major lineages of Phyllantheae had already emerged when Epicephala colonized these plants ~30 mya. Sequential radiation of Epicephala on an already diverged host lineage has likely provided opportunities for the moth pollinators to establish new mutualistic associations in distant host lineages. Thus, specialization to moth pollination occurred multiple times independently in Phyllantheae as Epicephala spread onto a broad range of the Phyllantheae lineage.

Our results also indicate that colonization of new host lineages by the pollinators sometimes results in a loss of mutualistic traits. A derived clade of Epicephala has completely lost the pollinating behavior after colonizing herbaceous species of Phyllanthus. These plants regularly attain full seed set through ant pollination (Fig. 6.1); thus, time and energetic costs required during pollination probably outweighed the benefit of assuring seed set in these moth lineages. At the same time, effective pollination by ants probably swamped the mutualistic effect of pollination by moths; thus, selection did not favor these Phyllanthus to specialize to moth pollination.

Taken together, the overall evolutionary history of Phyllantheae and Epicephala provides two general implications for the coevolutionary dynamics of mutualisms. First, although species associations are phylogenetically conserved in most coevolving interactions (Thompson 2005), rare shifts by a partner possessing the mutualistic trait can give rise to new mutualisms in phylogenetically distant partner lineages. In this sense, the active pollination behavior in Epicephala has been of critical importance for the establishment and maintenance of the Phyllantheae–Epicephala mutualism and thus represents a key innovation in this association. Second, the outcome of a species interaction can vary greatly depending on the community context in which it occurs (Thompson and Pellmyr 1992; Thompson and Cunningham 2002; Westerbergh 2004); thus, transitions between mutualism and antagonism can occur repeatedly within a single phylogenetic lineage. This parallels findings in other mutualisms where derived parasitic taxa are nested within ancestrally mutualistic clades (Pellmyr et al. 1996b; Machado et al. 2001; Als et al. 2004). Of particular relevance to future studies is our finding that the mutualism arose independently in several Phyllantheae lineages, which provides outstanding opportunities for comparative analyses of character evolution, diversification rates, and factors affecting mutualism establishment and stability.