Macroevolution of the plant-hummingbird pollination system.

Plant-hummingbird interactions are considered a classic example of coevolution, a process in which mutually dependent species influence each other's evolution. Plants depend on hummingbirds for pollination, whereas hummingbirds rely on nectar for food. As a step towards understanding coevolution, this review focuses on the macroevolutionary consequences of plant-hummingbird interactions, a relatively underexplored area in the current literature. We synthesize prior studies, illustrating the origins and dynamics of hummingbird pollination across different angiosperm clades previously pollinated by insects (mostly bees), bats, and passerine birds. In some cases, the crown age of hummingbirds pre-dates the plants they pollinate. In other cases, plant groups transitioned to hummingbird pollination early in the establishment of this bird group in the Americas, with the build-up of both diversities coinciding temporally, and hence suggesting co-diversification. Determining what triggers shifts to and away from hummingbird pollination remains a major open challenge. The impact of hummingbirds on plant diversification is complex, with many tropical plant lineages experiencing increased diversification after acquiring flowers that attract hummingbirds, and others experiencing no change or even a decrease in diversification rates. This mixed evidence suggests that other extrinsic or intrinsic factors, such as local climate and isolation, are important covariables driving the diversification of plants adapted to hummingbird pollination. To guide future studies, we discuss the mechanisms and contexts under which hummingbirds, as a clade and as individual species (e.g. traits, foraging behaviour, degree of specialization), could influence plant evolution. We conclude by commenting on how macroevolutionary signals of the mutualism could relate to coevolution, highlighting the unbalanced focus on the plant side of the interaction, and advocating for the use of species-level interaction data in macroevolutionary studies.

Pollen as the link between floral phenotype and fitness.

Pollen plays a key role in plant reproductive biology. Despite the long history of research on pollen and pollination, recent advances in pollen-tracking methods and statistical approaches to linking plant phenotype, pollination performance, and reproductive fitness yield a steady flow of exciting new insights. In this introduction to the Special Issue "Pollen as the Link Between Phenotype and Fitness," we start by describing a general conceptual model linking functional classes of floral phenotypic traits to pollination-related performance metrics and reproductive fitness. We use this model as a framework for synthesizing the relevant literature, highlighting the studies included in the Special Issue, and identifying gaps in our understanding and opportunities for further development of the field. The papers that follow in this Special Issue provide new insights into the relationships between pollen production, presentation, flower morphology, and pollination performance (e.g., pollen deposition onto stigmas), the role of pollinators in pollen transfer, and the consequences of heterospecific pollen deposition. Several of the studies demonstrate exciting experimental and analytical approaches that should pave the way for continued work addressing the intriguing role of pollen in linking plant phenotypes to reproductive fitness.

Limited reproductive interference despite high rates of heterospecific pollen transfer among co-occurring bat-pollinated Burmeistera.

PREMISE: Many tropical plants are bat-pollinated, but these mammals often carry copious, multispecific pollen loads making bat-pollinated plants susceptible to heterospecific pollen deposition and reproductive interference. We investigated pollen transfer between sympatric bat-pollinated Burmeistera species and their response to heterospecific pollen deposition from each other. METHODS: We quantified conspecific and heterospecific pollen deposition for two populations of B. ceratocarpa, a recipient species in heterospecific pollen transfer interactions, that co-occur with different donor relatives (B. borjensis and B. glabrata). We then used a cross-pollination scheme using pollen mixtures to assess the species' responses to heterospecific pollen deposition in terms of fruit abortion and seed production. RESULTS: Burmeistera ceratocarpa received significantly more heterospecific pollen from its relatives at both sites than its own pollen was deposited on its relatives. However, heterospecific pollen deposition only affected seed production by B. borjensis and B. glabrata, but not by B. ceratocarpa, suggesting that early acting post-pollination barriers buffer the latter against reproductive interference. Crosses between sympatric and allopatric populations suggest that the study species are fully isolated in sympatry, while isolation between allopatric populations is strong but incomplete. CONCLUSIONS: We did not observe evidence of reproductive interference among our study species, because either heterospecific pollen deposition did not affect their seed production (B. ceratocarpa) or they receive heterospecific pollen only rarely (B. borjensis and B. glabrata). Frequent heterospecific pollen deposition might favor the evolution of barriers against foreign pollen (as in B. ceratocarpa) that alleviate the competitive costs of sharing low fidelity pollinators with co-occurring species.

Pollinator type strongly impacts gene flow within and among plant populations for six Neotropical species.

Animal pollinators directly affect plant gene flow by transferring pollen grains between individuals. Pollinators with restricted mobility are predicted to limit gene flow within and among populations, whereas pollinators that fly longer distances are likely to promote genetic cohesion. These predictions, however, remain poorly tested. We examined population genetic structure and fine-scale spatial genetic structure (FSGS) in six perennial understory angiosperms in Andean cloud forests of northwestern Ecuador. Species belong to three families (Gesneriaceae, Melastomataceae, and Rubiaceae), and within each family we paired one insect-pollinated with one hummingbird-pollinated species, predicting that insect-pollinated species have greater population differentiation (as quantified with the FST statistic) and stronger FSGS (as quantified with the SP statistic) than hummingbird-pollinated species. We confirmed putative pollinators through a literature review and fieldwork, and inferred population genetic parameters with a genome-wide genotyping approach. In two of the three species pairs, insect-pollinated species had much greater (>2-fold) population-level genetic differentiation and correspondingly steeper declines in fine-scale genetic relatedness. In the Gesneriaceae pair, however, FST and SP values were similar between species and to those of the other hummingbird-pollinated plants. In this pair, the insect pollinators are euglossine bees (as opposed to small bees and flies in the other pairs), which are thought to forage over large areas, and therefore may provide similar levels of gene flow as hummingbirds. Overall, our results shed light on how different animal pollination modes influence the spatial scale of plant gene flow, suggesting that small insects strongly decrease genetic cohesion.

Increased resolution in the face of conflict: phylogenomics of the Neotropical bellflowers (Campanulaceae: Lobelioideae), a rapid plant radiation.

BACKGROUND AND AIMS: The centropogonid clade (Lobelioideae: Campanulaceae) is an Andean-centred rapid radiation characterized by repeated convergent evolution of morphological traits, including fruit type and pollination syndromes. While previous studies have resolved relationships of lineages with fleshy fruits into subclades, relationships among capsular species remain unresolved. This lack of resolution has impeded reclassification of non-monophyletic genera, whose current taxonomy relies heavily on traits that have undergone convergent evolution. METHODS: Targeted sequence capture using a probe-set recently developed for the centropogonid clade was used to obtain phylogenomic data from DNA extracted from both silica-dried and herbarium leaf tissue. These data were used to infer relationships among species using concatenated and partitioned species tree methods, and to quantify gene tree discordance. KEY RESULTS: While silica-dried leaf tissue resulted in longer assembled sequence data, the inclusion of herbarium samples improved taxonomic representation. Relationships among baccate lineages are similar to those inferred in previous studies, although they differ for lineages within and among capsular clades. We improve the phylogenetic resolution of Siphocampylus, which forms ten groups of closely related species which we informally name. Two subclades of Siphocampylus and two individual species are rogue taxa whose placement differs widely across analyses. Gene tree discordance (including cytonuclear discordance) is rampant. CONCLUSIONS: This first phylogenomic study of the centropogonid clade considerably improves our understanding of relationships in this rapid radiation. Differences across analyses and the possibility of additional lineage discoveries still hamper a solid and stable reclassification. Rapid morphological innovation corresponds with a high degree of phylogenomic complexity, including cytonuclear discordance, nuclear gene tree conflict and well-supported differences between analyses based on different nuclear loci. Together, these results point to a potential role of hemiplasy underlying repeated convergent evolution. This hallmark of rapid radiations is probably present in many other species-rich Andean plant radiations.

Genome-wide ultraconserved elements resolve phylogenetic relationships and biogeographic history among Neotropical leaf-nosed bats in the genus Anoura (Phyllostomidae).

AnouraGray, 1838 are Neotropical nectarivorous bats and the most speciose genus within the phyllostomid subfamily Glossophaginae. However, Anoura species limits remain debated, and phylogenetic relationships remain poorly known, because previous studies used limited Anoura taxon sampling or focused primarily on higher-level relationships. Here, we conduct the first phylogenomic study of Anoura by analyzing 2039 genome-wide ultraconserved elements (UCEs) sequenced for 42 individuals from 8 Anoura species/lineages plus two outgroups. Overall, our results based on UCEs resolved relationships in the genus and supported (1) the monophyly of small-bodied Anoura species (previously genus Lonchoglossa); (2) monotypic status of A. caudifer; and (3) nested positions of "A. carishina", A. caudifer aequatoris, and A. geoffroyi peruana specimens within A. latidens, A. caudifer and A. geoffroyi, respectively (suggesting that these taxa are not distinct species). Additionally, (4) phylogenetic networks allowing reticulate edges did not explain gene tree discordance better than the species tree (without introgression), indicating that a coalescent model accounting for discordance solely through incomplete lineage sorting fit our data well. Sensitivity analyses indicated that our species tree results were not adversely affected by varying taxon sampling across loci. Tree calibration and Bayesian coalescent analyses dated the onset of diversification within Anoura to around âˆ¼ 6-9 million years ago in the Miocene, with extant species diverging mainly within the past âˆ¼ 4 million years. We inferred a historical biogeographical scenario for Anoura of parapatric speciation fragmenting the range of a wide-ranging ancestral lineage centered in the Central to Northern Andes, along with Pliocene-Pleistocene dispersal or founder event speciation in Amazonia and the Brazilian Atlantic forest during the last âˆ¼ 2.5 million years.

Global patterns of population genetic differentiation in seed plants.

Evaluating the factors that drive patterns of population differentiation in plants is critical for understanding several biological processes such as local adaptation and incipient speciation. Previous studies have given conflicting results regarding the significance of pollination mode, seed dispersal mode, mating system, growth form and latitudinal region in shaping patterns of genetic structure, as estimated by FST values, and no study to date has tested their relative importance together across a broad scale. Here, we assembled a 337-species data set for seed plants from publications with data on FST from nuclear markers and species traits, including variables pertaining to the sampling scheme of each study. We used species traits, while accounting for sampling variables, to perform phylogenetic multiple regressions. Results demonstrated that FST values were higher for tropical, mixed-mating, non-woody species pollinated by small insects, indicating greater population differentiation, and lower for temperate, outcrossing trees pollinated by wind. Among the factors we tested, latitudinal region explained the largest portion of variance, followed by pollination mode, mating system and growth form, while seed dispersal mode did not significantly relate to FST . Our analyses provide the most robust and comprehensive evaluation to date of the main ecological factors predicted to drive population differentiation in seed plants, with important implications for understanding the basis of their genetic divergence. Our study supports previous findings showing greater population differentiation in tropical regions and is the first that we are aware of to robustly demonstrate greater population differentiation in species pollinated by small insects.

Floral reorientation: the restoration of pollination accuracy after accidents.

Plants sometimes suffer mechanical injury. The nonlethal collapse of a flowering stalk, for example, can greatly reduce plant fitness if it leads to 'incorrect' floral orientation and thus reduced visitation or poor pollination. When floral orientation is important for accurate pollination, as has been suggested for bilaterally symmetrical flowers, we predict that such flowers should have developmental and/or behavioural mechanisms for restoring 'correct' orientation after accidents. We made observations and conducted experiments on 23 native and cultivated flowering plant species in Australia, South America, North America and Europe. We found that flowers with bilateral symmetry usually have the capacity to reorient after accidents, and that this is manifested through rapid bending and/or rotation of pedicels or sexual organs or slower peduncle bending. Floral reorientation restores pollination accuracy and fit with pollinators. However, experimental floral misorientation in eight species with radially symmetrical flowers showed that, with one exception, they had little capacity to reorient their flowers, in line with expectations that the orientation of radially symmetrical flowers does not substantially affect pollination accuracy. Our results suggest that quick corrective reorientation of bilaterally symmetrical flowers is adaptive, highlighting a little-studied aspect of plant-pollinator interactions and plant evolution.

Utility of targeted sequence capture for phylogenomics in rapid, recent angiosperm radiations: Neotropical Burmeistera bellflowers as a case study.

Targeted sequence capture is a promising approach for large-scale phylogenomics. However, rapid evolutionary radiations pose significant challenges for phylogenetic inference (e.g. incomplete lineages sorting (ILS), phylogenetic noise), and the ability of targeted nuclear loci to resolve species trees despite such issues remains poorly studied. We test the utility of targeted sequence capture for inferring phylogenetic relationships in rapid, recent angiosperm radiations, focusing on Burmeistera bellflowers (Campanulaceae), which diversified into ~130 species over less than 3 million years. We compared phylogenies estimated from supercontig (exons plus flanking sequences), exon-only, and flanking-only datasets with 506-546 loci (~4.7 million bases) for 46 Burmeistera species/lineages and 10 outgroup taxa. Nuclear loci resolved backbone nodes and many congruent internal relationships with high support in concatenation and coalescent-based species tree analyses, and inferences were largely robust to effects of missing taxa and base composition biases. Nevertheless, species trees were incongruent between datasets, and gene trees exhibited remarkably high levels of conflict (~4-60% congruence, ~40-99% conflict) not simply driven by poor gene tree resolution. Higher gene tree heterogeneity at shorter branches suggests an important role of ILS, as expected for rapid radiations. Phylogenetic informativeness analyses also suggest this incongruence has resulted from low resolving power at short internal branches, consistent with ILS, and homoplasy at deeper nodes, with exons exhibiting much greater risk of incorrect topologies due to homoplasy than other datasets. Our findings suggest that targeted sequence capture is feasible for resolving rapid, recent angiosperm radiations, and that results based on supercontig alignments containing nuclear exons and flanking sequences have higher phylogenetic utility and accuracy than either alone. We use our results to make practical recommendations for future target capture-based studies of Burmeistera and other rapid angiosperm radiations, including that such studies should analyze supercontigs to maximize the phylogenetic information content of loci.

A gradient of pollination specialization in three species of Bolivian Centropogon.

PREMISE: Closely related plant species with overlapping ranges often experience competition for pollination services. Such competition can select for divergence in floral traits that attract pollinators or determine pollen placement. While most species in Centropogon (Campanulaceae: Lobelioideae) have flowers that suggest adaptation to bat or hummingbird pollination, actual pollinators are rarely documented, and a few species have a mix of traits from both pollination syndromes. We studied the pollination biology of a "mixed-syndrome" species and its co-occurring congeners to examine the relationship between floral traits and visitation patterns for Centropogon. METHODS: Fieldwork at two sites in Bolivian cloud forests involved filming floral visitors, quantifying pollen transfer, and measuring floral traits. Stamen exsertion, which determines pollen placement, was measured from herbarium specimens across the geographic range of these species to test for character displacement. RESULTS: Results show a generalization gradient, from primarily bat pollination in white-flowered Centropogon incanus, to bat pollination with secondary hummingbird pollination in the cream-flowered C. brittonianus, to equal reliance on both pollinators in the red-flowered, mixed-syndrome C. mandonis. Pollen transfer between these species is further reduced by differences in stamen exsertion that are accentuated in zones of sympatry, a pattern consistent with character displacement. CONCLUSIONS: Our results demonstrate that key differences in floral color and shape mediate a gradient of specialization in Bolivian Centropogon. Interspecific pollen transfer is further reduced by potential character displacement of a key trait. Broadly, our results have implications for understanding the hyper-diversity of Andean cloud forests, in which multiple species of the same genus frequently co-occur.

Repeated evolution of vertebrate pollination syndromes in a recently diverged Andean plant clade.

Although specialized interactions, including those involving plants and their pollinators, are often invoked to explain high species diversity, they are rarely explored at macroevolutionary scales. We investigate the dynamic evolution of hummingbird and bat pollination syndromes in the centropogonid clade (Lobelioideae: Campanulaceae), an Andean-centered group of ∼550 angiosperm species. We demonstrate that flowers hypothesized to be adapted to different pollinators based on flower color fall into distinct regions of morphospace, and this is validated by morphology of species with known pollinators. This supports the existence of pollination syndromes in the centropogonids, an idea corroborated by ecological studies. We further demonstrate that hummingbird pollination is ancestral, and that bat pollination has evolved ∼13 times independently, with ∼11 reversals. This convergence is associated with correlated evolution of floral traits within selective regimes corresponding to pollination syndrome. Collectively, our results suggest that floral morphological diversity is extremely labile, likely resulting from selection imposed by pollinators. Finally, even though this clade's rapid diversification is partially attributed to their association with vertebrate pollinators, we detect no difference in diversification rates between hummingbird- and bat-pollinated lineages. Our study demonstrates the utility of pollination syndromes as a proxy for ecological relationships in macroevolutionary studies of certain species-rich clades.

Phylogenetic relationships of Burmeistera (Campanulaceae: Lobelioideae): Combining whole plastome with targeted loci data in a recent radiation.

The field of molecular systematics has benefited greatly with the advent of high-throughput sequencing (HTS), making large genomic datasets commonplace. However, a large number of targeted Sanger sequences produced by many studies over the last two decades are publicly available and should not be overlooked. In this study, we elucidate the phylogenetic relationships of the plant genus Burmeistera (Campanulaceae: Lobelioideae), while investigating how to best combine targeted Sanger loci with HTS data. We sequence, annotate, and analyze complete to nearly complete plastomes for a subset of the genus. We then combine these data with a much larger taxonomic dataset for which only Sanger sequences are available, making this the most comprehensively sampled study in the genus to date. We show that using a phylogeny inferred from the species with plastome data as a topological constraint for the larger dataset increases the resolution of our data and produces a more robust evolutionary hypothesis for the group. We then use the resulting phylogeny to study the evolution of morphological traits thought to be important in Burmeistera, and assess their usefulness in the current taxonomic classification of the genus. The main morphological character used to delimit subgeneric sections, the presence or absence of hairs on the apex of the two ventral anthers, shows a complex evolutionary history with many changes in the tree, suggesting that this character should not be used for taxonomic classification. Although it is too soon to propose a new subgeneric classification for Burmeistera, our results highlight some morphological traits shared by whole clades that could potentially be used in future taxonomic work.

The Complexity of Background Clutter Affects Nectar Bat Use of Flower Odor and Shape Cues.

Given their small size and high metabolism, nectar bats need to be able to quickly locate flowers during foraging bouts. Chiropterophilous plants depend on these bats for their reproduction, thus they also benefit if their flowers can be easily located, and we would expect that floral traits such as odor and shape have evolved to maximize detection by bats. However, relatively little is known about the importance of different floral cues during foraging bouts. In the present study, we undertook a set of flight cage experiments with two species of nectar bats (Anoura caudifer and A. geoffroyi) and artificial flowers to compare the importance of shape and scent cues in locating flowers. In a training phase, a bat was presented an artificial flower with a given shape and scent, whose position was constantly shifted to prevent reliance on spatial memory. In the experimental phase, two flowers were presented, one with the training-flower scent and one with the training-flower shape. For each experimental repetition, we recorded which flower was located first, and then shifted flower positions. Additionally, experiments were repeated in a simple environment, without background clutter, or a complex environment, with a background of leaves and branches. Results demonstrate that bats visit either flower indiscriminately with simple backgrounds, with no significant difference in terms of whether they visit the training-flower odor or training-flower shape first. However, in a complex background olfaction was the most important cue; scented flowers were consistently located first. This suggests that for well-exposed flowers, without obstruction from clutter, vision and/or echolocation are sufficient in locating them. In more complex backgrounds, nectar bats depend more heavily on olfaction during foraging bouts.

Phylogeny, classification, and fruit evolution of the species-rich Neotropical bellflowers (Campanulaceae: Lobelioideae).

UNLABELLED: • PREMISE OF THE STUDY: The species-rich Neotropical genera Centropogon, Burmeistera, and Siphocampylus represent more than half of the ∼1200 species in the subfamily Lobelioideae (Campanulaceae). They exhibit remarkable morphological variation in floral morphology and habit. Limited taxon sampling and phylogenetic resolution, however, obscures our understanding of relationships between and within these genera and underscores our uncertainty of the systematic value of fruit type as a major diagnostic character.• METHODS: We inferred a phylogeny from five plastid DNA regions (rpl32-trnL, ndhF-rpl32, rps16-trnK, trnG-trnG-trns, rbcL) using maximum-likelihood and Bayesian inference. Ancestral character reconstructions were applied to infer patterns of fruit evolution.• KEY RESULTS: Our results demonstrate that the majority of species in the genera Centropogon, Burmeistera, and Siphocampylus together form a primarily mainland Neotropical clade, collectively termed the "centropogonids." Caribbean Siphocampylus, however, group with other Caribbean lobelioid species. We find high support for the monophyly of Burmeistera and the polyphyly of Centropogon and mainland Siphocampylus. The ancestral fruit type of the centropogonids is a capsule; berries have evolved independently multiple times.• CONCLUSIONS: Our plastid phylogeny greatly improves the phylogenetic resolution within Neotropical Lobelioideae and highlights the need for taxonomic revisions in the subfamily. Inference of ancestral character states identifies a dynamic pattern of fruit evolution within the centropogonids, emphasizing the difficulty of diagnosing broad taxonomic groups on the basis of fruit type. Finally, we identify that the centropogonids, Lysipomia, and Lobelia section Tupa form a Pan-Andean radiation with broad habitat diversity. This clade is a prime candidate for investigations of Neotropical biogeography and morphological evolution.

Competition for hummingbird pollination shapes flower color variation in Andean solanaceae.

One classic explanation for the remarkable diversity of flower colors across angiosperms involves evolutionary shifts among different types of pollinators with different color preferences. However, the pollinator shift model fails to account for the many examples of color variation within clades that share the same pollination system. An alternate explanation is the competition model, which suggests that color divergence evolves in response to interspecific competition for pollinators, as a means to decrease interspecific pollinator movements. This model predicts color overdispersion within communities relative to null assemblages. Here, we combine morphometric analyses, field surveys, and models of pollinator vision with a species-level phylogeny to test the competition model in the primarily hummingbird-pollinated clade Iochrominae (Solanaceae). Results show that flower color as perceived by pollinators is significantly overdispersed within sites. This pattern is not simply due to phylogenetic history: phylogenetic community structure does not deviate from random expectations, and flower color lacks phylogenetic signal. Moreover, taxa that occur in sympatry occupy a significantly larger volume of color space than those in allopatry, supporting the hypothesis that competition in sympatry drove the evolution of novel colors. We suggest that competition among close relatives may commonly underlie floral divergence, especially in species-rich habitats where congeners frequently co-occur.

Competition drives specialization in pollination systems through costs to male fitness.

Specialization in pollination systems played a central role in angiosperm diversification, yet the evolution of specialization remains poorly understood. Competition through interspecific pollen transfer may select for specialization through costs to male fitness (pollen lost to heterospecific flowers) or female fitness (heterospecific pollen deposited on stigmas). Previous theoretical treatments of pollination focused solely on seed set, thus overlooking male fitness. Here we use individual-based models that explicitly track pollen fates to explore how competition affects the evolution of specialization. Results show that plants specialize on different pollinators when visit rates are high enough to remove most pollen from anthers; this increases male fitness by minimizing pollen loss to foreign flowers. At low visitation, plants generalize, which minimizes pollen left undispersed in anthers. A model variant in which plants can also evolve differences in sex allocation (pollen/ovule production) produces similar patterns of specialization. At low visitation, plants generalize and allocate more to female function. At high visitation, plants specialize and allocate equally to both sexes (in line with sex-allocation theory). This study demonstrates that floral specialization can be driven by selection through male function alone and more generally highlights the importance of community context in the ecology and evolution of pollination systems.

Fur versus feathers: pollen delivery by bats and hummingbirds and consequences for pollen production.

One floral characteristic associated with bat pollination (chiropterophily) is copious pollen production, a pattern we confirmed in a local comparison of hummingbird- and bat-adapted flowers from a cloud forest site in Ecuador. Previous authors have suggested that wasteful pollen transfer by bats accounted for the pattern. Here we propose and test a new hypothesis: bats select for increased pollen production because they can efficiently transfer larger amounts of pollen, which leads to a more linear male fitness gain curve for bat-pollinated plants. Flight cage experiments with artificial flowers and flowers of Aphelandra acanthus provide support for this hypothesis; in both instances, the amount of pollen delivered to stigmas by birds is not related to the amount of pollen removed from anthers on the previous visit, while the same function for bats increases linearly. Thus, increased pollen production will be linearly related to increased male reproductive success for bat flowers, while for bird flowers, increased pollen production leads to rapidly diminishing fitness returns. We speculate that fur takes up and holds more pollen than feathers, which seem to readily shed excess grains. Our gain-curve hypothesis may also explain why evolutionary shifts from bird to bat pollination seem more common than shifts in the opposite direction.

Going to great lengths: selection for long corolla tubes in an extremely specialized bat-flower mutualism.

In a hypothesis that has remained controversial since its inception, Darwin suggested that long-tubed flowers and long-tongued pollinators evolved together in a coevolutionary race, with each selecting for increasing length in the other. Although the selective pressures that flowers impose on tongue length are relatively straightforward, in that longer tongues allow access to more nectar, selective pressures that pollinators impose on flower length are less clear. Here, we test for such selective pressures in the highly specialized mutualism between the nectar bat Anoura fistulata, which can extend its tongue twice as far as other nectar bats, and Centropogon nigricans, which has flowers of a similar length (8-9 cm). We used flight cage experiments to examine the effects of artificially manipulated flower lengths on (i) bat behaviour and (ii) pollen transfer. Increased length produced longer visits, but did not affect the force bats applied during visits. In the second experiment, flower length increased both the male and female components of flower function: long male flowers delivered more pollen grains and long female flowers received more pollen grains. However, pollen transfer was not correlated with visit duration, so the mechanism behind differences in pollen transfer remains unclear. By demonstrating that bats select for increasing flower length, these results are consistent with the hypothesis that A. fistulata evolved its remarkable tongue in a coevolutionary race with long-tubed flowers similar to that envisioned by Darwin.

A generalized pollination system in the tropics: bats, birds and Aphelandra acanthus.

BACKGROUND AND AIMS: A number of different types of flower-visiting animals coexist in any given habitat. What evolutionary and ecological factors influence the subset of these that a given plant relies on for its pollination? Addressing this question requires a mechanistic understanding of the importance of different potential pollinators in terms of visitation rate (pollinator 'quantity') and effectiveness at transferring pollen (pollinator 'quality') is required. While bat-pollinated plants typically are highly specialized to bats, there are some instances of bat-pollinated plants that use other pollinators as well. These generalized exceptions tend to occur in habitats where bat 'quantity' is poor due to low or fluctuating bat densities. METHODS: Aphelandra acanthus occurs in tropical cloud forests with relatively high densities of bat visitors, yet displays a mix of floral syndrome characteristics, suggesting adaptation to multiple types of pollinators. To understand its pollination system better, aspects of its floral phenology and the 'quantity' and 'quality' components of pollination by its floral visitors are studied here. KEY RESULTS: Flowers were found to open and senesce throughout the day and night, although anther dehiscence was restricted to the late afternoon and night. Videotaping reveals that flowers are visited nocturnally by bats and moths, and diurnally by hummingbirds. Analysis of pollen deposition shows that bats regularly transfer large amounts of conspecific pollen, while hummingbirds occasionally transfer some pollen, and moths rarely do so. CONCLUSIONS: Hummingbirds and bats were comparable in terms of pollination 'quantity', while bats were the most effective in terms of 'quality'. Considering these components together, bats are responsible for approx. 70 % of A. acanthus pollination. However, bats also transferred remarkably large amounts of foreign pollen along with the conspecific grains (three of four grains were foreign). It is suggested that the negative effects of interspecific pollen transfer may decrease bat 'quality' for A. acanthus, and thus select for generalization on multiple pollinators instead of specialization on bats.