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Rethinking the pollination syndromes in Hymenaea (Leguminosae): the role of anthesis in the diversification

Abstract

Floral traits associated with functional groups of pollinators have been largely employed to understand mechanisms of floral diversification. Hymenaea is a monophyletic legume genus widely recognized to being bat-pollinated, with nocturnal anthesis and copious nectar. The most of species has short-paniculate inflorescences, white and robust flowers, congruent with a bat-pollination syndrome. However, other Hymenaea species show a different floral pattern (e.g., long-paniculate inflorescences and smaller flowers) which we report here as being bird pollinated. We examined the floral traits and visitors of Hymenaea oblongifolia var. latifolia and identified evolutionary shifts in floral traits associated with potential pollinators of Hymenaea species. Floral traits of H. oblongifolia var. latifolia differ from those expected for bat-pollinated flowers in species of sect. Hymenaea, and we observed hummingbirds collecting nectar legitimately. Our phylogenetic analysis did not support the monophyly of the taxonomic sections and suggests that bat pollination is ancestral in Hymenaea, with bird pollination evolving later. The transition coupling with shifts in the timing of anthesis and other floral traits. Pollinator-mediated evolutionary divergence hypothesis partially explains the Hymenaea diversification in the Neotropics. It is congruent with those species shifting from traits linked traditionally to bat pollination to hummingbird pollination.

Key words
Bat pollination; ethological isolation; floral traits; hummingbirds; legume

INTRODUCTION

Pollination syndrome concept is based on the notion that suites of floral traits reflect adaptations to a specific pollinator or group of most effective pollinators (Stebbins 1970STEBBINS GL. 1970. Adaptive radiation of reproductive characters in angiosperms I: pollination mechanisms. Annu Rev Ecol Syst 1: 307-326., Faegri & van der Pijl 1979FAEGRI K & VAN DER PIJL L. 1979. The Principles of Pollination Ecology, 3nd ed., New York: Pergamon Press, Oxford, 244 p., Grant 1994GRANT V. 1994. Modes and origins of mechanical and ethological isolation in angiosperms. P Natl Acad Sci USA 91: 3-10.). It is a proxy for understanding floral trait diversification and evolution towards specialized pollination systems (Fenster et al. 2004FENSTER CB, ARMBRUSTER WS, WILSON P, DUDASH MR & THOMSON JT. 2004. Pollination syndromes and floral specialization. Annu Rev Ecol Evol S 35: 375-403., Armbruster et al. 2014ARMBRUSTER WS, SHI XQ & HUANG SQ. 2014. Do specialized flowers promote reproductive isolation? Realized pollination accuracy of three sympatric Pedicularis species. Ann Bot 113: 331-340., Rosas-Guerrero et al. 2014ROSAS-GUERRERO V, AGUILAR R, MARTEN-RODRIGUEZ S, ASHWORTH L, LOPEZARAIZA-MIKEL M, BASTIDA JM & QUESADA M. 2014. A quantitative review of pollination syndromes: do floral traits predict effective pollinators? Ecol Lett 17: 388-400.). This specialized interaction of floral traits and pollinators is a key component that can drive convergent floral evolution in distinct lineages (Johnson 2010JOHNSON SD. 2010. The pollination niche and its role in the diversification and maintenance of the southern African flora. Philos T Roy Soc B 365: 499-516., Papadopulos et al. 2013PAPADOPULOS AST ET AL. 2013. Convergent evolution of floral signals underlies the success of Neotropical orchids. P R Soc B 280: 20130960., Newman et al. 2014NEWMAN E, MANNING JC & ANDERSON B. 2014. Matching floral and pollinator traits through guild convergence and pollinator ecotype formation. Ann Bot 113: 373-384., Ferreira et al. 2015FERREIRA C, MARUYAMA PK & OLIVEIRA PE. 2015. Convergence beyond flower morphology? Reproductive biology of hummingbird-pollinated plants in the Brazilian Cerrado. Plant Biol 18: 316-324.). On the other hand, multiple functional groups of pollinators can drive ancestral polymorphic populations to different floral traits (i.e., divergent evolution processes) in an idiosyncratic geographic-ecological context (Shemske & Bradshaw 1999, Eaton et al. 2012EATON DAR, FENSTER CB, HEREFORD J, HUANG SQ & REE RH. 2012. Floral diversity and community structure in Pedicularis (Orobanchaceae). Ecology 93: S182-S194., Armbruster 2014ARMBRUSTER WS. 2014. Floral specialization and angiosperm diversity: phenotypic divergence, fitness trade-offs and realized pollination accuracy. AoB Plants 6: plu003.). In other possible scenario, the conservatism of floral traits is intuitively understood as a direct result of the evolutionary history in the context of sharing of the common ancestry without selective pressures exerted by pollinators (i.e., phylogenetic inertia; Sakazono et al. 2012SAKAZONO S, HIRAMATSU M, HUANG KL, HUANG CL & OKUBO H. 2012. Phylogenetic Relationship between Degree of Self-compatibility and Floral Traits in Lilium longiflorum Thunb. (Liliaceae). J Jpn Soc Hortic Sci 81: 80-90., Papadopoulos et al. 2013).

Hymenaea L., a Neotropical monophyletic genus of subfamily Detarioideae in the Leguminosae, is a good biological model to investigate the mechanisms that drive the floral evolution in plants, mainly due to its great variation in floral and inflorescence attributes. In the first revision of the genus, two sections were proposed in accordance to morphological traits (i.e., sect. Hymenaea and sect. Trachylobium; Lee & Langenheim 1975LEE YT & LANGENHEIM JH. 1975. A systematic revision of the genus Hymenaea L. (Leguminosae; Caesalpinioideae; Detarieae). Oakland: University of California Publications in Botany, 109 p.). Hymenaea has long been considered entirely bat-pollinated, because most species have floral traits related to chiropterophily (e.g., nocturnal anthesis, white and robust petals, and copious nectar production; Langenheim et al. 1973LANGENHEIM JH, LEE YT & MARTIN SS. 1973. An evolutionary and ecological perspective of Amazonian Hylea species of Hymenaea (Leguminosae: Caesalpinioidea). Manaus, Amazonas. Acta Amazon 3: 5-38., Lee & Langenheim 1975LEE YT & LANGENHEIM JH. 1975. A systematic revision of the genus Hymenaea L. (Leguminosae; Caesalpinioideae; Detarieae). Oakland: University of California Publications in Botany, 109 p., Arroyo 1981ARROYO MTK. 1981. Breeding systems and pollination biology in Leguminosae. In: Polhill RM and Raven PH (Eds). Adv Legum Systemat 723-769., Gibbs et al. 1999GIBBS PE, OLIVEIRA PE & BIANCHI MB. 1999. Postzygotic control of selfing in Hymenaea stigonocarpa (Leguminosae--Caesalpinioideae), a bat-pollinated tree of the Brazilian cerrados. Int J Plant Sci 160: 72-78., Dunphy et al. 2004DUNPHY BK, HAMRICK JL & SCHWAGERL J. 2004. A comparison of direct and indirect measures of gene flow in the bat-pollinated tree Hymenaea courbaril in the dry forest life zone of south-western Puerto Rico. Int J Plant Sci 165: 427-436., Paiva & Machado 2008PAIVA EAS & MACHADO SR. 2008. The floral nectary of Hymenaea stigonocarpa (Fabaceae, Caesalpinioideae): structural aspects during floral development. Ann Bot 101: 125-133., Fleming et al. 2009FLEMING TH, GEISELMAN C & KRESS WJ. 2009. The evolution of bat pollination: a phylogenetic perspective. Ann Bot 104: 1017-1043., Domingos-Melo et al. 2019DOMINGOS-MELO A, MILET-PINHEIRO P, NAVARRO DM & MACHADO IC. 2019. It’s raining fragrant nectar in the Caatinga: evidence of nectar olfactory signaling in bat-pollinated flowers. Ecology: 10.1002/ecy.2914.). This generalization is supported by empirical evidence in natural populations of H. stigonocarpa Mart. ex Hayne, H. courbaril L, and H. cangaceira R.B. Pinto, Mansano & A.M.G. Azevedo, which have been observed to be pollinated by Phyllostomid bats (sect. Hymenaea; Arroyo 1981ARROYO MTK. 1981. Breeding systems and pollination biology in Leguminosae. In: Polhill RM and Raven PH (Eds). Adv Legum Systemat 723-769., Gibbs et al. 1999GIBBS PE, OLIVEIRA PE & BIANCHI MB. 1999. Postzygotic control of selfing in Hymenaea stigonocarpa (Leguminosae--Caesalpinioideae), a bat-pollinated tree of the Brazilian cerrados. Int J Plant Sci 160: 72-78., Dunphy et al. 2004DUNPHY BK, HAMRICK JL & SCHWAGERL J. 2004. A comparison of direct and indirect measures of gene flow in the bat-pollinated tree Hymenaea courbaril in the dry forest life zone of south-western Puerto Rico. Int J Plant Sci 165: 427-436., Lacerda et al. 2008LACERDA AEB, KANASHIRO M & SEBBENN AM. 2008. Long-pollen Movement and Deviation of Random Mating in a Low-density Continuous Population of a Tropical Tree Hymenaea courbaril in the Brazilian Amazon. Biotropica 40: 462-470., Moraes & Sebbenn 2011MORAES MLT & SEBBENN AM. 2011. Pollen dispersal between isolated trees in the Brazilian savannah: a case study of the Neotropical tree Hymenaea stigonocarpa. Biotropica 40: 462-470., Domingos-Melo et al. 2019DOMINGOS-MELO A, MILET-PINHEIRO P, NAVARRO DM & MACHADO IC. 2019. It’s raining fragrant nectar in the Caatinga: evidence of nectar olfactory signaling in bat-pollinated flowers. Ecology: 10.1002/ecy.2914.). All of these species are members of sect. Hymenaea, but no data are available in the literature about the pollination mode in sect. Trachylobium.

Phyllostomid bat diversity is particularly important in tropical rainforests surrounding the equator, which suggests that this diversity could be explained by environmental triggers such as warm temperatures, low seasonality and wet conditions (Stevens 2011, 2013, Alroy 2019). Furthermore, a rapid expansion of potential geographical ranges after evolving has been proposed for phyllostomid lineages (Weber et al. 2014), but with geographic gaps due to fluctuations in fruit and flower resources. These limitations, driven by rainfall and temperature seasonality, in specialists frugivorous and nectarivorous bats (Stevens 2013), are similar with nectarivorous birds (e.g., hummingbirds; McGuire et al. 2014MCGUIRE JA ET AL. 2014. Molecular phylogenetics and diversification of hummingbirds. Curr Biol 24: 910-916.), which share some floral attributes with nectarivorous bats (e.g., dilute nectar, large pollen grains). Nectarivorous birds and bats, have a high diversity associated with Neotropical rainforests, but they are ethologically separated (i.e., diurnal vs. nocturnal foragers). The high diversity associated to Neotropical rainforests is congruent with the geographic distribution and time of diversification of Hymenaea (Lee & Langemheim 1975, Mackinder 2005MACKINDER B. 2005. Detarieae. In: Lewis G, Schrire B, Mackinder B and Lock M (Eds). Legumes of the world, Kew: The Royal Botanical Gardens, p. 69-71.). The genus has its center of distribution associated to Amazonian hylaea, in which Neotropical Trachylobium species are restricted to the Amazonian domain, except by the disjunct distribution of H. oblongifolia Huber, which presents a variety restricted to the Atlantic Rainforest (Lee & Langenheim 1975LEE YT & LANGENHEIM JH. 1975. A systematic revision of the genus Hymenaea L. (Leguminosae; Caesalpinioideae; Detarieae). Oakland: University of California Publications in Botany, 109 p.). Unfortunately, the Atlantic Rainforest – a hotspot of diversity and physiographic domain of Neotropical realm – is one of the most fragmented and endangered biomes of Brazil due to intense deforestation (SOS Mata Atlântica and INPE 2016). The habitat fragmentation might affect the fitness of plant populations and pollinator guilds services (Elmqvist et al. 2003ELMQVIST T, FOLKE C & NYSTRÖM M. 2003. Response diversity, ecosystem change, and resilience. Front Ecol Environ 1: 488-494.).

Based on the morphology of floral traits used to delimit the sections recognized by Lee & Langenheim (1975)LEE YT & LANGENHEIM JH. 1975. A systematic revision of the genus Hymenaea L. (Leguminosae; Caesalpinioideae; Detarieae). Oakland: University of California Publications in Botany, 109 p., the chiropterophily widely attributed to Hymenaea does not explain the suites of floral traits observed in the genus. Here, we predict that the bat pollinated generalization reflects a misclassification for sect. Trachylobium. This hypothesis is grounded in the proposition of pollination syndromes (sensu Faegri & van der Pijl), excluding a priori the distinct floral patterns being a result of stochastic processes (e.g., drift, environment filters). In the present study, we examined the floral traits and potential pollinators of Hymenaea oblongifolia var. latifolia Lee & Langenh. (sect. Trachylobium; sensu Lee & Langenheim 1975LEE YT & LANGENHEIM JH. 1975. A systematic revision of the genus Hymenaea L. (Leguminosae; Caesalpinioideae; Detarieae). Oakland: University of California Publications in Botany, 109 p.) – an endemic Atlantic Rainforest species, with records only to Bahia and Pernambuco States, Northeastern, Brazil (Flora do Brasil 2020FLORA DO BRASIL. 2020. Em construção. Hymenaea. Jardim Botânico do Rio de Janeiro. Available in: <http://floradobrasil.jbrj.gov.br/reflora/floradobrasil/FB78710>. Acessado em: 15 Jul. 2020.
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), in which the floral traits are distinct from the traditionally chiropterophilous species. In addition, we investigated the evolution of the pollination syndromes in the genus based on a phylogenetic framework of Hymenaea and related genera of the subfamily Detarioideae (LPWG 2017LPWG - LEGUME PHYLOGENY WORKING GROUP. 2017. A new subfamily classification of the Leguminosae based on a taxonomically comprehensive phylogeny. Taxon 66: 44-77.). In addition, we review the morphology of other Hymenaea species and outgroups (based on Fougère-Danezan et al. 2007FOUGÈRE-DANEZAN M, MAUMONT S & BRUNEAU A. 2007. Relationships among resin-producing Detarieae s.l. (Leguminosae) as inferred by molecular data. Syst Bot 32: 748-761., 2010FOUGÈRE-DANEZAN M, HERENDEEN PS, MAUMONT S & BRUNEAU A. 2010. Morphological evolution in the variable resin-producing Detarieae (Fabaceae): do morphological characters retain a phylogenetic signal? Ann Bot 105: 311-325., Bruneau et al. 2008BRUNEAU A, MERCURE M, LEWIS GP & HERENDEEN PS. 2008. Phylogenetic patterns and diversification in the caesalpinioid legumes. Botany 86: 697-718.) to build a matrix of floral traits used for identifying pollination syndromes. In this sense, we addressed the following questions: (1) Are the morphological sections (Lee & Langenheim 1975LEE YT & LANGENHEIM JH. 1975. A systematic revision of the genus Hymenaea L. (Leguminosae; Caesalpinioideae; Detarieae). Oakland: University of California Publications in Botany, 109 p.) phylogenetically supported? (2) What is the pollination syndrome of Hymenaea oblongifolia var. latifolia Y.T. Lee & Langenh. based on morphological and field observations? (3) Is the divergence of floral traits associated to distinct potential pollinators in the genus? (4) Are the pollination syndromes a consequence of divergent evolution driven by distinct pollinator guilds or are they a result of phylogenetic inertia?

MATERIALS AND METHODS

Hymenaea L. and focal species

The diversity of Hymenaea L. is typically centered in the tropical rainforest, except for H. verrucosa, which is distributed in Sub-Saharan Africa (Mackinder 2005MACKINDER B. 2005. Detarieae. In: Lewis G, Schrire B, Mackinder B and Lock M (Eds). Legumes of the world, Kew: The Royal Botanical Gardens, p. 69-71., Fougère-Danezan et al. 2007FOUGÈRE-DANEZAN M, MAUMONT S & BRUNEAU A. 2007. Relationships among resin-producing Detarieae s.l. (Leguminosae) as inferred by molecular data. Syst Bot 32: 748-761., Bruneau et al. 2008BRUNEAU A, MERCURE M, LEWIS GP & HERENDEEN PS. 2008. Phylogenetic patterns and diversification in the caesalpinioid legumes. Botany 86: 697-718., LPWG 2017LPWG - LEGUME PHYLOGENY WORKING GROUP. 2017. A new subfamily classification of the Leguminosae based on a taxonomically comprehensive phylogeny. Taxon 66: 44-77.). Hymenaea comprises 20 recognized species, occurring in several biomes, like tropical rainforests, seasonally dry tropical forests and woodlands (SDTFW), and savannas (Mackinder 2005MACKINDER B. 2005. Detarieae. In: Lewis G, Schrire B, Mackinder B and Lock M (Eds). Legumes of the world, Kew: The Royal Botanical Gardens, p. 69-71., Fougère-Danezan et al. 2007FOUGÈRE-DANEZAN M, MAUMONT S & BRUNEAU A. 2007. Relationships among resin-producing Detarieae s.l. (Leguminosae) as inferred by molecular data. Syst Bot 32: 748-761., Bruneau et al. 2008BRUNEAU A, MERCURE M, LEWIS GP & HERENDEEN PS. 2008. Phylogenetic patterns and diversification in the caesalpinioid legumes. Botany 86: 697-718., Souza et al. 2014SOUZA IM, FUNCH LS & QUEIROZ LP. 2014. Morphological analyses suggest a new taxonomic circumscription for Hymenaea courbaril L. (Leguminosae, Caesalpinioideae). PhytoKeys: 10.3897/phytokeys.@.7408., Lima & Pinto 2015LIMA HC & PINTO RB. 2015. Hymenaea. In: Lista de Espécies da Flora do Brasil. Jardim Botânico do Rio de Janeiro. http://floradobrasil.jbrj.gov.br/jabot/floradobrasil/FB22971/. (acessado 5 Nov 2015).
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, Ribeiro et al. 2015RIBEIRO RD, CARDOSO DBOS & LIMA HC. 2015. A New Species of Hymenaea (Leguminosae: Caesalpinioideae) with a revised identification key to the genus in the Brazilian Atlantic forest. Syst Bot 40: 151-156., LPWG 2017LPWG - LEGUME PHYLOGENY WORKING GROUP. 2017. A new subfamily classification of the Leguminosae based on a taxonomically comprehensive phylogeny. Taxon 66: 44-77., Aguilar et al. 2018AGUILAR FR, POVEDA LJA & SANTAMARÍA-AGUILAR D. 2018. Hymenaea osanigraseminae: Un nuevo guapinol (Fabaceae) del Pacífico central y sur de Costa Rica. Phytoneuron 20: 1-12.). In the first revision of the genus, Hymenaea was traditionally divided into two sections based on morphological criteria: (i) sect. Hymenaea, with short-paniculate inflorescences, large flowers, and additional mass of nectariferous tissue; and (ii) sect. Trachylobium with long-paniculate inflorescences, smaller flowers, without additional mass of nectariferous tissue (Lee & Langenheim 1975LEE YT & LANGENHEIM JH. 1975. A systematic revision of the genus Hymenaea L. (Leguminosae; Caesalpinioideae; Detarieae). Oakland: University of California Publications in Botany, 109 p.).

We investigated the floral biology and potential pollinators of Hymenaea oblongifolia var. latifolia, one of the only three species of the sect. Trachylobium, with a disjunct distribution in the Amazon and Atlantic Rainforest (Lee & Langenheim 1975LEE YT & LANGENHEIM JH. 1975. A systematic revision of the genus Hymenaea L. (Leguminosae; Caesalpinioideae; Detarieae). Oakland: University of California Publications in Botany, 109 p.), and for which no information (in the genus) over on the floral biology and pollinators is available yet. Despite the wide distribution through the ombrophilous forests in the Brazil, H. oblongifolia is divided into four taxonomic varieties (i.e., H. oblongifolia var. oblongifolia; H. oblongifolia var. davisii [Sandwith] Y.T. Lee & Langenh.; H. oblongifolia var. latifolia; and H. oblongifolia var. palustris [Ducke] Y.T. Lee & Langenh.), with the variety under study is restricted to the Atlantic Rainforest where it naturally occurs as a very large canopy tree (Lee & Langenheim 1975LEE YT & LANGENHEIM JH. 1975. A systematic revision of the genus Hymenaea L. (Leguminosae; Caesalpinioideae; Detarieae). Oakland: University of California Publications in Botany, 109 p., Lima & Pinto 2015LIMA HC & PINTO RB. 2015. Hymenaea. In: Lista de Espécies da Flora do Brasil. Jardim Botânico do Rio de Janeiro. http://floradobrasil.jbrj.gov.br/jabot/floradobrasil/FB22971/. (acessado 5 Nov 2015).
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) at low population densities (0.29 ind/ha; Thomas et al. 2009THOMAS WW, JARDIM JG, FIASCHI P, MARIANO NE & AMORIM AM. 2009. Composição florística e estrutura do componente arbóreo de uma área transicional de Floresta Atlântica no sul da Bahia Brasil. Braz J Bot 32: 65-78.). The species is 15–40 m tall with paniculate inflorescences (14–22 cm long), flowers 14–16 mm long, with a campanulate hypanthium and a stalk-like base 1–2 mm long; the four sepals are brownish, fleshy, and widely ovate; the five petals are white and spatulate, with 5–7 mm long claws; the ten stamens are 14–23 mm long, with anthers c. 3 mm long; the ovary is oblong, pubescent throughout and hirsute at the base, with a c. 2 mm long stipe at the base (Souza et al. 2016SOUZA IM, FUNCH LS & QUEIROZ LP. 2016. Flora da Bahia: Leguminosae - Hymenaea (Caesalpinioideae: Detarieae). Sitientibus 16: 10.13102/scb1092.).

We studied a population located in the municipality of Ibirapitanga, Bahia State, Brazil (13°58’S, 39°28’W, at 342 m a.s.l.), within the Atlantic Forest phytogeographic domain. The southern coastal sub-region of Bahia State consists of a fragmented agricultural landscape containing cacao plantations (cabruca) and primary and secondary rainforest fragments (Landau et al. 2008LANDAU EC, HIRSCH A & MUSINSKY J. 2008. Vegetation cover and land use in the Atlantic coastal forest of southern Bahia, Brazil, based on satellite imagery: a comparison among municipalities. In: Thomas WW (Ed). The Atlantic coastal forest of northeastern Brazil, New York: The New York Botanical Garden Press, p. 221-244.) subject to a tropical climate (type Af; Alvares et al. 2013ALVARES CA, STAPE JL, SENTELHAS PC, GONÇALVES JL & SPAROVEK G. 2013. Köppen’s climate classification map for Brazil. Meteorol Z 22: 711-728.) without a dry season, with intense rainfall throughout the year and mean monthly temperatures varying between 21.6 (July) and 25.9 °C (February).

Floral biology of the focal species

Field observations of floral attributes were performed from the canopy of one tree (ca. 40 m tall, including the canopy), during three days (January 19–21, 2015), totaling 41 hours of diurnal and nocturnal observations. We conducted observations using climbing techniques (Fig. 1), to identify the exact moment of flower opening and availability of floral resources, comprising all the floral activity range. Only one plant of H. oblongifolia var. latifolia was observed due: (i) the tree size and the necessity of climbing techniques; (ii) the low population density (Thomas et al. 2009THOMAS WW, JARDIM JG, FIASCHI P, MARIANO NE & AMORIM AM. 2009. Composição florística e estrutura do componente arbóreo de uma área transicional de Floresta Atlântica no sul da Bahia Brasil. Braz J Bot 32: 65-78.); and (iii) the asynchrony among five individuals in the studied population (i.e., only one individual with flower production during the focal observations). In addition, the aggregate floral production of H. oblongifolia var. latifolia (December to January, Souza & Funch 2016SOUZA IM & FUNCH LS. 2016. Synchronization of leafing and reproductive phenological events in Hymenaea L. species (Leguminosae, Caesalpinioideae): the role of photoperiod as the trigger. Braz J Bot 40: 125-136.) restricted the sampling effort, once January we performed the first focal observations for the species, and February the population did not present flowers anymore.

Figure 1
Hymenaea oblongifolia var. latifolia Lee & Langenh. in a fragmente of ombrophilous forest, Ibirapitanga, Bahia, Brazil. (a) Individual focal plant in its habitat; (b and c) Hummingbird (Hylocharis sapphrina) making contact with the anthers of H. oblongifolia var. latifolia during its visits (red arrow); (d, e and f) Sequence of anthesis of H. oblongifolia var. latifolia (floral bud at the beginning of anthesis, flower at the middle of the anthesis, and flower at end of anthesis, respectively). Red bar = 1 cm.

The timing and duration of anthesis and pollen availability were analyzed by observing 20 floral buds isolated in voile bags, and 20 non-isolated floral buds. Pollen grains were collected directly from the anthers of 40 flowers every two hours from 06:00 to 12:00 h. Laboratory analyses of pollen viability were performed using lactophenol (Willis 1999WILLIS J. 1999. The role of genes of large effect on inbreeding depression in Mimulus guttatus. Evolution 53: 1678-1691.). Receptivity of the stigma was determined by dipping the stigmas of 40 flowers in hydrogen peroxide every two hours (from 06:00 to 12:00 h) to detect esterase activity. Nectar sugar concentrations were sampled in the field every hour, throughout the floral cycle, between 06:00 and 12:00 h, using a hand-held refractometer. The presence of osmophores (i.e., odoriferous glands – usually present in chiropterophilous flowers and absent in ornithophilous ones) was verified by immersing the flowers in 1% neutral-red solution (10 min) with subsequent washing in a 5% solution of glacial acetic acid (Dafni et al. 2005DAFNI A, KEVAN PG & HUSBAND BC. 2005. Practical Pollination Biology, Canada: Enviroquest Ltd, 590 p.). Finally, we investigated the pigments presence with absorption ranges within the ultra-violet spectrum by maintaining the flowers in an atmosphere of ammonium hydroxide for five minutes (Scogin et al. 1977SCOGIN RY, YOUNG DA & JONES CE. 1977. Anthochlor pigments and pollination biology: II. The ultraviolet patterns of Coreopsis gigantea (Asteraceae). B Torrey Bot Club 104: 155-159.).

Flower visitors and potential pollinators of the focal species

We performed surveys of floral visitors during 3 days (from 05:00 to 22:00 on the first day, and from 04:00 to 16:00 in the second and third days) in a tree canopy by observing the harvesting of floral resources and foraging behaviors during visits. The observed visitors were ranked as: (i) potential pollinators, who performed legitimate visits; or (ii) pollen or nectar thieves, who performed illegitimate visits but collected resources without damaging the flower (Inouye 1980INOUYE DW. 1980. The terminology of floral larceny. Ecology 61: 1251-1253.).

DNA extraction, amplification and sequencing

The taxonomic sampling included 13 species of Hymenaea (i.e., 10 species of sect. Hymenaea and three species of sect. Trachylobium) and six outgroups belonging to Guibourtia Benn., Peltogyne Vogel, and Goniorrhachis Taub. based on previous phylogenetic studies of Deatrioideae (Fougère-Danezan et al. 2007FOUGÈRE-DANEZAN M, MAUMONT S & BRUNEAU A. 2007. Relationships among resin-producing Detarieae s.l. (Leguminosae) as inferred by molecular data. Syst Bot 32: 748-761., 2010, Bruneau et al. 2008BRUNEAU A, MERCURE M, LEWIS GP & HERENDEEN PS. 2008. Phylogenetic patterns and diversification in the caesalpinioid legumes. Botany 86: 697-718., de la Estrella et al. 2018DE LA ESTRELLA M, FOREST F, KLITGÅRD B, LEWIS GP, MACKINDER BA, DE QUEIROZ LP, WIERINGA JJ & BRUNEAU A. 2018. A new phylogeny-based tribal classification of subfamily Detarioideae, an early branching clade of florally diverse tropical arborescent legumes. Sci Rep 8: 6884.).

We obtained sequences previously published in studies from the plastid trnL intron, matK gene and the nuclear ribosomal internal transcribed spacer (ITS) (Table I; Bruneau et al. 2001BRUNEAU A, FOREST F, HERENDEEN P, KLITGAARD B & LEWIS G. 2001. Phylogenetic Relationships in the Caesalpinioideae (Leguminosae) as Inferred from Chloroplast trnL Intron Sequences. Syst Bot 26: 487-514., 2008, Fougère-Danezan et al. 2003FOUGÈRE-DANEZAN M, MAUMONT S & BRUNEAU A. 2003. Phylogenetic relationships in resin-producing Detarieae inferred from molecular data and preliminary results for a biogeographic hypothesis. In: Klitgaard BB and Bruneau A (Eds). Advances in legume systematics, Part 10, Kew: Royal Botanic Gardens, p. 161-180., 2007, de la Estrella et al. 2018DE LA ESTRELLA M, FOREST F, KLITGÅRD B, LEWIS GP, MACKINDER BA, DE QUEIROZ LP, WIERINGA JJ & BRUNEAU A. 2018. A new phylogeny-based tribal classification of subfamily Detarioideae, an early branching clade of florally diverse tropical arborescent legumes. Sci Rep 8: 6884.) available in the GenBank database (http://www.ncbi.nlm.nih.gov/genbank). We selected sequences that could be validated by voucher specimens in herbarium. In addition, samples of a few species were obtained from specimens collected in the field (preserved in silica gel) and from herbarium material (Table I). Vouchers were deposited in the HUEFS – Herbário da Universidade Estadual de Feira de Santana.

Table I
List of taxa (Hymenaea, Guibourtia and Peltogyne) used in the phylogenetic inference, markers, vouchers and GenBank accesses.

We extracted total genomic DNA from fresh leaves (preserved in silica gel) and herbarium samples using, respectively, modified CTAB protocol of Doyle & Doyle (1987)DOYLE JJ & DOYLE JL. 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull 19: 11-15. and DNeasy Plant Mini Kit (QIAGEN GmbH, Hilden, Germany). PCR reactions were performed using the TopTaq Master Mix Kit (QIAGEN GmbH, Hilden, Germany; according to the manufacturer’s protocol) for a final volume of 10 μL. For herbarium samples, PCR reactions also included 2 μL of TBT-PAR [trealose, bovine serum albumin (BSA), polysorbate-20 (Tween-20)] (Samarakoon et al. 2013), and for ITS they also included 0.4 μL of DMSO 99.5% (dimethyl sulfoxide). Amplifications were purified using PEG 11% (Paithankar & Prasad 1991PAITHANKAR KR & PRASAD KS. 1991. Precipitation of DNA by polyethylene glycol and ethanol. Nucleic Acids Res 19(6): 1346.), and then sequenced bidirectionally using the Big Dye Terminator v3.1 Cycle Sequencing Kit according to the manufacturer’s protocol and using the same primers as for the amplification (Appendix 1). Sequencing products were cleaned using isopropanol 80% and ethanol 70%, and analyzed on a 3130xl Genetic Analyzer (Applied Biosystems/HITACHI, Tokyo, Japan) at the Laboratório de Sistemática Molecular de Plantas of the Universidade Estadual de Feira de Santana.

Alignment, phylogenetic reconstruction and divergence time estimation

We aligned sequences in MUSCLE using the default settings (Edgar 2004EDGAR RC. 2004. Muscle: Multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32: 1792-1797.) and with manual adjustments for visual improvement in program MEGA version 10.0.4 (Kumar et al. 2018KUMAR S, STECHER G, LI M, KNYAZ C & TAMURA K. 2018. MEGA X: Molecular Evolutionary Genetics Analysis across computing platforms. Mol Biol Evol 35: 1547-1549.). We coded gaps as a fifth character state, and performed Bayesian analyses using MrBayes v.3.2.5 (Ronquist et al. 2012RONQUIST F ET AL. 2012. MrBayes 3.2: Efficient Bayesian phylogenetic inference and model choice across a large model space. Syst Biol 61: 539-542.) on the concatenated data matrix (all regions). We selected the best-fit substitution models for each plastid datasets and ITS partitions based on Maximum Likelihood Criterion (MrModeltest v.2.3; Nylander 2004NYLANDER JAA. 2004. MrModeltest v. 2. Program distributed by the author. Uppsala: Evolutionary Biology Center, Uppsala University.). The best models identified were: (i) GTR for the matK gene, ITS1 and ITS2; (ii) F81+G for the trnL-intron; and (iii) K80 for the 5.8S of the nuclear ribosomal locus.

Divergence time estimates were inferred using the concatenated data matrix for the 13 Hymenaea taxa and outgroups used in the phylogenetic reconstruction. To estimate divergence time we used BEAST v.1.8.2 (Drummond et al. 2012DRUMMOND AJ, SUCHARD MA, XIE D & RAMBAUT A. 2012. Bayesian Phylogenetics with BEAUti and the BEAST 1.7. Mol Biol Evol 29: 1969-1973.) with a GTR substitution model, an uncorrelated lognormal relaxed clock model, a tree prior with a Yule speciation model, and a random starting tree. For the analyses, two nodes were constrained. The stem node representing the Most Recent Common Ancestor (MRCA) of the clade, that includes the genera Peltogyne, Guibourtia and Hymenaea, was set at 46.1 Mya (normal distribution; mean = 46.0; stdev = 2.65) based on the divergence time estimated by Bruneau et al. (2008)BRUNEAU A, MERCURE M, LEWIS GP & HERENDEEN PS. 2008. Phylogenetic patterns and diversification in the caesalpinioid legumes. Botany 86: 697-718.. The MRCA of the genus Hymenaea was calibrated at 24 Mya (lognormal prior; offset = 23.0; mean = 0; stdev = 1) based on a fossil flower preserved in amber from the Dominican Republic (Hueber & Langenheim 1986HUEBER FM & LANGENHEIM J. 1986. Dominican amber tree had African ancestors. Geotimes 31: 8-10.). Because this flower has morphological features of sect. Hymenaea and this section was reconstructed as paraphyletic (see Results), we set the crown node of the MRCA of the genus Hymenaea because it matches the age estimated for the same node by Bruneau et al. (2008: mean = 24.7; stdev = 1.369). Four independents Metropolis-coupled Markov Chain Monte Carlo (MCMC) were run for 10 million generations, and parameters and trees were sampled every 5000 generations. Convergence and stationarity were checked in Tracer v1.6 (Rambaut & Drummond 2013RAMBAUT A & DRUMMOND AJ. 2013. Tracer. Version 1.6. http://beast.bio.ed.ac.uk/Tracer/ (accessed 2 Jun 2017).
http://beast.bio.ed.ac.uk/Tracer/...
). All parameters had ESS (effective sample size) values > 200. Trees sampled after exclusion of 25% burn-in of each run were combined with LogCombiner (Rambaut & Drummond 2010aRAMBAUT A & DRUMMOND AJ. 2010a. LogCombiner: MCMC output combiner. Version 1.5.4.). The maximum clade credibility (MCC) tree was estimated using mean ages and 95% highest posterior density (HPD) intervals of node ages using TreeAnnotator (Rambaut & Drummond 2010bRAMBAUT A & DRUMMOND AJ. 2010b. TreeAnnotator: MCMC output analysis. Version 1.5.4.). The MCC tree was visualized and edited with FigTree version 1.4.2 (Rambaut 2014RAMBAUT A. 2014. Figtree, a graphical viewer of phylogenetic trees. http://tree.bio.ed.ac.uk/software/figtree/ (accessed Jun 02 2017).
http://tree.bio.ed.ac.uk/software/figtre...
).

Evolution of pollination systems

Pollination systems (i.e., bee, bird and bat) were scored for the sampled species of Hymenaea, Guibourtia, Peltogyne (the closest outgroups) and Goniorrhachis, based on the pollination syndromes inference (morphological criteria according to Faegri & van der Pijl 1979FAEGRI K & VAN DER PIJL L. 1979. The Principles of Pollination Ecology, 3nd ed., New York: Pergamon Press, Oxford, 244 p.), and confirmed by field observations for Peltogyne pauciflora Benth. and Goniorrhachis marginata Taub. (I.M. Souza, unpublished data). Despite the recognition of varieties for some species of Hymenaea, such as the focal species in the present study (i.e., H. oblongifolia var. oblongifolia; H. oblongifolia var. davisi.; H. oblongifolia var. latifolia; H. oblongifolia var. palustris), here we assume that missing lineages (i.e., infraspecific categories) will not affect further character reconstructions mainly due to the conserved floral pattern at species level (Lee & Langenheim 1975LEE YT & LANGENHEIM JH. 1975. A systematic revision of the genus Hymenaea L. (Leguminosae; Caesalpinioideae; Detarieae). Oakland: University of California Publications in Botany, 109 p.).

The match between the morpho-functional interpretation of pollinator mode (i.e., flower morphology, anthesis and odor) and empirical field observations of pollination mode is supported by earliest studies with H. courbaril and H. stigonocarpa (Arroyo 1981ARROYO MTK. 1981. Breeding systems and pollination biology in Leguminosae. In: Polhill RM and Raven PH (Eds). Adv Legum Systemat 723-769., Gibbs et al. 1999GIBBS PE, OLIVEIRA PE & BIANCHI MB. 1999. Postzygotic control of selfing in Hymenaea stigonocarpa (Leguminosae--Caesalpinioideae), a bat-pollinated tree of the Brazilian cerrados. Int J Plant Sci 160: 72-78., Dunphy et al. 2004DUNPHY BK, HAMRICK JL & SCHWAGERL J. 2004. A comparison of direct and indirect measures of gene flow in the bat-pollinated tree Hymenaea courbaril in the dry forest life zone of south-western Puerto Rico. Int J Plant Sci 165: 427-436.) and by our own field observations of H. eriogyne, H. martiana and H. velutina. We treated pollination syndromes as categorical states (standard data; Table III) and modeled using the mk model of Lewis (2001)LEWIS PO. 2001. A likelihood approach to estimating phylogeny from discrete morphological character data. Syst Biol 50: 913-925. as implemented by Ronquist et al. (2012)RONQUIST F ET AL. 2012. MrBayes 3.2: Efficient Bayesian phylogenetic inference and model choice across a large model space. Syst Biol 61: 539-542., optimized onto 1,000 trees sampled at stationarity to account for phylogenetic uncertainty, and presented at the MCC tree in Mesquite v.3.40 (Maddison & Maddison 2018MADDISON WP & MADDISON DR. 2018. Mesquite: a modular system for evolutionary analysis. Version 3.40. http://www.mesquiteproject.org.
http://www.mesquiteproject.org...
).

Table II
Floral visitors of Hymenaea oblongifolia var. latifolia Y.T. Lee & Langenh. in a rain forest area, Ibirapitanga, Bahia State, Brazil. Flower visitor classification follows Inouye (1980)INOUYE DW. 1980. The terminology of floral larceny. Ecology 61: 1251-1253. terminology.
Table III
Inflorescence and floral traits of Hymenaea from herbarium, field data and literature, used to infer about potential pollinators in the reconstruction of character state. Legend: Inflor = inflorescence; ds = dish-shaped; cs = cup-shaped; int = intermediate (10 < int < 20 mm long); lar = large (lar ≥ 20 mm long); Sym = symmetry; act = actinomorphy; szyg = slightly zygomorphy; zyg = zygomorphy; Noc = nocturnal; Diu = diurnal; pres = present; abs = absent; Pol = pollination syndrome; chi = chiropterophily; orn = ornithophily; Hab = habitat; wet = wet forest; dry = dry forest.

RESULTS

Floral biology of H. oblongifolia var. latifolia

The species has flexible pedicels and curved flowers, slightly zygomorphic. At the end of anthesis, the base of the petal remains erect, extending the hypanthium cavity, and the reproductive organs are projected forward (Fig. 1). Anthesis is diurnal, occurring from 04:00 to 07:00 h, and flowers lasted for circa 12 hours. Some buds that had not reached anthesis until c. 07:00 h remained partially opened, finishing that process at the next sunrise. The anthers became dehiscent shortly before 06:00 h and pollen grains are available until c. 11:00 h. The stigma became receptive from 06:00 h. The species showed high pollen viability (100%) during the floral cycle. No scent was perceived, and osmophores were absent. Pigments reflecting UV were observed on large sections of the sepals and petals, and at the base of the stamens and pistil. Nectar production was observed between 08:00 and 12:00 h, and the sugar concentration varied from 8% to 23% (absolute values) in the same time interval.

Floral visitors and potential pollinators of H. oblongifolia var. latifolia

The flowers were visited by hummingbirds, perching birds, bees, butterflies, and beetles (Table II). The hummingbirds Discosura longicaudus, Eupetomena macroura, and Hylocharis sapphirina were first observed foraging in the canopy near 06:00 h, feeding on nectar (Fig. 1), and apparently disputing territories. Their visits were principally in the morning, until around 11:00 h, and they came into direct contact with the reproductive organs (stigma and stamens) during times of pollen grain availability and stigma receptivity (between 06:00 and 07:00 h). Pollen grains were deposited on the throats of the hummingbirds due to the exserted stamens, floral opening in relation to axis of the inflorescence, and flexible pedicel. In addition, the stigmatic surface is spatially distant from the set of anthers (i.e., positive hercogamy). Several flowers were visited when the hummingbird entered in the feeding territory, and perching on a branch or leaving the tree canopy. In the afternoon period, hummingbirds were only observed making short visits to occasional flowers.

The other floral visitors did not make contact with the sexual organs of the flowers and were considered illegitimate. Bananaquit birds (Coereba flaveola) made intense visits during the morning hours but came into contact with the flowers only laterally (i.e., piercing the flower). Apis mellifera bees were observed foraging on the flowers at around 05:00 h and during the entire day, but they only made contact with the anthers and collected pollen grains (they did not contact de stigma). Carpenter bees (Xylocopa sp.) made occasional visits between 10:00 and 11:00 h and were observed feeding on nectar laterally in the flowers. Butterflies were observed only in the early morning (around 07:15 h), landing on the petals and introducing their proboscis into the hypanthium cavity to feed on the nectar. Flies and small beetles collected pollen grains but they did not contact the stigma of the flower.

Floral traits and pollination syndrome evolution

The Bayesian analyses of the concatenated dataset did not support the monophyly of morphological sections (Fig. 2) and recovered two clades: (i) one major clade associated with distinct ecological preferences (Fig. 2; Table III), with chiropterophilous species from distinct habitats (i.e., dry and wet forests); and (ii) a second clade aggregating only species from wet forests, but which have distinct morphological traits and, consequently, distinct pollination syndromes (Fig. 2). The age of the most recent common ancestor of the genus (MRCA) was estimated at between 32.97 Mya (mean stem age) and 23.95 Mya (mean crown age) (Fig. 2).

Figure 2
Phylogenetic analysis of Hymenaea clade: outgroups are colored in gray, Hymenaea sect. Hymenaea (sensu Lee & Langenheim 1975LEE YT & LANGENHEIM JH. 1975. A systematic revision of the genus Hymenaea L. (Leguminosae; Caesalpinioideae; Detarieae). Oakland: University of California Publications in Botany, 109 p.) is in black, and Hymenaea sect. Trachylobium (sensu Lee & Langenheim 1975LEE YT & LANGENHEIM JH. 1975. A systematic revision of the genus Hymenaea L. (Leguminosae; Caesalpinioideae; Detarieae). Oakland: University of California Publications in Botany, 109 p.) is in green. Summary of the Bayesian majority-rule consensus tree sampled, with optimization of pollination syndromes, and Maximum clade credibility tree of Hymenaea L. obtained from BEAST analyses. Numbers above branches are divergence time estimates. Black arrow refesr to the calibration point for the genus Hymenaea (Hueber & Langenheim 1986HUEBER FM & LANGENHEIM J. 1986. Dominican amber tree had African ancestors. Geotimes 31: 8-10.). Absent node mean an equivocal reconstruction node. Numbers on the nodes refer to the Bayesian posterior probabilities (only values > 95%).

Reconstruction of the pollination syndrome indicated that the MRCA of the genus Hymenaea was probably pollinated by bats (bat-pollination = 37.5%, bird-pollination = 0.4%, equivocal reconstruction = 62.1%) (Fig. 2). Species with hummingbird pollination diverged later (ca. 11.95 Mya; Fig. 2), but internal resolution within the clade is too low to determine if this type of pollination evolved once or more times. Bird-pollinated species of Hymenaea differs from bat-pollinated ancestors by a shift in inflorescence arrangement, in shape and size of the flower, arrangement of the stamens and pistil, degree of asymmetry, time of anthesis and loss of odor (Table III).

DISCUSSION

Hymenaea has been associated to bat pollination due to the nocturnal flowers of the majority of species studied so far (e.g., Arroyo 1981ARROYO MTK. 1981. Breeding systems and pollination biology in Leguminosae. In: Polhill RM and Raven PH (Eds). Adv Legum Systemat 723-769., Gibbs et al. 1999GIBBS PE, OLIVEIRA PE & BIANCHI MB. 1999. Postzygotic control of selfing in Hymenaea stigonocarpa (Leguminosae--Caesalpinioideae), a bat-pollinated tree of the Brazilian cerrados. Int J Plant Sci 160: 72-78., Dunphy et al. 2004DUNPHY BK, HAMRICK JL & SCHWAGERL J. 2004. A comparison of direct and indirect measures of gene flow in the bat-pollinated tree Hymenaea courbaril in the dry forest life zone of south-western Puerto Rico. Int J Plant Sci 165: 427-436.). However, we report, for the first time, diurnal anthesis and legitimate visits by hummingbirds to the flowers of one species, Hymenaea oblongifolia Huber, which occurs in the Atlantic Rainforest. Hymenaea oblongifolia is restricted to wet habitats and exhibits a disjunct distribution, occurring in the Amazonian and Atlantic Rainforests (Lee & Langenheim 1975LEE YT & LANGENHEIM JH. 1975. A systematic revision of the genus Hymenaea L. (Leguminosae; Caesalpinioideae; Detarieae). Oakland: University of California Publications in Botany, 109 p., Lima & Pinto 2015LIMA HC & PINTO RB. 2015. Hymenaea. In: Lista de Espécies da Flora do Brasil. Jardim Botânico do Rio de Janeiro. http://floradobrasil.jbrj.gov.br/jabot/floradobrasil/FB22971/. (acessado 5 Nov 2015).
http://floradobrasil.jbrj.gov.br/jabot/flo...
). The species includes four taxonomic varieties, of which Hymenaea oblongifolia var. latifolia (the focal species) is endemic to the Atlantic Rainforest (sect. Trachylobium; Lee & Langenheim 1975LEE YT & LANGENHEIM JH. 1975. A systematic revision of the genus Hymenaea L. (Leguminosae; Caesalpinioideae; Detarieae). Oakland: University of California Publications in Botany, 109 p.). In addition, we present a preliminary analysis of the evolution of pollination syndromes in the genus Hymenaea, and reveal that the morphological sections proposed by Lee & Langenheim (1975)LEE YT & LANGENHEIM JH. 1975. A systematic revision of the genus Hymenaea L. (Leguminosae; Caesalpinioideae; Detarieae). Oakland: University of California Publications in Botany, 109 p. are not supported phylogenetically. Here, the “morphological sections” were reinterpreted as ‘floral morphs or groups’. We suggest that divergent evolution, driven by distinct pollinator guilds, might explain the distinct floral patterns observed in the genus, therefore contradicting the phylogenetic inertia hypothesis.

Bird pollination

The floral attributes of H. oblongifolia var. latifolia do not match the typical chiropterophily syndrome, as reported for H. courbaril, H. stigonocarpa and H. cangaceira, all of them with robust and strongly scented flowers, with a landing platform, rigid pedicels, and nocturnal anthesis (Arroyo 1981ARROYO MTK. 1981. Breeding systems and pollination biology in Leguminosae. In: Polhill RM and Raven PH (Eds). Adv Legum Systemat 723-769., Gibbs et al. 1999GIBBS PE, OLIVEIRA PE & BIANCHI MB. 1999. Postzygotic control of selfing in Hymenaea stigonocarpa (Leguminosae--Caesalpinioideae), a bat-pollinated tree of the Brazilian cerrados. Int J Plant Sci 160: 72-78., Domingos-Melo et al. 2019DOMINGOS-MELO A, MILET-PINHEIRO P, NAVARRO DM & MACHADO IC. 2019. It’s raining fragrant nectar in the Caatinga: evidence of nectar olfactory signaling in bat-pollinated flowers. Ecology: 10.1002/ecy.2914.). On the other hand, H. oblongifolia var. latifolia shows several morphofunctional floral traits that match those expected for bird-pollinated species (Muchhala 2006MUCHHALA N. 2006. The pollination biology of Burmeistera (Campanulaceae): Specialization and syndromes. Am J Bot 93: 1081-1089.), such as delicate and odorless flowers, without landing platform, a narrow tube aperture (composed of the hypanthium and corolla base), stamens and pistil clustered and projected beyond the perianth, flexible pedicels and diurnal anthesis.

The floral structure and architecture of H. oblongifolia var. latifolia enables different animals to illegitimately access floral rewards (i.e., without contacting simultaneously the reproductive organs). The stamens and pistil are clustered and projected beyond the perianth/corolla/petals so that insects (collecting pollen or nectar) generally do not come into contact with the anthers and stigma during their visits because of their small body sizes and foraging behaviors. This exposure of the reproductive organs (exserted pistil and stamens) requires a precise approach to the flower and for nectar collection to effect pollination, as exhibited here only by hummingbirds. The correspondence between the floral display and the attributes of the animal (e.g., behavior and body size) contributes to the plant pollination success and as has already been well documented for different plant species. For example, Tillandsia macropetala Wawra is a bromeliad species with bat pollinated flowers for which have been recorded o be visited by different insects, which despite accessing the floral reward, because of their small body size they did not make contact with the anthers and stigma during their visits (Aguilar-Rodríguez et al. 2014AGUILAR-RODRÍGUEZ PA, MACSWINEY GONZÁLEZ MC, KRÖMER T, GARCÍA-FRANCO JG, KNAUER A & KESSLER M. 2014. First record of bat-pollination in the species-rich genus Tillandsia (Bromeliaceae). Ann Bot 113: 1047-1055.); and Burmeistera (Campanulaceae) has many species with bat and hummingbird pollinated flowers, and a floral architecture that limits and ensures the pollinator fidelity, but illegitimate visits by several moths have nonetheless been recorded (Muchhala 2006MUCHHALA N. 2006. The pollination biology of Burmeistera (Campanulaceae): Specialization and syndromes. Am J Bot 93: 1081-1089.).

Bird-pollinated flowers do not always display all the traits typically associated with ornithophily, and hummingbirds often visit a wide spectrum of floral types while foraging for nectar (Temeles et al. 2002TEMELES EJ, LINHART YB, MASONJONES M & MASONJONES HD. 2002. The role of flower width in hummingbird bill length-flower length relationships. Biotropica 34: 68-80., Micheneau et al. 2006MICHENEAU C, FOURNEL J & PAILLER T. 2006. Bird pollination in an angraecoid orchid on Reunion Island (Mascarene Archipelago, Indian Ocean). Ann Bot 97: 965-974., Navarro et al. 2008NAVARRO L, GUITIAN P & AYENSA G. 2008. Pollination ecology of Disterigma stereophyllum (Ericaceae) in south-western Colombia. Plant Biol 10: 512-518., Las-Casas et al. 2012LAS-CASAS FMG, AZEVEDO JÚNIOR SM & DIAS MM. 2012. The community of hummingbirds (Aves: Trochilidae) and the assemblage of flowers in a caatinga vegetation. Braz J Biol 72: 51-58., Marques et al. 2015MARQUES JS, TAGLIATI MC & FARIA APG. 2015. Diurnal versus nocturnal pollination success in Billbergia horrida Regel (Bromeliaceae) and the first record of chiropterophily for the genus. An Acad Bras Cienc 87: 835-842.). Here, Discosura longicaldus (Gmelin, 1788) and Hylocharis sapphirine (Gmelin, 1788) (hummingbirds) were identified, for the first time, as pollinators of H. oblongifolia var. latifolia – a component of the canopy trees of a tropical rainforest. So far, only D. longicaudus has been reported as a floral visitor of Parkia nitida Miq. in the Amazonian rainforest (Hopkins 1984), but its contribution to the pollination of trees in the Atlantic Rainforest remains unexplored.

Floral adaptations related to hummingbird pollination are principally (Cronk & Ojeda 2008CRONK Q & OJEDA I. 2008. Bird-pollinated flowers in an evolutionary and molecular context. J Exp Bot 59: 715-727.) supported by: (i) attraction mechanisms, often food rewards (such as the dilute nectar of H. oblongifolia var. latifolia), and (ii) specific pollination mechanisms, often related to the spatial and temporal presentation of the reproductive organs (e.g., exserted stamens and pistil). The timing of anthesis is another relevant trait for identifying potential pollinators, implying ethological isolation (Grant 1994GRANT V. 1994. Modes and origins of mechanical and ethological isolation in angiosperms. P Natl Acad Sci USA 91: 3-10.). In Hymenaea, many species belonging to floral morph typical of the sect. Hymenaea (6 of the 17 total species) exhibit nocturnal anthesis (i.e., H. stigonocarpa, Gibbs et al. 1999GIBBS PE, OLIVEIRA PE & BIANCHI MB. 1999. Postzygotic control of selfing in Hymenaea stigonocarpa (Leguminosae--Caesalpinioideae), a bat-pollinated tree of the Brazilian cerrados. Int J Plant Sci 160: 72-78.; H. courbaril, Dunphy et al. 2004DUNPHY BK, HAMRICK JL & SCHWAGERL J. 2004. A comparison of direct and indirect measures of gene flow in the bat-pollinated tree Hymenaea courbaril in the dry forest life zone of south-western Puerto Rico. Int J Plant Sci 165: 427-436.; H. cangaceira, Domingos-Melo et al. 2019DOMINGOS-MELO A, MILET-PINHEIRO P, NAVARRO DM & MACHADO IC. 2019. It’s raining fragrant nectar in the Caatinga: evidence of nectar olfactory signaling in bat-pollinated flowers. Ecology: 10.1002/ecy.2914.; H. eriogyne, H. martiana and H. velutina, I.M. Souza, personal observations), restricting plant-pollinator interactions to nocturnal foraging animals, which when associated with other floral traits, indicate typical attributes of chiropterophily. Bat-pollination seems to be a predominant feature in the floral morph Hymenaea, and this has been generalized for the entire genus (Lee & Langenheim 1975LEE YT & LANGENHEIM JH. 1975. A systematic revision of the genus Hymenaea L. (Leguminosae; Caesalpinioideae; Detarieae). Oakland: University of California Publications in Botany, 109 p., Arroyo 1981ARROYO MTK. 1981. Breeding systems and pollination biology in Leguminosae. In: Polhill RM and Raven PH (Eds). Adv Legum Systemat 723-769., Gibbs et al. 1999GIBBS PE, OLIVEIRA PE & BIANCHI MB. 1999. Postzygotic control of selfing in Hymenaea stigonocarpa (Leguminosae--Caesalpinioideae), a bat-pollinated tree of the Brazilian cerrados. Int J Plant Sci 160: 72-78., Dunphy et al. 2004DUNPHY BK, HAMRICK JL & SCHWAGERL J. 2004. A comparison of direct and indirect measures of gene flow in the bat-pollinated tree Hymenaea courbaril in the dry forest life zone of south-western Puerto Rico. Int J Plant Sci 165: 427-436., Domingos-Melo et al. 2019DOMINGOS-MELO A, MILET-PINHEIRO P, NAVARRO DM & MACHADO IC. 2019. It’s raining fragrant nectar in the Caatinga: evidence of nectar olfactory signaling in bat-pollinated flowers. Ecology: 10.1002/ecy.2914.).

Trachylobium group, composed only of three species (i.e., H. oblongifolia, H. parvifolia and H. verrucose), is morphofunctionally distinct from floral morph Hymenaea by presenting long-paniculate inflorescences with smaller, slightly zygomorphic and horizontally positioned flowers on flexuous pedicels, without an additional mass of nectariferous tissue (see section description proposed by Lee & Langenheim 1975LEE YT & LANGENHEIM JH. 1975. A systematic revision of the genus Hymenaea L. (Leguminosae; Caesalpinioideae; Detarieae). Oakland: University of California Publications in Botany, 109 p.). Given the strong morphological floral similarities among the species of this group, it is possible that bird pollination (reported here for H. oblongifolia var. latifolia) is a general feature of this floral morph, as well as is bat pollination for the morphological sect. Hymenaea (i.e., ecological signatures).

Finally, bat-pollination is typically encountered in tropical regions (Faegri & van der Pijl 1979FAEGRI K & VAN DER PIJL L. 1979. The Principles of Pollination Ecology, 3nd ed., New York: Pergamon Press, Oxford, 244 p., Cronk & Ojeda 2008CRONK Q & OJEDA I. 2008. Bird-pollinated flowers in an evolutionary and molecular context. J Exp Bot 59: 715-727.), and there are many similarities between bat and bird-pollinated species (Sazima et al. 1999SAZIMA M, BUZATO S & SAZIMA I. 1999. Bat-pollinated flower assemblage and bat visitors at two Atlantic forest sites in Brazil. Ann Bot 83: 705-712., Fleming et al. 2005FLEMING TH, MUCHHALA N & ORNELAS JF. 2005. New World nectar-feeding vertebrates: community patterns and processes. In: Sánchez-Cordero V and Medellín RA (Eds). Contribuciones mastozoológicas en homenaje a Bernardo Villa. Mexico City: Instituto de Biología a Instituto de Ecología, UNAM, p. 161-184.). Nectars with low-sugar concentrations are found in both bat- and bird-pollinated species (Faegri & van der Pijl 1979FAEGRI K & VAN DER PIJL L. 1979. The Principles of Pollination Ecology, 3nd ed., New York: Pergamon Press, Oxford, 244 p., Gibbs et al. 1999GIBBS PE, OLIVEIRA PE & BIANCHI MB. 1999. Postzygotic control of selfing in Hymenaea stigonocarpa (Leguminosae--Caesalpinioideae), a bat-pollinated tree of the Brazilian cerrados. Int J Plant Sci 160: 72-78.). However, bird-pollinated species often exhibit the combination of diurnal anthesis and dilute nectar (with sugar concentrations varying between 20% and 26%) (Hainsworth & Wolf 1972HAINSWORTH FR & WOLF LL. 1972. Crop volume, nectar concentration and hummingbird energetics. Comp Biochem Phys A 42: 359-366., Baker 1975BAKER H. 1975. Sugar concentration in nectars from hummingbird flowers. Biotropica 7: 37-41., Cruden et al. 1983CRUDEN RW, HERMANN SM & PETERSON S. 1983. Patterns of nectar production and plant animal coevolution. In: Bentley B and Elias T (Eds). The Biology of Nectaries, New York: Columbia University Press, New York, USA, p. 126-152., Ackermann & Weigend 2006ACKERMANN M & WEIGEND M. 2006. Nectar, floral morphology and pollination syndrome in Loasaceae subfam. Loasoideae (Cornales). Ann Bot 98: 503-514., Cronk & Ojeda 2008CRONK Q & OJEDA I. 2008. Bird-pollinated flowers in an evolutionary and molecular context. J Exp Bot 59: 715-727.) as was found here for H. oblongifolia var. latifolia.

Evolution of floral traits and pollination syndromes

Pollinators exert selective pressures on reproductive traits of plants (e.g., morphological and ecological floral attributes; Dudash et al. 2011DUDASH MR, HASSLER C, STEVENS PM & FENSTER CB. 2011. Experimental floral and inflorescence trait manipulations affect pollinator preference and function in a hummingbird-pollinated plant. Am J Bot 98: 275-282., Temeles et al. 2013TEMELES EJ ET AL. 2013. Pollinator mediated selection in a specialized hummingbird-Heliconia system in the Eastern Caribbean. J Evolution Biol 26: 347-356., Suzuki & Ohashi 2014SUZUKI MF & OHASHI K. 2014. How does a floral colour-changing species differ from its non-colour-changing congener? - a comparison of trait combinations and their effects on pollination. Funct Ecol 28: 549-560., Ferreira et al. 2015FERREIRA C, MARUYAMA PK & OLIVEIRA PE. 2015. Convergence beyond flower morphology? Reproductive biology of hummingbird-pollinated plants in the Brazilian Cerrado. Plant Biol 18: 316-324.), contributing indirectly to floral evolution (Shemske & Bradshaw 1999, Eaton et al. 2012EATON DAR, FENSTER CB, HEREFORD J, HUANG SQ & REE RH. 2012. Floral diversity and community structure in Pedicularis (Orobanchaceae). Ecology 93: S182-S194., Armbruster 2014ARMBRUSTER WS. 2014. Floral specialization and angiosperm diversity: phenotypic divergence, fitness trade-offs and realized pollination accuracy. AoB Plants 6: plu003.). In the present study, the MRCA of Hymenaea was probably bat-pollinated (i.e., approximately 24 Mya) and the bird pollination emerging later (i.e., approximately 11.95 Mya); The morphological sections (i.e., Hymenaea and Trachylobium) proposed by Lee & Langenheim (1975)LEE YT & LANGENHEIM JH. 1975. A systematic revision of the genus Hymenaea L. (Leguminosae; Caesalpinioideae; Detarieae). Oakland: University of California Publications in Botany, 109 p. were not phylogenetically supported; and the transitions in pollination syndromes were accompanied by shifts in specific floral traits (e.g., morphology of flowers and inflorescence, timing of anthesis, presence/absence of floral scent). These results reinforce the notion that distinct floral patterns observed in the genus might be a result of divergent evolution processes driven independently by distinct pollinator guilds (i.e., hummingbirds vs. nectar-feeding bats).

Plants pollinated by vertebrates share certain morphofunctional floral traits, such as dilute nectar and large pollen grains with striate-verrucate ornamentation (Banks & Rudall 2016BANKS H & RUDALL PJ. 2016. Pollen structure and function in caesalpinioid legumes. Am J Bot 103: 423-436.). Different degrees of evolutionary specialization to vertebrate pollination by tropical and subtropical plants have been associated with taxonomic richness, body mass, and hovering ability as the result of the substantial adaptive radiation of tropical hummingbirds and nectar-feeding bats (Fleming & Muchhala 2008FLEMING TH & MUCHHALA N. 2008. Nectar-feeding bird and bat niches in two worlds: pantropical comparisons of vertebrate pollination systems. J Biogeogr 35: 764-780.). New World specialized nectarivorous vertebrates are largely glossophagine bats (family Phyllostomidae, with c. 38 species) and hummingbirds (family Trochilidae, c. 338 species; Fleming et al. 2005FLEMING TH, MUCHHALA N & ORNELAS JF. 2005. New World nectar-feeding vertebrates: community patterns and processes. In: Sánchez-Cordero V and Medellín RA (Eds). Contribuciones mastozoológicas en homenaje a Bernardo Villa. Mexico City: Instituto de Biología a Instituto de Ecología, UNAM, p. 161-184., 2009, McGuire et al. 2014MCGUIRE JA ET AL. 2014. Molecular phylogenetics and diversification of hummingbirds. Curr Biol 24: 910-916.). Boundaries between bird and bat-pollination have been hypothesized to be related to floral morphology and reward accessibility (Muchhala 2003MUCHHALA N. 2003. Exploring the boundary between pollination syndromes: bats and hummingbirds as pollinators of Burmeistera cyclostigmata and B. tenuiflora (Campanulaceae). Oecologia 134: 373-380.), or primarily to absolute nectar volumes and sugar concentrations (Ackermann & Weigend 2006ACKERMANN M & WEIGEND M. 2006. Nectar, floral morphology and pollination syndrome in Loasaceae subfam. Loasoideae (Cornales). Ann Bot 98: 503-514.). These are the results of divergent evolution processes, in which distinct pressures on floral traits were driven by distinct, ethologically isolated (i.e., diurnal hummingbirds vs. nocturnal nectar-feeding bats; Fleming et at. 2005) pollinators guilds. In addition, for the paraphyletic group composed of species considered to be bird pollinated, we might infer a process of convergent evolution driven by different birds in the geographic ranges (i.e., H. oblongifolia in Atlantic and Amazonian forests, H. parvifolia in Amazonian forests, and H. verrucosa in wet forests in Africa). These results are supported by a pollinator-mediated divergent evolution hypothesis.

Glossophaginae and hummingbirds radiated almost synchronously during the Early Miocene, approximately 22 Mya ago (Datzmann et al. 2010DATZMANN T, VON HELVERSEN O & MAYER F. 2010. Evolution of nectarivory in phyllostomid bats (Phyllostomidae Gray, 1825, Chiroptera: Mammalia). BMC Evol Biol 10: 165., McGuire et al. 2014MCGUIRE JA ET AL. 2014. Molecular phylogenetics and diversification of hummingbirds. Curr Biol 24: 910-916.) – which is highly congruent with age estimates for the appearance of bat pollination and the early diversification of Hymenaea. Different groups of plants (e.g., Tiquilia Pers., Boraginaceae, Moore & Jansen 2006MOORE JM & JANSEN RK. 2006. Molecular evidence for the age, origin, and evolutionary history of the American desert plant genus Tiquilia (Boraginaceae). Mol Phylogenet Evol 39: 668-687., Agave L., Agavaceae, Good-Avila et al. 2006GOOD-AVILA SV, SOUZA V, GAUT BS & EGUIARTE LE. 2006. Timing and rate of speciation in Agave (Agavaceae). P Natl Acad Sci USA 103: 9124-9129., Heliotropium sect. Cochranea (Miers) Kuntze, Heliotropiaceae, Luebert & Wen 2008LUEBERT F & WEN J. 2008. Phylogenetic analysis and evolutionary diversification of Heliotropium sect. Cochranea (Heliotropiaceae) in the Atacama Desert. Syst Bot 33: 390-402., Cactaceae, Arakaki et al. 2011ARAKAKI M ET AL. 2011. Contemporaneous and recent radiations of the world’s major succulent plants lineages. P Natl Acad Sci USA 108: 8379-8384.) diversified during drier and cooler global conditions after the Middle Miocene Climatic Transition that promoted the expansion of dry biomes throughout the tropics (Egan & Crandall 2008EGAN AN & CRANDALL KA. 2008. Divergence and diversification in North American Psoraleeae (Fabaceae) due to climate change. BMC Biol 6: 55., Couvreur et al. 2011COUVREUR TLP ET AL. 2011. Early evolutionary history of the flowering plant family Annonaceae: Steady diversification and boreotropical geodispersal. J Biogeogr 38: 664-680., Janssens et al. 2016JANSSENS SB, GROENINCKX I, DE BLOCK PJ, VERSTRAETE B, SMETS EF & DESSEIN S. 2016. Dispersing towards Madagascar: Biogeography and evolution of the Madagascan endemics of the Spermacoceae tribe (Rubiaceae). Mol Phylogenet Evol 95: 58-66.).

Environmental factors may drive floral diversification among species (Koski & Ashman 2016KOSKI MH & ASHMAN TL. 2016. Macroevolutionary patterns of ultraviolet floral pigmentation explained by geography and associated bioclimatic factors. New Phytol 211: 708-718.), with selective forces acting on floral traits (e.g., color, timing of anthesis, ultraviolet pigmentation; see Schemske & Bierzychudek 2001SCHEMSKE DW & BIERZYCHUDEK P. 2001. Evolution of flower color in the desert annual Linanthus parryae: Wright revisited. Evolution 55: 1269-1282., Warren & Mackenzie 2001, Coberly & Rausher 2003COBERLY LC & RAUSHER MD. 2003. Analysis of a chalcone synthase mutant in Ipomoea purpurea reveals a novel function for flavonoids: amelioration of heat stress. Mol Eco 12: 1113-1124., Koski & Ashman 2016KOSKI MH & ASHMAN TL. 2016. Macroevolutionary patterns of ultraviolet floral pigmentation explained by geography and associated bioclimatic factors. New Phytol 211: 708-718.) favoring adaptation to a particular pollinator group. For example, the rainforests present low inter-annual rainfall fluctuations and a greater abundance of hummingbird species than dry habitats, implying a larger number of bird pollinated plant species (Fleming et al. 2005FLEMING TH, MUCHHALA N & ORNELAS JF. 2005. New World nectar-feeding vertebrates: community patterns and processes. In: Sánchez-Cordero V and Medellín RA (Eds). Contribuciones mastozoológicas en homenaje a Bernardo Villa. Mexico City: Instituto de Biología a Instituto de Ecología, UNAM, p. 161-184.). Abiotic and biotic factors may also have been important in Hymenaea, where bat pollinated species have colonized both rainforest and seasonally dry vegetation, whereas the bird pollinated species seem to be restricted to rainforests, with its higher proportion of flower-visiting birds than seasonally dry habitats (Machado & Lopes 2004MACHADO IC & LOPES AV. 2004. Floral traits and pollination systems in the caatinga, a Brazilian tropical dry forest. Ann Bot 94: 365-376., Fleming et al. 2005FLEMING TH, MUCHHALA N & ORNELAS JF. 2005. New World nectar-feeding vertebrates: community patterns and processes. In: Sánchez-Cordero V and Medellín RA (Eds). Contribuciones mastozoológicas en homenaje a Bernardo Villa. Mexico City: Instituto de Biología a Instituto de Ecología, UNAM, p. 161-184., Las-Casas et al. 2012LAS-CASAS FMG, AZEVEDO JÚNIOR SM & DIAS MM. 2012. The community of hummingbirds (Aves: Trochilidae) and the assemblage of flowers in a caatinga vegetation. Braz J Biol 72: 51-58.).

In conclusion, we assume that shifts in particular floral traits throughout the diversification of Hymenaea have implicated in the distinct floral patterns observed in genus: (i) inflorescence dense, with large and robust flowers, and nocturnal anthesis; and (ii) inflorescence laxa, with small and delicate flowers, and diurnal anthesis. These patterns are the result of divergent evolution processes, in which pollinators ethologically isolated have driven distinct pressures on floral traits. On the other hand, for the paraphyletic group composed by supposed bird pollinated species, we inferred a process of convergent evolution. These evidences are supported by pollinator-mediated flower divergent evolution hypothesis – common in different lineages of angiosperm (Valente et al. 2012VALENTE LM, MANNING JC, GOLDBLATT P & VARGAS P. 2012. Did pollination shifts drive diversification in Southern African Gladiolus? Evaluating the model of pollinator-driven speciation. Am Nat 180: 83-98., van der Niet & Johnson 2012VAN DER NIET T & JOHNSON SD. 2012. Phylogenetic evidence for pollinator-driven diversification of angiosperms. Trends Ecol Evol 27: 353-361., Schiestl & Johnson 2013SCHIESTL FP & JOHNSON SD. 2013. Pollinator-mediated evolution of floral signals. Trends Ecol Evol 28: 307-315., van der Niet et al. 2014VAN DER NIET T, PEAKALL R & JOHNSON SD. 2014. Pollinator-driven ecological speciation in plants: new evidence and future perspectives. Ann Bot 113: 199-211.). Finally, we reinforce the necessity of increasing the collection efforts on flower-pollinator interactions should be mobilized to assess the contribution of the divergent evolution in Hymenaea species diversity in the Neotropics.

ACKNOWLEDGMENTS

This paper is part of the PhD Thesis of IMS in the Pos-Graduate Program in Botany of the Universidade Estadual de Feira de Santana. Constructive suggestions and questions by two anonymous referees greatly improved the final version of the manuscript. This work was sponsored by the Programa de Pesquisa em Biodiversidade do Semiárido (PPBIO), and the Sistema Nacional de Pesquisa em Biodiversidade (SISBIOTA, processes CNPq 563084/2010-3 and FAPESB PES0053/2011). This study was partially financed by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brazil (CAPES) - Finance Code 001: IMS was supported by a CAPES grant. FMH was supported by a CNPq-302381/2020-1 grant. LPQ was supported by a CNPq-Pq1A grant.

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Appendix 1


Primers used for PCR amplification and sequencing, and PCR conditions.

Publication Dates

  • Publication in this collection
    22 Oct 2021
  • Date of issue
    2021

History

  • Received
    25 Nov 2019
  • Accepted
    31 Aug 2020
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