Contrasting patterns of foraging behavior in neotropical stingless bees using pollen and honey metabarcoding.

Stingless bees are major flower visitors in the tropics, but their foraging preferences and behavior are still poorly understood. Studying stingless bee interactions with angiosperms is methodologically challenging due to the high tropical plant diversity and inaccessibility of upper canopy flowers in forested habitats. Pollen DNA metabarcoding offers an opportunity of assessing floral visitation efficiently and was applied here to understand stingless bee floral resources spectra and foraging behavior. We analyzed pollen and honey from nests of three distantly related stingless bee species, with different body size and social behavior: Melipona rufiventris, Scaptotrigona postica and Tetragonisca angustula. Simultaneously, we evaluate the local floristic components through seventeen rapid botanical surveys conducted at different distances from the nests. We discovered a broad set of explored floral sources, with 46.3 plant species per bee species in honey samples and 53.67 in pollen samples. Plant families Myrtaceae, Asteraceae, Euphorbiaceae, Melastomataceae and Malpighiaceae dominated the records, indicating stingless bee preferences for abundant resources that flowers of these families provide in the region. Results also reinforce the preference of stingless bees for forest trees, even if only available at long distances. Our high-resolution results encourage future bee-plant studies using pollen and honey metabarcoding in hyper-diverse tropical environments.

Genomic insights into rapid speciation within the world's largest tree genus Syzygium.

Species radiations, despite immense phenotypic variation, can be difficult to resolve phylogenetically when genetic change poorly matches the rapidity of diversification. Genomic potential furnished by palaeopolyploidy, and relative roles for adaptation, random drift and hybridisation in the apportionment of genetic variation, remain poorly understood factors. Here, we study these aspects in a model radiation, Syzygium, the most species-rich tree genus worldwide. Genomes of 182 distinct species and 58 unidentified taxa are compared against a chromosome-level reference genome of the sea apple, Syzygium grande. We show that while Syzygium shares an ancient genome doubling event with other Myrtales, little evidence exists for recent polyploidy events. Phylogenomics confirms that Syzygium originated in Australia-New Guinea and diversified in multiple migrations, eastward to the Pacific and westward to India and Africa, in bursts of speciation visible as poorly resolved branches on phylogenies. Furthermore, some sublineages demonstrate genomic clines that recapitulate cladogenetic events, suggesting that stepwise geographic speciation, a neutral process, has been important in Syzygium diversification.

Evolutionary lability in floral ontogeny affects pollination biology in Trimezieae.

PREMISE: There is little direct evidence linking floral development and pollination biology in plants. We characterize both aspects in plain and ornamented flowers of Trimezieae (Iridaceae) to investigate how changes in floral ontogeny may affect their interactions with pollinators through time. METHODS: We examined floral ontogeny in 11 species and documented pollination biology in five species displaying a wide range of floral morphologies. We coded and reconstructed ancestral states of flower types over the tribal phylogeny to estimate the frequency of transition between different floral types. RESULTS: All Trimezieae flowers are similar in early floral development, but ornamented flowers have additional ontogenetic steps compared with plain flowers, indicating heterochrony. Ornamented flowers have a hinge pollination mechanism (newly described here) and attract more pollinator guilds, while plain flowers offer less variety of resources for a shorter time. Although the ornamented condition is plesiomorphic in this clade, shifts to plain flowers have occurred frequently and abruptly during the past 5 million years, with some subsequent reversals. CONCLUSIONS: Heterochrony has resulted in labile morphological changes during flower evolution in Trimezieae. Counterintuitively, species with plain flowers, which are endemic to the campo rupestre, are derived within the tribe and show a higher specialization than the ornamented species, with the former being visited by pollen-collecting bees only.

Is dispersal mode a driver of diversification and geographical distribution in the tropical plant family Melastomataceae?

Species of plants with different life history strategies may differ in their seed dispersal mechanisms, impacting their distribution and diversification patterns. Shorter or longer distance dispersal is favored by different dispersal modes, facilitating (or constraining) population isolation, which can, in turn, impact speciation and species range sizes. While these associations are intuitive, few studies have explicitly tested these hypotheses for large clades of angiosperms. The plant family Melastomataceae is found on disparate habitats with different dispersal modes, representing a good model to address these questions. In this study, we reconstruct the phylogeny of Melastomataceae and gather data on their dispersal mode and range size to test the impact of dispersal mode on diversification and range size evolution. We found that abiotic dispersal is ancestral in the family, while biotic dispersal evolved multiple times. Species richness distribution is very similar across dispersal modes, although abiotically dispersed species tend to be relatively more diverse in seasonal environments. Range sizes across dispersal modes are not significantly different, although biotically dispersed species have slightly wider distributions. Model comparisons indicate that factors other than dispersal mode might have driven diversification heterogeneity. We did not find evidence for the role of dispersal mode driving diversification rates or range size in the Melastomataceae, suggesting a complex macroevolutionary scenario for this diverse angiosperm family. The bulk of changes to biotic dispersal coinciding with an increase in passerine diversification suggests a possible "past" key innovation in Melastomataceae. Future studies should investigate the role of other diversification drivers in the family and the relatively higher diversity of abiotically dispersed species in open habitats.

Fast diversification through a mosaic of evolutionary histories characterizes the endemic flora of ancient Neotropical mountains.

Mountains are among the most biodiverse areas on the globe. In young mountain ranges, exceptional plant species richness is often associated with recent and rapid radiations linked to the mountain uplift itself. In ancient mountains, however, orogeny vastly precedes the evolution of vascular plants, so species richness has been explained by species accumulation during long periods of low extinction rates. Here we evaluate these assumptions by analysing plant diversification dynamics in the campo rupestre, an ecosystem associated with pre-Cambrian mountaintops and highlands of eastern South America, areas where plant species richness and endemism are among the highest in the world. Analyses of 15 angiosperm clades show that radiations of endemics exhibit fastest rates of diversification during the last 5 Myr, a climatically unstable period. However, results from ancestral range estimations using different models disagree on the age of the earliest in situ speciation events and point to a complex floristic assembly. There is a general trend for higher diversification rates associated with these areas, but endemism may also increase or reduce extinction rates, depending on the group. Montane habitats, regardless of their geological age, may lead to boosts in speciation rates by accelerating population isolation in archipelago-like systems, circumstances that can also result in higher extinction rates and fast species turnover, misleading the age estimates of endemic lineages.

Biogeography and early diversification of Tapinotaspidini oil-bees support presence of Paleocene savannas in South America.

Worldwide distributed tropical savannas were established only in the Miocene, with climatic cooling and rise of C4 grasses. However, there is evidence for an earlier presence of savanna-like vegetation in southern parts of South America. Here we investigated the biogeographic history of a clade of solitary bees which have endemic groups in areas covered by savannas and other types of open vegetation as well as forested areas. We hypothesized that these bees originated in savanna-like biomes and that shifts to forested areas and floral host shifts increased species diversification along their evolutionary history. We reconstructed a comprehensive phylogeny for Tapinotaspidini bees based majorly on original DNA sequences. We then used macroevolutionary tools to estimate ancestral range area and reconstructed ancestral habitat (open versus forested) and host plant association to analyze the effects of shifts in vegetation type and flower hosts on their diversification. Tapinotaspidini bees originated in the Paleocene and in a savanna-type, Cerrado-like, which is reinforced by reconstruction of open vegetation as the most probable ancestral area, thus bringing additional evidence to a much earlier origin of this vegetation type in South America. Shifts to forested areas occurred at least three times in a period of 30 Ma and were responsible for slight increases in diversification rates. Malpighiaceae is the ancestral floral host; host broadening occurred only in the Miocene and at least in three occasions. Host shifts, i.e. from Malpighiaceae to other oil families, occurred in the Eocene and Miocene. Both host broadening and host shifts did not significantly alter diversification rates, however exploitation of other oil sources were important in occupying new habitats. The link between biomes and host plant shifts and changes in diversification rate brings us additional insights into the evolution of bees and associated flora in South America.

Calyx (con)fusion in a hyper-diverse genus: Parallel evolution of unusual flower patterns in Eugenia (Myrtaceae).

Eugenia has a pantropical distribution and comprises ca. 1000 species found mostly in the Neotropics. Recent DNA based phylogenies show that unusual flower morphology of 'eugenioid' collections, e.g. fused calices that open by tearing, consistently emerged within Eugenia. These results emphasize a demand to revaluate flower morphology in a phylogenetic context within the genus. A reassessment of calyx fusion in Eugenia and traditionally related genera is here focused on clarification of the systematic relevance of this apparently recurrent characteristic. Twenty-four Eugenia species with some level of calyx fusion in the bud were newly used (one nuclear and four plastid markers) in conjunction with a representative sample of previously sequenced species to recover a time-calibrated Eugenia phylogeny of 86 accessions. Development of the fused calyx was analysed using scanning electron microscopy, differing patterns were re-coded and subsequently phylogenetic character reconstruction was performed. Eugenia was recovered as monophyletic including the traditionally segregated genera Calycorectes and Catinga. Ancestral character reconstruction uncovered free calyx lobes as the ancestral condition. Five development patterns leading to calyx fusion are reported in Eugenia including species with apparently six petals, which contrast with the standard tetramerous flowers. This condition is interpreted as the petaloid pattern, where two external fused calyx lobes cover the bud while two internal calyx lobes are free and petaloid. The fused calyx condition is homoplastic and evolved independently, several times in Eugenia, as did the different development patterns. Data presented here show that systematic incongruence resulting from multiple, independent origins of the fused calyx in Eugenia is further aggravated by an inability to distinguish parallelism and convergence within the recovered patterns.

Advanced understanding of phylogenetic relationships, morphological evolution and biogeographic history of the mega-diverse plant genus Myrcia and its relatives (Myrtaceae: Myrteae).

Myrcia is the largest exclusively Neotropical genus of the plant family Myrtaceae with c. 770 species. Although several studies have elucidated the relationships within particular sections of the genus, to date no phylogeny has been produced that includes a broad taxonomic and geographic representation. Here we present a phylogenetic hypothesis of Myrcia and close relatives comprising 253 species and based on two nuclear and seven plastid markers. We combine previously available sequence data with 234 new sequences of the genus Myrcia for this study. We use this phylogeny to investigate the evolution of selected morphological traits and to infer the biogeographic history of the genus. Our results yield a highly supported phylogenetic tree where the Myrceugenia group is sister to the Myrcia and Plinia groups. Five Myrcia species previously considered unplaced emerge in a newly circumscribed clade. The monophyly of two Myrcia sections previously considered uncertain, Aulomyrcia and Gomidesia, are confirmed with strong support. Flowers with free calyx lobes, 2-locular ovaries, and anthers with symmetrical thecae are ancestral features of Myrcia. The Myrcia sect. Gomidesia is highly supported and recovered as monophyletic, with asymmetric anthers that retain their curvature after dehiscence as a morphological synapomorphy. The Atlantic Forest is the most likely ancestral area of the genus and most of its internal clades, from where multiple lineages colonized different regions of South and Central America, in particular the Brazilian Cerrado through multiple unidirectional range expansions. The southern Atlantic Forest is the ancestral area for Myrcia sect. Gomidesia, with lineages reaching the northern Atlantic Forest, Cerrado, Yungas, and other savanna vegetation of South America. Our results provide a solid backbone for further evolutionary and taxonomic work and clarify several previously uncertain relationships in this mega-diverse plant group, and shed light on its geographical range evolution.

Harnessing the potential of integrated systematics for conservation of taxonomically complex, megadiverse plant groups.

The value of natural history collections for conservation science research is increasingly recognized, despite their well-documented limitations in terms of taxonomic, geographic, and temporal coverage. Specimen-based analyses are particularly important for tropical plant groups for which field observations are scarce and potentially unreliable due to high levels of diversity-amplifying identification challenges. Specimen databases curated by specialists are rich sources of authoritatively identified, georeferenced occurrence data, and such data are urgently needed for large genera. We compared entries in a monographic database for the large Neotropical genus Myrcia in 2007 and 2017. We classified and quantified differences in specimen records over this decade and determined the potential impact of these changes on conservation assessments. We distinguished misidentifications from changes due to taxonomic remodeling and considered the effects of adding specimens and georeferences. We calculated the potential impact of each change on estimates of extent of occurrence (EOO), the most frequently used metric in extinction-risk assessments of tropical plants. We examined whether particular specimen changes were associated with species for which changes in EOO over the decade were large enough to change their conservation category. Corrections to specimens previously misidentified or lacking georeferences were overrepresented in such species, whereas changes associated with taxonomic remodeling (lumping and splitting) were underrepresented. Among species present in both years, transitions to less threatened status outnumbered those to more threatened (8% vs 3%, respectively). Species previously deemed data deficient transitioned to threatened status more often than to not threatened (10% vs 7%, respectively). Conservation scientists risk reaching unreliable conclusions if they use specimen databases that are not actively curated to reflect changing knowledge.

Empleo del Potencial de la Sistemática Integrada para la Conservación de Grupos Botánicos Complejos y Megadiversos Resumen Cada vez se reconoce más el valor que tienen las colecciones de historia natural para la investigación dentro de la ciencia de la conservación, a pesar de las limitaciones en la documentación adecuada de la cobertura taxonómica, geográfica y temporal. Los análisis con base en especímenes son de particular importancia en los grupos de plantas tropicales, para los cuales las observaciones en el campo son escasas y potencialmente de poca confianza debido la gran cantidad de retos de identificación que amplifican la diversidad. Las bases de datos de especímenes que son curadas por especialistas son fuentes abundantes de datos identificados autoritativamente y de distribución georeferenciada, por lo que es urgente la necesidad de dichos datos para géneros extensos. Comparamos las entradas en una base de datos monográfica para el género neotropical extenso Myrcia en 2007 y en 2017. Clasificamos y cuantificamos las diferencias en los registros de especímenes durante esta década y determinamos el impacto potencial de estos cambios sobre las evaluaciones de conservación. Separamos las identificaciones erróneas de los cambios causados por la remodelación taxonómica y consideramos los efectos de la adición de especímenes y de georeferencias. Calculamos el impacto potencial de cada cambio sobre las estimaciones de la extensión de la distribución (EOO, en inglés), la medida que se utiliza con mayor frecuencia en las valoraciones del riesgo de extinción de las plantas tropicales. Examinamos si los cambios en un espécimen particular estuvieron asociados con especies para las cuales los cambios en la EOO durante la década fueron lo suficientemente amplios para cambiarles la categoría de conservación. Las correcciones hechas a especímenes previamente mal identificados o carentes de georeferencias estuvieron sobre-representadas en dichas especies, mientras que los cambios asociados con la remodelación taxonómica (agrupamiento y separación) estuvieron sub-representados. Entre las especies presentes en ambos años, las transiciones hacia un estado de conservación con menor amenaza superaron en cantidad a aquellas hacia un estado de mayor amenaza (8% vs 3%, respectivamente). Las especies que con anterioridad se clasificaban como deficientes de datos tuvieron más transiciones hacia el estado de amenazadas que hacia el estado de no amenazadas (10% vs 7%, respectivamente). Los científicos de la conservación corren el riesgo de llegar a conclusiones poco confiables si utilizan bases de datos de especímenes que no sean actualizadas para reflejar el conocimiento cambiante.

Floral uniformity through evolutionary time in a species-rich tree lineage.

Changes in floral morphology are expected across evolutionary time and are often promoted as important drivers in angiosperm diversification. Such a statement, however, is in contrast to empirical observations of species-rich lineages that show apparent conservative floral morphologies even under strong selective pressure to change from their environments. Here, we provide quantitative evidence for prolific speciation despite uniform floral morphology in a tropical species-rich tree lineage. We analyse floral disparity in the environmental and phylogenetic context of Myrcia (Myrtaceae), one of the most diverse and abundant tree genera in Neotropical biomes. Variation in floral morphology among Myrcia clades is exceptionally low, even among distantly related species. Discrete floral specialisations do occur, but these are few, present low phylogenetic signal, have no strong correlation with abiotic factors, and do not affect overall macroevolutionary dynamics in the lineage. Results show that floral form and function may be conserved over large evolutionary time scales even in environments full of opportunities for ecological interactions and niche specialisation. Species accumulation in diverse lineages with uniform flowers apparently does not result from shifts in pollination strategies, but from speciation mechanisms that involve other, nonfloral plant traits.

Floral heterochrony promotes flexibility of reproductive strategies in the morphologically homogeneous genus Eugenia (Myrtaceae).

Background and Aims: Comparative floral ontogeny represents a valuable tool to understand angiosperm evolution. Such an approach may elucidate subtle changes in development that discretely modify floral architecture and underlie reproductive lability in groups with superficial homogeneous morphology. This study presents a comparative survey of floral development in Eugenia (Myrtaceae), one of the largest genera of angiosperms, and shows how previously undocumented ontogenetic trends help to explain the evolution of its megadiversity in contrast to its apparent flower uniformity. Methods: Using scanning electron microscopy, selected steps of the floral ontogeny of a model species (Eugenia punicifolia) are described and compared with 20 further species representing all ten major clades in the Eugenia phylogenetic tree. Additional floral trait data are contrasted for correlation analysis and character reconstructions performed against the Myrtaceae phylogenetic tree. Key results: Eugenia flowers show similar organ arrangement patterns: radially symmetrical, (most commonly) tetramerous flowers with variable numbers of stamens and ovules. Despite a similar general organization, heterochrony is evident from size differences between tissues and structures at similar developmental stages. These differences underlie variable levels of investment in protection, subtle modifications to symmetry, herkogamic effects and independent androecium and gynoecium variation, producing a wide spectrum of floral display and contributing to fluctuations in fitness. During Eugenia's bud development, the hypanthium (as defined here) is completely covered by stamen primordia, unusual in other Myrtaceae. This is the likely plesiomorphic state for Myrteae and may have represented a key evolutionary novelty in the tribe. Conclusions: Floral evolution in Eugenia depends on heterochronic patterns rather than changes in complexity to promote flexibility in floral strategies. The successful early establishment of Myrteae, previously mainly linked to the key innovation of fleshy fruit, may also have benefitted from changes in flower structure.

Myrteae phylogeny, calibration, biogeography and diversification patterns: Increased understanding in the most species rich tribe of Myrtaceae.

Myrteae (c. 2500 species; 51 genera) is the largest tribe of Myrtaceae and an ecologically important groups of angiosperms in the Neotropics. Systematic relationships in Myrteae are complex, hindering conservation initiatives and jeopardizing evolutionary modelling. A well-supported and robust phylogenetic hypothesis was here targeted towards a comprehensive understanding of the relationships within the tribe. The resultant topology was used as a base for key evolutionary analyses such as age estimation, historical biogeography and diversification rate patterns. One nuclear (ITS) and seven chloroplast (psbA-trnH, matK, ndhF, trnl-trnF, trnQ-rps16, rpl16 and rpl32-trnL) DNA regions for 115 taxa representing 46 out of the 51 genera in the tribe were accessed and analysed using maximum likelihood and Bayesian inference tools for phylogenetic reconstruction. Dates of diversification events were estimated and contrasted using two distinct fossil sets (macro and pollen) in BEAST. The subsequent dated phylogenies were compared and analysed for biogeographical patterns using BioGeoBEARS and diversification rates using BAMM. Myrteae phylogeny presents strong statistical support for three major clades within the tribe: Australasian group, Myrtus group and Main Neotropical Lineage. Dating results from calibration using macrofossil are an average of 20 million years older and show an early Paleocene origin of Myrteae, against a mid-Eocene one from the pollen fossil calibration. Biogeographic analysis shows the origin of Myrteae in Zealandia in both calibration approaches, followed by a widespread distribution throughout the still-linked Gondwana continents and diversification of Neotropical endemic lineages by later vicariance. Best configuration shift indicates three points of acceleration in diversification rates, all of them occurring in the Main Neotropical Lineage. Based on the reconstructed topology, several new taxonomic placements were recovered, including: the relative position of Myrtus communis, the placement of the Blepharocalyx group, the absence of generic endemism in the Caribbean, and the paraphyletism of the former Pimenta group. Distinct calibration approaches affect biogeography interpretation, increasing the number of necessary long distance dispersal events in the topology with older nodes. It is hypothesised that biological intrinsic factors such as modifications of embryo type and polyploidy might have played a role in accelerating shifts of diversification rates in Neotropical lineages. Future perspectives include formal subtribal classification, standardization of fossil calibration approaches and better links between diversification shifts and trait evolution.

Floral cost vs. floral display: Insights from the megadiverse Myrtales suggest that energetically expensive floral parts are less phylogenetically constrained.

PREMISE OF THE STUDY: Floral display describes the effect of flower size combined with the number of flowers per inflorescence. There is strong evidence that a floral-display trade-off operates under energetic constraint, with few-flowered inflorescences likely to have larger flowers than many-flowered inflorescences. Flower size can be estimated by different variables; thus, we propose that the variable for flower size that is most highly (negatively) correlated with the number of flowers per inflorescence will also be the best estimate of floral cost. Ranking the correlation with the phylogenetic signal of the variable can provide additional insight into the evolution of floral display. METHODS: The Myrtales were chosen as a model order based on age, worldwide distribution, and diversity of reproductive strategies. Ninety-nine species representing all families and one quarter of generic diversity across its geographic and ecological range were sampled to reconstruct a phylogeny based on rbcL and ndhF sequences. Correlation coefficients were calculated for flower size variables vs. the number of flowers per inflorescence. Phylogenetic signal was measured for all variables and for floral display. KEY RESULTS: Flowers per inflorescence showed significant negative correlation with the following flower size variables (weakest to strongest): filament length < anther size < flower depth < flower diameter. As the correlation of each character with number of flowers per inflorescence rose (suggesting increased cost), the values for phylogenetic signal diminished (suggesting less constraint). CONCLUSIONS: We conclude that energetically costly floral characters appear to be less phylogenetically constrained, while low-cost floral characters maintain higher levels of phylogenetic inertia.