Research Projects

Regional diversity is a product of the number of species found in a given location (alpha diversity) and the turnover in species composition across space and in different environments or habitats (beta diversity). In the lowland Amazon, there exists substantial turnover in species composition among terra firme, flooded and white-sand forests (Fine et al., 2010, Annals of the Missouri Botanical Garden). My dissertation work addressed the processes responsible for this compositional turnover in tree species on different soil types in the Western Amazon.  I performed a phylogenetically-controlled reciprocal transplant experiment with 20 species from six tree genera in nutrient-poor white sand forests and nutrient-rich clay forests. I manipulated the presence of herbivores to see if the evolutionary tradeoff between defense investment and growth rate could explain the maintenance of habitat specialization.  The results were clear and indicate that habitat specialization in these forests cannot be explained by soil factors alone.  Clay specialist species outperformed white-sand specialists in both soil types when protected from herbivores.  However, when unprotected, white-sand specialists dominated in white-sand forests and clay specialists dominated in clay forests. Herbivores, by preferentially attacking clay forest plants in white sand soils, thereby magnify habitat boundaries and increase the potential for habitat specialization in plants (Fine et al. 2004, Science). In addition, white-sand and clay specialist tree species exhibited a growth-defense tradeoff; functional traits associated with white-sand or clay specialization were not plastic responses to different soil types, but instead fixed traits shaped by natural selection that have evolved repeatedly by convergent evolution.  This fundamental tradeoff, mediated by herbivores, represents an important mechanism of plant coexistence that has been largely overlooked in studies of plant habitat specialization and niche assembly (Fine et al. 2006, Ecology, Fine and Baraloto 2016, Biotropica).

While evidence suggests that habitat specialization can be well explained by growth-defense tradeoffs, these insights remain to be integrated with large-scale distributional data and phylogenetic data in order to understand the general importance of habitat specialization at much larger taxonomic and geographic scales. Together with two collaborators, Chris Baraloto (INRA-France) and John Lokvam (Utah), I was awarded an NSF grant in Ecology (2008-2011) and Chris Baraloto was awarded a grant from ANR-France (2013-2018) to address the evolution of habitat specialization within five widespread Amazonian tree lineages in three countries spanning the entire Amazon basin. Together we have installed 116 0.1 ha plots in white sand, terra firme and flooded forest in Peru,  Brazil and French Guiana to test for patterns of habitat specialization within each country.  We have invested a great deal of time and energy to produce accurate, standardized species inventory data. All morphospecies from the 116 plots have been vouchered, and five important and species-rich lineages have been DNA-barcoded. All vouchers from the 80 plots in Peru and French Guiana were brought to the Missouri Botanical Garden (the largest Neotropical plant collection in the world), where we standardized the identification of lineages between Peru and French Guiana. In our 80 plots from just Peru and French Guiana, we have found more than 2300 tree morphospecies – and only a 6.5% overlap between countries. Initial results suggest that species from our focal tree lineages exhibit strong habitat associations (Lamarre et al. 2012, Ecology), but the most important papers summarizing our tree inventory results have yet to be published, although one is in in second revision (Baraloto et al., in review Ecological Monographs). 

We evaluated the contributions of abiotic and biotic mechanisms of performance tradeoffs by quantifying traits that confer flood tolerance, nutrient use efficiency, and chemical defense using a reciprocal transplant experiment that includes both habitat generalist and specialist tree species from each of our five focal lineages. Reciprocal transplant experiments with twenty species each in French Guiana and in Peru were conducted between 2009-2012. We found that habitat specialists outperformed non-specialists in each habitat, especially in seasonally-flooded and white-sand forests (Fortunel et al. 2016, Ecology Letters).

We have measured soil characteristics and many physiological plant traits in the different habitat types and found evidence consistent with discrete and divergent plant strategies being favored in each habitat type (Fortunel, et al., 2012, Functional Ecology; Fortunel et al. 2014, Journal of Ecology, Fortunel et al. 2014, New Phytologist).   We have also conducted multi-trophic inventories in the same plots where we have tree species abundance data, functional traits, and climate and soil variables.  Students affiliated with our project developed innovative ways to sample insects in the tree plots using flight intercept traps (Lamarre et al. 2012, ZooKeys), light traps, fruit traps and tree sapling insect traps (Lopes et al. 2019, Entomologia Experimentalis et Applicata.). During his Ph.D. dissertation, working in Peru and French Guiana, Greg Lamarre collected more than 200,000 insects to compare diversity patterns between regions and across habitats. We found strong patterns of functional and phylogenetic betadiversity across habitats in insect communities (Lamarre et al. 2016, Journal of Animal Ecology, Lamarre et al. 2016, Biotropica).  Similarly, in collaboration with Kabir Peay (Stanford), we surveyed fungal diversity in our Peruvian plots. We found that fungal and tree diversity were well-correlated, and that beta-diversity of fungal pathogens and tree beta-diversity were also correlated (Peay et al., 2013, The ISME Journal). These findings are consistent with what one would expect if fungal pathogens were responsible for causing density-dependent mortality in trees according to the Janzen-Connell hypothesis, one of the leading theories explaining the exceptional diversity of tropical forests.  In part C (see below), I describe our efforts to further investigate density dependent mortality and its role in driving patterns of commonness and rarity in tropical trees.

     During my Ph.D. work, I started research on the genera Protium, Crepidospermum and Tetragastris (the tribe Protieae from the family Burseraceae), which together arguably constitute one of the most ecologically important tree clades of the Neotropics. These three genera have been formally named as one monophyletic group – Protium (Daly and Fine, 2019 Brittonia). Encompassing ca.140 species, this group can achieve local diversities of 25-30 species per square km in Peru, Ecuador, Colombia and Brazil and more than 80 species have been recorded across the Amazon and Guianas. Moreover, in terms of abundance, in the most recent count from the Amazon Tree Diversity Network (1140+ plots across South America, ter Steege et al. 2013 Science) – the Protieae were the second most dominant clade, ranking only behind Eschweilera (Lecythidaceae).

Together with four collaborators (Susan Pell, Brooklyn Botanical Garden; Doug Daly, New York Botanical Garden; Andrea Weeks, George Mason; Brian Moore, UC Davis), I was awarded an NSF-grant in Systematics and Biological Inventories (2009-2012) to continue this research and to reconstruct the phylogeny of the Anacardiaceae-Burseraceae lineage, in order to improve our knowledge of infrafamilial classification and generic delimitation. We collected as many species of this lineage as possible, traveling to Vietnam, Papua New Guinea, Indonesia, Brazil, Colombia, Ecuador, Peru, French Guiana, and Cuba, and organizing colleagues to collect from many other countries so we could obtain material silica-dried in the field, which yields high-quality DNA sequences. For Protium, we were able to collect vouchers for 102 of the 140 species in the group, and our sampling had reasonably good geographic coverage, although we lacked  samples from the Andes (see below).  We have reconstructed a fossil-calibrated multilocus phylogeny based on three nuclear and two chloroplast genes and use these phylogenies to conduct biogeographic and habitat reconstructions, as well as investigate community phylogenetic structure within regions and lineage through time plots (Fine et al, 2014, Evolution). In addition, we were able to compare and contrast the different tempos and modes of diversification between the two sister families, Anacardiaceae and Burseraceae (Weeks et al. 2015, Frontiers in Genetics).

Our findings have implications for the origins of tropical diversity.  Although the Burseraceae is a classic “Boreotropical” clade with fossils from the Paleocene and Eocene of Europe and North America, we find that the oldest common ancestor to all Neotropical Protieae is reconstructed to be South American.  The Protieae radiated in the Amazon and then secondarily dispersed to Central America, the Caribbean, the Brazilian Atlantic Forest and the Chocó biogeographic region (Ecuador and Colombia).  This surprising result may be explained by the contraction of lowland rain forest habitat in Central America during the Oligocene and early Miocene – meaning that although the Protieae once lived in North America and likely dispersed to South America via Central America and/or the Caribbean – these progenitors must have gone extinct soon afterwards and thus all extant Caribbean and Central American taxa come from Amazonian ancestors. In the Miocene, dispersal among biogeographic regions in the Neotropics occurred in several lineages within the clade. This has resulted in little geographic structure across the phylogeny but consistent patterns were found towards the tips suggesting many instances of recent in-situ speciation within regions. We found that habitat specialization to sandy soil and flooded forest habitats has occurred many times independently, especially in the Amazon. That this accumulation of Amazonian diversity occurred in the Miocene agrees with recent paleogeographic reconstructions that indicate that this period involved major changes in the Western Amazon (Fine et al, 2014, Evolution).  Interestingly, the Anacardiaceae exhibit very different patterns than the Burseraceae, with multiple transitions between wet and dry habitats, and temperate and tropical biomes found in the Anacardiaceae, suggesting that for each family, the combination of dispersal ability and physiological adaptation drives diversification in very different ways (Weeks et al. 2015, Frontiers in Genetics).

My phylogenetic results with the tropical family Burseraceae support the hypothesis that edaphic heterogeneity can drive parapatric speciation, i.e. steep environmental gradients allow for evolutionary divergence between adjacent populations in the presence of limited gene flow. In order to investigate speciation mechanisms, we began an intensive study on one species, Protium subserratum Engl. that inhabits white-sand, brown-sand and clay forests throughout its range. This taxon includes several morphologically-distinct ecotypes that are associated with different habitats and geographic regions. When we began our research, it was unknown whether these ecotypes were genetically-based or simply plastic responses to different environments.

First, we reconstructed the genealogical relationships of populations from both white-sand forest and terra firme (brown sand and clay) of Protium subserratum and its close relatives using chloroplast and single-copy nuclear DNA markers from the hundreds of trees that I have collected with colleagues and georeferenced from multiple sites in the Peruvian Amazon, Brazilian Amazon, Guyana, and French Guiana. Protium subserratum is a morphologically variable taxon that has several different morphotypes, (some linked to soil type) and it is the sister species to two white-sand specialists, one of which is a new species Protium alvarezianum Daly and P. Fine that we recently published (Daly and Fine 2011, Systematic Botany).  In addition, we have found that edaphic specialization has evolved multiple times just within Protium subserratum (Fine et al. 2013, Journal of Biogeography) and that soil type is a much more important factor than distance in structuring phylogeographic patterns, a result consistent with the predictions of ecological speciation.

My former graduate student, Dr. Tracy Misiewicz, developed a microsatellite library for Protium subserratum to quantify the level of gene flow between white sand and clay lineages and to investigate patterns of spatial genetic structure within populations (Misiewicz et al., 2012, American Journal of Botany; Barbosa et al. 2013, PLoS ONE).  These microsatellites have allowed us to quantify the extent of gene flow between populations of Protium subserratum on the two soil types. We have found a small number of hybrids (Misiewicz and Fine, 2014, Molecular Ecology), consistent with the hypothesis that divergence with gene flow is occurring in this tree species complex.  This is a major finding and could indicate that ecological speciation across environmental gradients is a common phenomenon that may explain the high prevalence of habitat specialist trees in the Amazonian flora (Misiewicz et al., 2020, Ecology and Evolution).  

Since then, recently-graduated PhD student Gabriel Damasco has been investigating another species complex (Protium heptaphyllum) that occurs in white-sand and clay habitats in the Amazon basin, the Cerrado, and the Atlantic Forest. He has used DDRAD approaches and Next-Generation Sequencing (NGS) to resolve the taxonomy of this extremely common Protium species (one of the top 20 most common species in the Amazon basin, according to ter Steege et al. 2013, Science). He has found that this taxon actually represents multiple species, casting doubt on its hyperdominance (Damasco et al., 2019, Taxon, Damasco et al., in review). Another of my PhD students, Ricardo Perdiz (at INPA in Manaus) also did a molecular phylogenetic study on the Protium aracouchini species complex using next-generation sequencing techniques. His results are just beginning to be published (Perdiz et al. 2020, Phytokeys). In addition, Tracy and Gabriel have worked to develop the protocols that will make it possible for us to use NGS approaches to reconstruct the entire Protium phylogeny.  After several successful NGS runs, we now have more than 40 species of Protium sequenced, with hundreds of phylogenetically-informative loci.

Future Directions:

In January 2018. I received funding from the National Geographic Society to conduct expeditions to Peru and Colombia and to collect existing and discover new Andean species and also to use NGS approaches to investigate their phylogenetic relationships.  I have made trips to Antioquia, Colombia and Cusco and Madre de Dios, Peru in 2018 and to Antioquia, Colombia and Parque-Nacional Yanachaga-Chemillen in Peru in 2019.  In the new field expeditions, I was able to collect specimens from 9 different undescribed species of Protium and also make important collections of 3 newly described montane species of Protium that had not been included in our phylogeny.

My collaborator Douglas Daly of the NYBG, Tracy Misiewicz and I will soon submit a proposal to the NSF Systematics and Biological Inventories panel to obtain funding for the systematic monograph of the genus Protium, proposing to conduct the molecular phylogenetics and taxonomic revision of all named and undescribed Protium species. We will use NGS to reconstruct a phylogeny of all Protium species. In the years ahead, my laboratory will use this new phylogeny of Protium to investigate the role of morphological traits, ecological specialization, and biogeographic history on species diversification. In particular, chemical defense data from Protium species will be correlated with insect herbivore and fungal pathogen assemblages and phylogenies (see next section). In our speciation studies, we plan to use next-generation sequencing techniques on species complexes like Protium subserratum to identify a large number of fixed single nucleotide polymorphisms (SNPs) in multiple populations from different soil types across the range. My former PhD student Tracy Misiewicz received an NSF postdoctoral fellowship to study the Protium subserratum species complex and sequence collections across all of Amazonia. These techniques generate on the order of hundreds or thousands of diagnostic SNP loci, dramatically improving the resolution of population genetic parameters.  This will allow her to use powerful coalescent models, as well as quantify natural selection at different loci from different populations across the edaphic gradient.

Biologists have long hypothesized that natural enemies are driving the origin and maintenance of diversity in their host plants, especially in the extraordinarily diverse tropical rainforests. Plant diversification has been thought to be driven by the evolution of novel defenses that deter specialist enemies, which allows plants to expand their ranges and diversify. Yet, the mechanism by which Ehrlich and Raven’s (1964) “escape and radiate” hypothesis leads to speciation has never been fully elucidated. I propose that when range expansion involves entering a new habitat type, this could accelerate defense evolution if habitats contain different assemblages of herbivores and/or divergent resource availabilities that affect plant defense allocation. Gene flow between populations across a habitat boundary could disrupt locally-adapted defenses, and if intermediate phenotypes were unfit, this would cause selection for reinforcement of divergent traits and reproductive isolation between populations with different specialist enemies and novel defense strategies. Very little is known about the species of fungal pathogens and herbivores that attack tropical plants, how specialized they are and their geographic distributions – critical information to evaluate the potential of natural enemies to promote diversification in plants.

In January 2013, I was awarded an NSF CAREER grant entitled: “Natural enemies, chemical defenses and the diversification of Amazonian trees” to investigate the natural enemies that attack 45 species of Protium in Peru and Brazil and simultaneously characterize the chemical defenses in these plants.  Since 2013, together with Dr. Diego Salazar and Dr. John Lokvam and teams of Peruvian and Brazilian field assistants, we have been studying intensively the insect herbivore assemblages and chemical defense investment from 858 individuals from 34 species of Protieae (Burseraceae) from Iquitos, Peru, and 175 individuals from 16 species of Protieae from Manaus, Brazil (14 of these species are also found in Peru). In this project we have collected almost 4500 feeding records and 3500 insect herbivores (mostly hemipteran phloem-feeders, lepidopterans, and coleopterans, in order of importance) feeding directly on these plants. In the laboratory, using volatile chemical (GC-MS) and non-volatile chemical analysis (HPLC with diode-array, evaporative light-scattering and mass spectrometric detection) (See Lokvam et al., 2015, Chemoecology for a description of some of these methods) we have identified and quantified more than 600 different volatile and non-volatile secondary metabolites found in these 36 species. Our recently published species-level phylogeny of Protium (Fine et al. 2014, Evolution) includes all of the Peruvian and Brazilian species in our sample, allowing us to test the hypothesis that phylogenetic relatedness may predict chemical similarity in host plants and/or similarity in natural enemy assemblage.

We used a combination of ecological, phylogenetic, metabolomic, and statistical learning tools to accomplish the most comprehensive analysis of chemically mediated plant-herbivore interactions to date and test Ehrlich and Raven’s (1964) Escape and Radiate theory at a macroevolutionary scale (Salazar et al., 2018, Nature Ecology & Evolution). Our results show, for the first time: (a) that the most effective anti-herbivore plant secondary metabolites are more conserved across the phylogeny, (b) that there is high variability in the ecological and evolutionary value of specific plant secondary metabolites, and (c) that generalist herbivores play a major role in the evolution of plant chemical diversity. The results of this work challenge longstanding ecological hypotheses concerning the evolution of plant chemical defenses and suggest that plant chemical diversity arises from the cumulative result of a multitude of plant-herbivore interactions rather than the outcome of a chemical defense escalation driven by a small set of specialized herbivores. We believe that our findings represent a fundamental advance in the understanding of plant-insect interactions and chemical defense theory (Salazar et al., 2018, Nature Ecology & Evolution).

Our detailed chemical characterization database of Protium species allows us to test other major ecological hypotheses. For example, density-dependent enemy attacks have been proposed to be a major driver for the local coexistence of chemically divergent congeneric species. At the same time, we expect local soil conditions to favor the coexistence of species sharing similar functional traits related to resource use strategies, while environmental heterogeneity would promote the diversity of these traits at both local and large spatial scales. To test how traits were related to local coexistence, we used presence-absence data for 36 species from the plant genus Protium (Burseraceae), collected in 19 plots (2 ha each) in the Peruvian Amazon. We characterized the presence-absence of 189 plant secondary metabolites for 28 of these species, and 14 functional traits associated with resource use strategies for 16 species. Using this information, we found that secondary metabolites were significantly more dissimilar than null expectations for species co-occurring within plots, whereas functional traits were significantly more similar (Vleminckx et al. 2018, Frontiers in Plant Science). These results were consistent with the hypothesis that density-dependent enemy attacks contribute to the local coexistence of congeneric species displaying divergent chemical defenses, whereas local habitat conditions filter species with similar traits related to resource use strategies (Vleminckx et al. 2018, Frontiers in Plant Science).

Future Directions

We have only just begun to publish our findings on the insect herbivore fauna and chemical diversity of Protium and its relationship to insect herbivores.  In the next year or so, we will submit articles on the geographic variation in chemistry and herbivore faunas – comparing our Iquitos, Peru and Manaus, Brazil sites.

Regarding the relative abundance of trees, a recent study has shown that a small set of “hyperdominant” tree species are extremely abundant, in the Amazon basin (ter Steege et al. 2013, Science). This pattern raises questions concerning the mechanisms that diversify and maintain tropical diversity. We were recently funded by NSF (with my former postdoc Diego Salazar now an Assistant Professor at FIU and my collaborator Kabir Peay at Stanford) to sample root defenses and soil pathogens, and to conduct manipulative experiments with plant seedlings. This grant will begin in 2021. We propose to test the hypothesis that plant chemical defenses, by protecting plants against insect herbivores and soil pathogens, mitigate the effects of negative density dependent interactions and consequently, influence the commonness and rarity of tree species. In the laboratory, metabolomic approaches will assess the diversity of plant secondary metabolites in leaves and roots that are likely mediating plant-natural enemy interactions. In the field, a combination of observational and experimental approaches will quantify and test these plant-defense-enemy interactions and their effect on host plant species abundances in Protium. Moreover, the study will thoroughly catalogue and test the role of plant secondary metabolites as defenses against soil pathogens, an extremely understudied interaction in natural tropical systems.