Parallel evolution and cryptic diversification in a common and widespread Amazonian tree, Protium subserratum.

The lowland Amazon rainforest houses some of the greatest tree diversity on Earth. While the vast majority of these species are rare, a small number are common and widespread and thus considered to play a disproportionate role in many of the global ecosystem services provided by the Amazon. However, the extent to which dominant Amazonian tree species actually include multiple clades, each on their own unique evolutionary trajectory, is unknown. Here we investigate the extent to which lineage divergence may be occurring within Protium subserratum (Burseraceae), a common and widespread tree species that is monophyletic with populations exhibiting genotypic and phenotypic differences associated with soil and geography. Utilizing a combination of phylogenomic and population genomic methods with sampling from across the range, we found that P. subserratum contains at least eight distinct clades. Specialization onto white-sand soils has evolved independently at least twice within the species; however, phenotype is not correlated with soil type. Finally, cryptic diversity at the base of the Andes is associated with elevational shifts. Together these results lend support to the hypothesis that common and widespread Amazon tree species may not represent evolutionary cohesive units. Instead, these dominant species may more commonly represent species complexes, undergoing evolutionary transitions on a trajectory to become multiple range-restricted, specialist species.

De novo transcriptome assembly and comparative transcriptomic analysis provide molecular insights into low temperature stress response of Canarium album.

A de novo transcriptome analysis was performed in C. album, a temperature sensitive fruit tree in China, after treatment with varied temperatures. A total number of 168,385 transcripts were assembled, comprising of 109,439 unigenes, of which 70,530 were successfully annotated. Compared with control check group (CK), which was treated under 25 °C, the chilling stress (4 °C) treated group (CT), showed about 2810 up-regulated and 2567 down-regulated genes. Whereas, group treated under freezing (- 3 °C) stress (FT) showed an up-regulation and a down-regulation of 1748 and 1459 genes, respectively. GO classification analysis revealed that DEGs related to metabolic processes, single-organism metabolic process, and catalytic activity are significantly enriched in both CT and FT conditions. KEGG pathway enrichment analysis for both CT and FT treatments showed an enrichment of genes encoding or related to glycine/serine and threonine metabolism, alpha-linolenic acid metabolism, carotenoid biosynthesis, photosynthesis-antenna proteins, and circadian rhythm. However, genes related to photosynthesis, carbon fixation in photosynthetic organisms, glutathione metabolism, pyruvate metabolism, nicotinate and nicotinamide metabolism were specifically enriched in CT condition. Nevertheless, FT treatment induced genes related to plant-pathogen interaction, linoleic acid metabolism, plant hormone signal transduction and pentose phosphate pathway. Many of the genes involved in plant hormone signal transduction showed significantly different expression in both FT and CT conditions. However, the change was more evident in FT. Here we present the first of the reports for a de novo transcriptomic analysis in C. album, suggesting that the plant shows differential responses in chilling and freezing temperatures, where the hormone signaling and transduction contribute greatly to FT responses. Our study thus paves way for future research regarding functions of these potentially identified genes.

Revisiting the hyperdominance of Neotropical tree species under a taxonomic, functional and evolutionary perspective.

Recent studies have leveraged large datasets from plot-inventory networks to report a phenomenon of hyperdominance in Amazonian tree communities, concluding that few species are common and many are rare. However, taxonomic hypotheses may not be consistent across these large plot networks, potentially masking cryptic diversity and threatened rare taxa. In the current study, we have reviewed one of the most abundant putatively hyperdominant taxa, Protium heptaphyllum (Aubl.) Marchand (Burseraceae), long considered to be a taxonomically difficult species complex. Using morphological, genomic, and functional data, we present evidence that P. heptaphyllum sensu lato may represent eight separately evolving lineages, each warranting species status. Most of these lineages are geographically restricted, and few if any of them could be considered hyperdominant on their own. In addition, functional trait data are consistent with the hypothesis that trees from each lineage are adapted to distinct soil and climate conditions. Moreover, some of the newly discovered species are rare, with habitats currently experiencing rapid deforestation. We highlight an urgent need to improve sampling and methods for species discovery in order to avoid oversimplified assumptions regarding diversity and rarity in the tropics and the implications for ecosystem functioning and conservation.

Trees and their seed networks: The social dynamics of urban fruit trees and implications for genetic diversity.

Trees are a traditional component of urban spaces where they provide ecosystem services critical to urban wellbeing. In the Tropics, urban trees' seed origins have rarely been characterized. Yet, understanding the social dynamics linked to tree planting is critical given their influence on the distribution of associated genetic diversity. This study examines elements of these dynamics (seed exchange networks) in an emblematic indigenous fruit tree species from Central Africa, the African plum tree (Dacryodes edulis, Burseraceae), within the urban context of Yaoundé. We further evaluate the consequences of these social dynamics on the distribution of the genetic diversity of the species in the city. Urban trees were planted predominantly using seeds sourced from outside the city, resulting in a level of genetic diversity as high in Yaoundé as in a whole region of production of the species. Debating the different drivers that foster the genetic diversity in planted urban trees, the study argues that cities and urban dwellers can unconsciously act as effective guardians of indigenous tree genetic diversity.

New microsatellite markers for Dacryodes edulis (Burseraceae), an indigenous fruit tree species from Central Africa.

Microsatellites were designed and characterized in the African fruit tree species Dacryodes edulis (Burseraceae). The fruits are commercialized throughout Central Africa and the species is present in forested environments as well as cultivated systems. The high variability of these markers makes them suitable to investigate the structure of genetic diversity in this important food tree species from Central Africa. From a genomic library obtained by next-generation sequencing, 21 new polymorphic microsatellite loci were developed. Tested on 95 individuals from four populations coming from three countries of the Congo Basin, the microsatellites displayed two to 20 alleles (mean 7.5; expected heterozygosity 0.003 to 0.937, mean 0.666). The transferability of microsatellites was effective for four other Dacryodes species (D. buettneri, D. igaganga, D. osika, D. pubescens). This set of newly developed microsatellite markers will be useful for assessing the genetic diversity and differentiation as well as gene flow patterns of D. edulis in tropical forests from Central Africa.

Leaf Transcriptome Assembly of Protium copal (Burseraceae) and Annotation of Terpene Biosynthetic Genes.

Plants in the Burseraceae are globally recognized for producing resins and essential oils with medicinal properties and have economic value. In addition, most of the aromatic and non-aromatic components of Burseraceae resins are derived from a variety of terpene and terpenoid chemicals. Although terpene genes have been identified in model plant crops (e.g., Citrus, Arabidopsis), very few genomic resources are available for non-model groups, including the highly diverse Burseraceae family. Here we report the assembly of a leaf transcriptome of Protium copal, an aromatic tree that has a large distribution in Central America, describe the functional annotation of putative terpene biosynthetic genes and compare terpene biosynthetic genes found in P. copal with those identified in other Burseraceae taxa. The genomic resources of Protium copal can be used to generate novel sequencing markers for population genetics and comparative phylogenetic studies, and to investigate the diversity and evolution of terpene genes in the Burseraceae.

Reconciling species diversity in a tropical plant clade (Canarium, Burseraceae).

The challenges associated with sampling rare species or populations can limit our ability to make accurate and informed estimates of biodiversity for clades or ecosystems. This may be particularly true for tropical trees, which tend to be poorly sampled, and are thought to harbor extensive cryptic diversity. Here, we integrate genomics, morphology, and geography to estimate the number of species in a clade of dioecious tropical trees (Canarium L.; Burseraceae) endemic to Madagascar, for which previous taxonomic treatments have recognized between one and 33 species. By sampling genomic data from even a limited number of individuals per taxon, we were able to clearly reject both previous hypotheses, and support instead an intermediate number of taxa. We recognize at least six distinct clades based on genetic structure and species delimitation analyses that correspond clearly with geographic and discrete morphological differences. Two widespread clades co-occur broadly throughout eastern wet forests, one clade is endemic to western dry forests, and several slightly admixed clades are more narrowly distributed in mountainous regions in the north. Multiple previously described taxa were recovered as paraphyletic in our analyses, some of which were associated with admixed individuals, suggesting that hybridization contributes to taxonomic difficulties in Canarium. An improved understanding of Canarium species diversity has important implications for conservation efforts and understanding the origins of diversity in Madagascar. Our study shows that even limited genomic sampling, when combined with geography and morphology, can greatly improve estimates of species diversity for difficult tropical clades.

Genetic analysis of Canarium album in different areas of China by improved RAPD and ISSR.

To lay the foundation of the classification of Canarium album (C. album), and C. album from Terminalia Chebula (T. chebula) in different areas of China, improved RAPD and ISSR analysis were performed to analyze polymorphism and genetic relationship. Ten samples were collected from different locations in China. A total of 221 fragments were detected by improved RAPD, out of which polymorphic fragments accounted for 82.3% with average amplification bands of 10.05 per primer. ISSR markers revealed a total of 147 alleles, where polymorphic fragments accounted for 83.5%, with average amplification bands of 7.35 per primer. The sizes of fragments ranged from 200 to 2500bp and from 150 to 2000bp in RAPD and ISSR markers, respectively. The similarity coefficient ranged from 0.46 to 0.86 with RAPD markers and 0.36 to 0.89 with ISSR markers. The results indicated that improved RAPD and ISSR methods are useful for genetic diversity study of C. album. Thus, this study provides us the theoretical basis for the breeding and classification of C. album in South and Southwest China.

Development and significance of SCAR marker QG12-5 for Canarium album (Lour.) Raeusch by molecular cloning from improved RAPD amplification.

Sequence-characterized amplified region (SCAR) is a valuable molecular marker for the genetic identification of any species. This marker is mainly derived from molecular cloning of random amplified polymorphic DNA (RAPD). We have previously reported the use of an improved RAPD technique for the genetic characterization of different samples of Canarium album (Lour.) Raeusch (C. album). In this study, DNA fragments were amplified using improved RAPD amplified from different samples of C. album. The amplified DNA fragment was excised, purified from an agarose gel and cloned into a pGM-T vector; subsequently, a positive clone, called QG12-5 was identified by PCR amplification and enzymatic digestion and sequenced by Sanger di-deoxy sequencing method. This clone was revealed consisting of 510 nucleotides of C. album. The SCAR marker QG12-5 was developed using specifically designed PCR primers and optimized PCR conditions. This SCAR marker expressed seven continuous "TATG" [(TATG)n] tandem repeats, which was found to characterize C. album. Subsequently, this novel SCAR marker was deposited in GenBank with accession No. KT359568. Therefore, we successfully developed a C. album-specific SCAR marker for the identification and authentication of different C. album species in this study.

Experimental evidence for an intraspecific Janzen-Connell effect mediated by soil biota.

The negative effect of soil pathogens on seedling survival varies considerably among conspecific individuals, but the underlying mechanisms are largely unknown. For variation between heterospecifics, a common explanation is the Janzen-Connell effect: negative density dependence in survival due to specialized pathogens aggregating on common hosts. We test whether an intraspecific Janzen-Connell effect exists, i.e., whether the survival chances of one population's seedlings surrounded by a different conspecific population increase with genetic difference, spatial distance, and trait dissimilarity between them. In a shade-house experiment, we grew seedlings of five populations of each of two subtropical tree species (Castanopsis fissa and Canarium album) for which we measured genetic distance using intersimple sequence repeat (ISSR) analysis and eight common traits/characters, and we treated them with soil material or soil biota filtrate collected from different populations. We found that the relative survival rate increased with increasing dissimilarity measured by spatial distance, genetic distance, and trait differences between the seedling and the population around which the soil was collected. This effect disappeared after soil sterilization. Our results provide evidence that genetic variation, trait similarity, and spatial distance can explain intraspecific variation in plant-soil biotic interactions and suggest that limiting similarity also occurs at the intraspecific level.

Investigating processes of neotropical rain forest tree diversification by examining the evolution and historical biogeography of the Protieae (Burseraceae).

Andean uplift and the collision of North and South America are thought to have major implications for the diversification of the Neotropical biota. However, few studies have investigated how these geological events may have influenced diversification. We present a multilocus phylogeny of 102 Protieae taxa (73% of published species), sampled pantropically, to test hypotheses about the relative importance of dispersal, vicariance, habitat specialization, and biotic factors in the diversification of this ecologically dominant tribe of Neotropical trees. Bayesian fossil-calibrated analyses date the Protieae stem at 55 Mya. Biogeographic analyses reconstruct an initial late Oligocene/early Miocene radiation in Amazonia for Neotropical Protieae, with several subsequent late Miocene dispersal events to Central America, the Caribbean, Brazil's Atlantic Forest, and the Chocó. Regional phylogenetic structure results indicate frequent dispersal among regions throughout the Miocene and many instances of more recent regional in situ speciation. Habitat specialization to white sand or flooded soils was common, especially in Amazonia. There was one significant increase in diversification rate coincident with colonization of the Neotropics, followed by a gradual decrease consistent with models of diversity-dependent cladogenesis. Dispersal, biotic interactions, and habitat specialization are thus hypothesized to be the most important processes underlying the diversification of the Protieae.

Evidence for ecological divergence across a mosaic of soil types in an Amazonian tropical tree: Protium subserratum (Burseraceae).

Soil heterogeneity is an important driver of divergent natural selection in plants. Neotropical forests have the highest tree diversity on earth, and frequently, soil specialist congeners are distributed parapatrically. While the role of edaphic heterogeneity in the origin and maintenance of tropical tree diversity is unknown, it has been posited that natural selection across the patchwork of soils in the Amazon rainforest is important in driving and maintaining tree diversity. We examined genetic and morphological differentiation among populations of the tropical tree Protium subserratum growing parapatrically on the mosaic of white-sand, brown-sand and clay soils found throughout western Amazonia. Nuclear microsatellites and leaf morphology were used to (i) quantify the extent of phenotypic and genetic divergence across habitat types, (ii) assess the importance of natural selection vs. drift in population divergence, (iii) determine the extent of hybridization and introgression across habitat types, (iv) estimate migration rates among populations. We found significant morphological variation correlated with soil type. Higher levels of genetic differentiation and lower migration rates were observed between adjacent populations found on different soil types than between geographically distant populations on the same soil type. PST -FST comparisons indicate a role for natural selection in population divergence among soil types. A small number of hybrids were detected suggesting that gene flow among soil specialist populations may occur at low frequencies. Our results suggest that edaphic specialization has occurred multiple times in P. subserratum and that divergent natural selection across edaphic boundaries may be a general mechanism promoting and maintaining Amazonian tree diversity.

Insect herbivores, chemical innovation, and the evolution of habit specialization in Amazonian trees.

Herbivores are often implicated in the generation of the extraordinarily diverse tropical flora. One hypothesis linking enemies to plant diversification posits that the evolution of novel defenses allows plants to escape their enemies and expand their ranges. 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. We evaluated this hypothesis by investigating two sister habitat specialist ecotypes of Protium subserratum (Burseraceae), a common Amazonian tree that occurs in white-sand and terra firme forests. We collected insect herbivores feeding on the plants, assessed whether growth differences between habitats were genetically based using a reciprocal transplant experiment, and sampled multiple populations of both lineages for defense chemistry. Protium subserratum plants were attacked mainly by chrysomelid beetles and cicadellid hemipterans. Assemblages of insect herbivores were dissimilar between populations of ecotypes from different habitats, as well as from the same habitat 100 km distant. Populations from terra firme habitats grew significantly faster than white-sand populations; they were taller, produced more leaf area, and had more chlorophyll. White-sand populations expressed more dry mass of secondary compounds and accumulated more flavone glycosides and oxidized terpenes, whereas terra firme populations produced a coumaroylquinic acid that was absent from white-sand populations. We interpret these results as strong evidence that herbivores and resource availability select for divergent types and amounts of defense investment in white-sand and terra firme lineages of Protium subserratum, which may contribute to habitat-mediated speciation in these trees.

Plant ontogeny, spatial distance, and soil type influence patterns of relatedness in a common Amazonian tree.

The formation of spatial genetic structure (SGS) may originate from different patterns of seed deposition in the landscape, and is mostly determined by seed dispersal limitation. After dispersal, mechanisms such as filtering by environmental factors or attack by herbivores/pathogens throughout plant development stages, and potentially either disrupt or intensify SGS patterns. We investigated how the genotype of Protium subserratum (Burseraceae), a common tree species in the Ducke Reserve, Brazil, is distributed across the landscape. We used seven microsatellite markers to assess the SGS among plants at different life stages and in different environments. By quantifying the patterns of relatedness among plants of different sizes, we inferred the ontogenetic stage in which SGS changes occurred, and compared these effects across soil types. Relatedness among seedlings decreased when distance between seedlings increased, especially for the youngest seedlings. However, this trend was not continued by older plants, as relatedness values were higher among neighboring individuals of the juvenile and adult size class. Contrasting relatedness patterns between seedlings and larger individuals suggests a trade-off between the negative effects of being near closely-related adults (e.g. due to herbivore and pathogen attack) and the advantage of being in a site favorable to establishment. We also found that soil texture strongly influenced density-dependence patterns, as young seedlings in clay soils were more related to each other than were seedlings in bottomland sandy soils, suggesting that the mechanisms that create and maintain patterns of SGS within a population may interact with environmental heterogeneity.

Microsatellite primers for an Amazonian lowland tropical tree, Protium subserratum (Burseraceae).

PREMISE OF THE STUDY: The first microsatellite primers were developed for Protium subserratum, a widespread Amazonian tree, to investigate genetic differentiation between populations found on clay, brown-sand, and white-sand soils. • METHODS AND RESULTS: Seventeen primer pairs were identified from two individuals of P. subserratum found on white-sand and brown-sand soil types. Polymorphism was analyzed in 63 individuals from a total of three populations, each found on a different soil type. The primers amplified tetra-, tri-, and dinucleotide repeats with three to 24 alleles per locus. Excluding monomorphic loci, observed and expected heterozygosities ranged from 0 to 0.852 and 0.036 to 0.901, respectively. • CONCLUSIONS: These new microsatellite markers will be useful in studies of genetic diversity, population differentiation, and gene flow across habitat types in P. subserratum.

Characterization of new microsatellite loci isolated from Santiria trimera (Burseraceae).

PREMISE OF THE STUDY: To study the genetic structure among three morphotypes of an African rainforest tree species, Santiria trimera, nuclear microsatellite markers were isolated and characterized. METHODS AND RESULTS: Seven polymorphic loci were isolated using a pyrosequencing-based protocol and successfully amplified on three different morphotypes of S. trimera. For six of the seven loci, there is at least one private allele for one of the three morphotypes. The mean effective number of alleles is about four for each of the three morphotypes. CONCLUSIONS: These microsatellite markers are promising to explore the genetic delimitation among sympatric morphotypes found in Gabonese forests and to study the spatial genetic structure within each gene pool.

Diversity gradients and phylogeographic patterns in Santiria trimera (Burseraceae), a widespread African tree typical of mature rainforests.

PREMISE OF THE STUDY: New insights into the history of the African rainforest can be gathered from the phylogeographic structures of their constituent species, but few studies have been performed in this ecosystem. We studied the phylogeographic structure of Santiria trimera, a primate- and bird-dispersed, dioecious tree typical of mature African rainforests. METHODS: We sequenced three chloroplast DNA (cpDNA) regions (trnL-F, rbcL, and rpl36-infA-rps8) in 377 individuals from 42 populations. RESULTS: Sequence chromatograms regularly displayed double peaks of unequal heights. Cloning of PCR products and sequencing of outgroup taxa led to assigning the taller peak in ambiguous sequence positions to cpDNA. A total of 14 polymorphic cpDNA sites and 12 haplotypes were detected. Populations from three distinct biogeographic regions, namely, Upper Guinea, Lower Guinea, and the volcanic island of São Tomé, did not share any haplotype, indicating allopatric divergence. In Lower Guinea, Gabonese forests had high diversity mainly from the sympatry of two genetically divergent morphotypes, whereas forests of eastern Cameroon were less diversified. The two haplotypes of the morphotype without stilt roots were distributed north and south of the Ogooué River, suggesting refuges on both sides of the river bed. CONCLUSIONS: The divergence between Upper and Lower Guinean rainforests is explained by the discontinuity of forest between those regions throughout most of the Quaternary. The distribution of rare endemic haplotypes concurred with proposed Pleistocene rainforest refuges in west and southwest Cameroon. Overall, phylogeographic structure is consistent with the biogeographic hypotheses largely based on patterns of species diversity.

Polymorphic microsatellite loci from Dacryodes edulis (Burseraceae), a Central African rainforest and fruit-tree species.

PREMISE OF THE STUDY: Microsatellite markers were isolated and characterized from Dacryodes edulis (Burseraceae), a tropical rainforests fruit tree of central Africa. METHODS AND RESULTS: Using an enrichment protocol, six microsatellites loci were developed from Dacryodes edulis. We investigated polymorphism using 45 trees from three widely separated populations in Cameroon. All loci were polymorphic, with the number of alleles ranging from 2 to 15. Polymorphism was widely variable among loci and expected heterozygosities ranged from 0.06 to 0.84 with a mean value of 0.49. CONCLUSIONS: These loci will be useful for the in-depth analysis of population structure and phylogeographic variation throughout the distribution range of Dacryodes edulis and other related taxa, Dacryodes buettneri and D. normandii, in which all loci were also amplified. Furthermore, they will offer the opportunity to study early domestication processes acting on the genetic diversity of Dacryodes edulis.

Colonization processes and the maintenance of genetic diversity: insights from a pioneer rainforest tree, Aucoumea klaineana.

Despite recurrent episodes of range expansion and contraction, forest trees often harbour high genetic diversity. Studies of temperate forest trees suggest that prolonged juvenile phase and high pollen flow are the main factors limiting founder effects. Here, we studied the local colonization process of a pioneer rainforest tree in central Africa, Aucoumea klaineana. We identified 87% of parents among trees up to 20-25 years old and could thus compare direct parentage structure data with classical population genetics estimators. In this species, genetic diversity was maintained during colonization. The absence of founder effects was explained by (i) local random mating and (ii) local recruitment, as we showed that 75% of the trees in the close neighbourhood participated in the recruitment of new saplings. Long-distance pollen flow contributed little to genetic diversity: pollen and seed dispersal was mainly within stand (128 and 118 m, respectively). Spatial genetic structure was explained by aggregated seed dispersal rather than by mother-offspring proximity as assumed in classical isolation-by-distance models. Hence, A. klaineana presents a genetic diversity pattern typical of forest trees but does not follow the classical rules by which this diversity is generally achieved. We suggest that while high local genetic variability is of general importance to forest tree survival, the proximate mechanisms by which it is achieved may follow very different scenarios.

Molecular phylogenetic analysis of Commiphora (Burseraceae) yields insight on the evolution and historical biogeography of an "impossible" genus.

Expansion of the arid zone of sub-Saharan tropical Africa during the Miocene is posited as a significant contributing factor in the evolution of contemporary African flora. Nevertheless, few molecular phylogenetic studies have tested this hypothesis using reconstructed historical biogeographies of plants within this zone. Here, we present a molecular phylogeny of Commiphora, a predominantly tropical African, arid-adapted tree genus, in order to test the monophyly of its taxonomic sections and identify clades that will help direct future study of this species-rich and geographically widespread taxon. We then use multiple fossil calibrations of Commiphora phylogeny to determine the timing of well-supported diversification events within the genus and interpret these age estimates to determine the relative contribution of vicariance and dispersal in the expansion of Commiphora's geographic range. We find that Commiphora is sister to Vietnamese Bursera tonkinensis and that its crown group radiation corresponds with the onset of the Miocene.