The phylogeny and global biogeography of Primulaceae based on high-throughput DNA sequence data.

The angiosperm family Primulaceae is morphologically diverse and distributed nearly worldwide. However, phylogenetic uncertainty has obstructed the identification of major morphological and biogeographic transitions within the clade. We used target capture sequencing with the Angiosperms353 probes, taxon-sampling encompassing nearly all genera of the family, tree-based sequence curation, and multiple phylogenetic approaches to investigate the major clades of Primulaceae and their relationship to other Ericales. We generated dated phylogenetic trees and conducted broad-scale biogeographic analyses as well as stochastic character mapping of growth habit. We show that Ardisia, a pantropical genus and the largest in the family, is not monophyletic, with at least 19 smaller genera nested within it. Neotropical members of Ardisia and several smaller genera form a clade, an ancestor of which arrived in the Neotropics and began diversifying about 20 Ma. This Neotropical clade is most closely related to Elingamita and Tapeinosperma, which are most diverse on islands of the Pacific. Both Androsace and Primula are non-monophyletic by the inclusion of smaller genera. Ancestral state reconstructions revealed that there have either been parallel transitions to an herbaceous habit in Primuloideae, Samolus, and at least three lineages of Myrsinoideae, or a common ancestor of nearly all Primulaceae was herbaceous. Our results provide a robust estimate of phylogenetic relationships across Primulaceae and show that a revised classification of Myrsinoideae and several other clades within the family is necessary to render all genera monophyletic.

Admixture may be extensive among hyperdominant Amazon rainforest tree species.

Admixture is a mechanism by which species of long-lived plants may acquire novel alleles. However, the potential role of admixture in the origin and maintenance of tropical plant diversity is unclear. We ask whether admixture occurs in an ecologically important clade of Eschweilera (Parvifolia clade, Lecythidaceae), which includes some of the most widespread and abundant tree species in Amazonian forests. Using target capture sequencing, we conducted a detailed phylogenomic investigation of 33 species in the Parvifolia clade and investigated specific hypotheses of admixture within a robust phylogenetic framework. We found strong evidence of admixture among three ecologically dominant species, E. coriacea, E. wachenheimii and E. parviflora, but a lack of evidence for admixture among other lineages. Accepted species were largely distinguishable from one another, as was geographic structure within species. We show that hybridization may play a role in the evolution of the most widespread and ecologically variable Amazonian tree species. While admixture occurs among some species of Eschweilera, it has not led to widespread erosion of most species' genetic or morphological identities. Therefore, current morphological based species circumscriptions appear to provide a useful characterization of the clade's lineage diversity.

By Animal, Water, or Wind: Can Dispersal Mode Predict Genetic Connectivity in Riverine Plant Species?

Seed dispersal is crucial to gene flow among plant populations. Although the effects of geographic distance and barriers to gene flow are well studied in many systems, it is unclear how seed dispersal mediates gene flow in conjunction with interacting effects of geographic distance and barriers. To test whether distinct seed dispersal modes (i.e., hydrochory, anemochory, and zoochory) have a consistent effect on the level of genetic connectivity (i.e., gene flow) among populations of riverine plant species, we used unlinked single-nucleotide polymorphisms (SNPs) for eight co-distributed plant species sampled across the Rio Branco, a putative biogeographic barrier in the Amazon basin. We found that animal-dispersed plant species exhibited higher levels of genetic diversity and lack of inbreeding as a result of the stronger genetic connectivity than plant species whose seeds are dispersed by water or wind. Interestingly, our results also indicated that the Rio Branco facilitates gene dispersal for all plant species analyzed, irrespective of their mode of dispersal. Even at a small spatial scale, our findings suggest that ecology rather than geography play a key role in shaping the evolutionary history of plants in the Amazon basin. These results may help improve conservation and management policies in Amazonian riparian forests, where degradation and deforestation rates are high.

A Biogeographic Barrier Test Reveals a Strong Genetic Structure for a Canopy-Emergent Amazon Tree Species.

Wallace's (1854) Riverine Barrier hypothesis is one of the earliest explanations for Amazon biotic diversification. Despite the importance of this hypothesis for explaining speciation in some animal groups, it has not been studied extensively for plant species. In this study we use a prominent Amazon tree, Buchenavia oxycarpa (Mart.) Eichler (Combretaceae), to evaluate Wallace's hypothesis along the Rio Negro, a major Amazon tributary that has driven allopatric speciation for several animal taxa. We sampled six individuals from sixteen localities along both river banks, and used a modified ddRADseq protocol to identify SNP markers. Our population genomic data revealed strong genetic structure for B. oxycarpa sampled across banks of the Rio Negro (ϕCT = 0.576, P < 0.001), supporting the hypothesis that the Rio Negro acted as a significant genetic barrier for B. oxycarpa. Our study shows that gene flow for this large and well-dispersed Amazon tree is impeded by riverine barriers, though this has not yet resulted in speciation. Future studies focused on species with different life histories, including species restricted to non-flooded forests, are needed to further advance our understanding of Amazon rivers as drivers of biotic diversification.

Genomic evidence of survival near ice sheet margins for some, but not all, North American trees.

Temperate species experienced dramatic range reductions during the Last Glacial Maximum, yet refugial populations from which modern populations are descended have never been precisely located. Climate-based models identify only broad areas of potential habitat, traditional phylogeographic studies provide poor spatial resolution, and pollen records for temperate forest communities are difficult to interpret and do not provide species-level taxonomic resolution. Here we harness signals of range expansion from large genomic datasets, using a simulation-based framework to infer the precise latitude and longitude of glacial refugia in two widespread, codistributed hickories (Carya spp.) and to quantify uncertainty in these estimates. We show that one species likely expanded from close to ice sheet margins near the site of a previously described macrofossil for the genus, highlighting support for the controversial notion of northern microrefugia. In contrast, the expansion origin inferred for the second species is compatible with classic hypotheses of distant displacement into southern refugia. Our statistically rigorous, powerful approach demonstrates how refugia can be located from genomic data with high precision and accuracy, addressing fundamental questions about long-term responses to changing climates and providing statistical insight into longstanding questions that have previously been addressed primarily qualitatively.

Target sequence capture in the Brazil nut family (Lecythidaceae): Marker selection and in silico capture from genome skimming data.

Reconstructing species trees from multi-loci datasets is becoming a standard practice in phylogenetics. Nevertheless, access to high-throughput sequencing may be costly, especially with studies of many samples. The potential high cost makes a priori assessments desirable in order to make informed decisions about sequencing. We generated twelve transcriptomes for ten species of the Brazil nut family (Lecythidaceae), identified a set of putatively orthologous nuclear loci and evaluated, in silico, their phylogenetic utility using genome skimming data of 24 species. We designed the markers using MarkerMiner, and developed a script, GoldFinder, to efficiently sub-select the best makers for sequencing. We captured, in silico, all designed 354 nuclear loci and performed a maximum likelihood phylogenetic analysis on the concatenated sequence matrix. We also calculated individual gene trees with maximum likelihood and used them for a coalescent-based species tree inference. Both analyses resulted in almost identical topologies. However, our nuclear-loci phylogenies were strongly incongruent with a published plastome phylogeny, suggesting that plastome data alone is not sufficient for species tree estimation. Our results suggest that using hundreds of nuclear markers (i.e. 354) will significantly improve the Lecythidaceae species tree. The framework described here will be useful, generally, for developing markers for species tree inference.

Tangled banks: A landscape genomic evaluation of Wallace's Riverine barrier hypothesis for three Amazon plant species.

Wallace's Riverine Barrier hypothesis is one of the earliest biogeographic explanations for Amazon speciation, but it has rarely been tested in plants. In this study, we used three woody Amazonian plant species to evaluate Wallace's Hypothesis using tools of landscape genomics. We generated unlinked single-nucleotide polymorphism (SNP) data from the nuclear genomes of 234 individuals (78 for each plant species) across 13 sampling sites along the Rio Branco, Brazil, for Amphirrhox longifolia (8,075 SNPs), Psychotria lupulina (9,501 SNPs) and Passiflora spinosa (14,536 SNPs). Although significantly different migration rates were estimated between species, the population structure data do not support the hypothesis that the Rio Branco-an allopatric barrier for primates and birds-is a significant genetic barrier for Amphirrhox longifolia, Passiflora spinosa or Psychotria lupulina. Overall, we demonstrated that medium-sized rivers in the Amazon Basin, such as the Rio Branco, are permeable barriers to gene flow for animal-dispersed and animal-pollinated plant species.

Scale and strength oak-mesophyte interactions in a transitional oak-hickory forest.

Forests in eastern North America are undergoing rapid compositional changes as they experience novel climate, disturbance, and pest conditions. One striking pattern is the replacement of canopy oaks (Quercus spp.) by mesic, fire-sensitive, shade-tolerant species like red maple (Acer rubrum). To gain insight into the successional patterns driving stand-level canopy oak replacement we ask two questions: (i) What is the spatial association of oak and mesophyte recruitment compared to oak and mesophyte overstory individuals, and (ii) How do oaks and mesophytes differentially respond to canopy openings. We analyzed census data from a 23 ha forest plot surveyed in 2003, 2008 and 2014. We show that oak recruits are negatively associated with overstory red maples and black cherries (Prunus serotina), while mesophytic recruits were positively associated with overstory oaks. Second, we found that proximity to a dead overstory tree increased growth and survival for black cherries, increased growth for red maples, but had no effect on oaks. Black cherries and red maples are therefore better suited than oaks to take advantage of canopy openings and the moderate light available under adult oaks. These same fine scale competitive processes are contributing to canopy oak replacement across eastern North America.

Complete plastome sequences from Bertholletia excelsa and 23 related species yield informative markers for Lecythidaceae.

PREMISE OF THE STUDY: The tropical tree family Lecythidaceae has enormous ecological and economic importance in the Amazon basin. Lecythidaceae species can be difficult to identify without molecular data, however, and phylogenetic relationships within and among the most diverse genera are poorly resolved. METHODS: To develop informative genetic markers for Lecythidaceae, we used genome skimming to de novo assemble the full plastome of the Brazil nut tree (Bertholletia excelsa) and 23 other Lecythidaceae species. Indices of nucleotide diversity and phylogenetic signal were used to identify regions suitable for genetic marker development. RESULTS: The B. excelsa plastome contained 160,472 bp and was arranged in a quadripartite structure. Using the 24 plastome alignments, we developed primers for 10 coding and non-coding DNA regions containing exceptional nucleotide diversity and phylogenetic signal. We also developed 19 chloroplast simple sequence repeats for population-level studies. DISCUSSION: The coding region ycf1 and the spacer rpl16-rps3 outperformed plastid DNA markers previously used for barcoding and phylogenetics. Used in a phylogenetic analysis, the matrix of 24 plastomes showed with 100% bootstrap support that Lecythis and Eschweilera are polyphyletic. The plastomes and primers presented in this study will facilitate a broad array of ecological and evolutionary studies in Lecythidaceae.

Comment on "Persistent effects of pre-Columbian plant domestication on Amazonian forest composition".

Levis et al (Research Articles, 3 March 2017, p. 925) concluded that pre-Columbian tree domestication has shaped present-day Amazonian forest composition. The study, however, downplays five centuries of human influence following European arrival to the Americas. We show that the effects of post-Columbian activities in Amazonia are likely to have played a larger role than pre-Columbian ones in shaping the observed floristic patterns.

Altitudinal gradients, biogeographic history and microhabitat adaptation affect fine-scale spatial genetic structure in African and Neotropical populations of an ancient tropical tree species.

The analysis of fine-scale spatial genetic structure (FSGS) within populations can provide insights into eco-evolutionary processes. Restricted dispersal and locally occurring genetic drift are the primary causes for FSGS at equilibrium, as described in the isolation by distance (IBD) model. Beyond IBD expectations, spatial, environmental or historical factors can affect FSGS. We examined FSGS in seven African and Neotropical populations of the late-successional rain forest tree Symphonia globulifera L. f. (Clusiaceae) to discriminate the influence of drift-dispersal vs. landscape/ecological features and historical processes on FSGS. We used spatial principal component analysis and Bayesian clustering to assess spatial genetic heterogeneity at SSRs and examined its association with plastid DNA and habitat features. African populations (from Cameroon and São Tomé) displayed a stronger FSGS than Neotropical populations at both marker types (mean Sp = 0.025 vs. Sp = 0.008 at SSRs) and had a stronger spatial genetic heterogeneity. All three African populations occurred in pronounced altitudinal gradients, possibly restricting animal-mediated seed dispersal. Cyto-nuclear disequilibria in Cameroonian populations also suggested a legacy of biogeographic history to explain these genetic patterns. Conversely, Neotropical populations exhibited a weaker FSGS, which may reflect more efficient wide-ranging seed dispersal by Neotropical bats and other dispersers. The population from French Guiana displayed an association of plastid haplotypes with two morphotypes characterized by differential habitat preferences. Our results highlight the importance of the microenvironment for eco-evolutionary processes within persistent tropical tree populations.

Wide but not impermeable: Testing the riverine barrier hypothesis for an Amazonian plant species.

Wallace's riverine barrier hypothesis postulates that large rivers, such as the Amazon and its tributaries, reduce or prevent gene flow between populations on opposite banks, leading to allopatry and areas of species endemism occupying interfluvial regions. Several studies have shown that two major tributaries, Rio Branco and Rio Negro, are important barriers to gene flow for birds, amphibians and primates. No botanical studies have considered the potential role of the Rio Branco as a barrier, while a single botanical study has evaluated the Rio Negro as a barrier. We studied an Amazon shrub, Amphirrhox longifolia (A. St.-Hil.) Spreng (Violaceae), as a model to test the riverine barrier hypothesis. Twenty-six populations of A. longifolia were sampled on both banks of the Rio Branco and Rio Negro in the core Amazon Basin. Double-digest RADseq was used to identify 8,010 unlinked SNP markers from the nuclear genome of 156 individuals. Data relating to population structure support the hypothesis that the Rio Negro acted as a significant genetic barrier for A. longifolia. On the other hand, no genetic differentiation was detected among populations spanning the narrower Rio Branco, which is a tributary of the Rio Negro. This study shows that the strength of riverine barriers for Amazon plants is dependent on the width of the river separating populations and species-specific dispersal traits. Future studies of plants with contrasting life history traits will further improve our understanding of the landscape genetics and allopatric speciation history of Amazon plant diversity.

Minimum sample sizes for population genomics: an empirical study from an Amazonian plant species.

High-throughput DNA sequencing facilitates the analysis of large portions of the genome in nonmodel organisms, ensuring high accuracy of population genetic parameters. However, empirical studies evaluating the appropriate sample size for these kinds of studies are still scarce. In this study, we use double-digest restriction-associated DNA sequencing (ddRADseq) to recover thousands of single nucleotide polymorphisms (SNPs) for two physically isolated populations of Amphirrhox longifolia (Violaceae), a nonmodel plant species for which no reference genome is available. We used resampling techniques to construct simulated populations with a random subset of individuals and SNPs to determine how many individuals and biallelic markers should be sampled for accurate estimates of intra- and interpopulation genetic diversity. We identified 3646 and 4900 polymorphic SNPs for the two populations of A. longifolia, respectively. Our simulations show that, overall, a sample size greater than eight individuals has little impact on estimates of genetic diversity within A. longifolia populations, when 1000 SNPs or higher are used. Our results also show that even at a very small sample size (i.e. two individuals), accurate estimates of FST can be obtained with a large number of SNPs (≥1500). These results highlight the potential of high-throughput genomic sequencing approaches to address questions related to evolutionary biology in nonmodel organisms. Furthermore, our findings also provide insights into the optimization of sampling strategies in the era of population genomics.

Impacts of biogeographic history and marginal population genetics on species range limits: a case study of Liriodendron chinense.

Species ranges are influenced by past climate oscillations, geographical constraints, and adaptive potential to colonize novel habitats at range limits. This study used Liriodendron chinense, an important temperate Asian tree species, as a model system to evaluate the roles of biogeographic history and marginal population genetics in determining range limits. We examined the demographic history and genetic diversity of 29 L. chinense populations using both chloroplast and nuclear microsatellite loci. Significant phylogeographic structure was recovered with haplotype clusters coinciding with major mountain regions. Long-term demographical stability was suggested by mismatch distribution analyses, neutrality tests, and ecological niche models (ENM) and suggested the existence of LGM refuges within mountain regions. Differences in genetic diversity between central and marginal populations were not significant for either genomic region. However, asymmetrical gene flow was inferred from central populations to marginal populations, which could potentially limit range adaptation and expansion of L. chinense.

Spatial Scales of Genetic Structure in Free-Standing and Strangler Figs (Ficus, Moraceae) Inhabiting Neotropical Forests.

Wind-borne pollinating wasps (Agaonidae) can transport fig (Ficus sp., Moraceae) pollen over enormous distances (> 100 km). Because of their extensive breeding areas, Neotropical figs are expected to exhibit weak patterns of genetic structure at local and regional scales. We evaluated genetic structure at the regional to continental scale (Panama, Costa Rica, and Peru) for the free-standing fig species Ficus insipida. Genetic differentiation was detected only at distances > 300 km (Jost´s Dest = 0.68 ± 0.07 & FST = 0.30 ± 0.03 between Mesoamerican and Amazonian sites) and evidence for phylogeographic structure (RST>>permuted RST) was only significant in comparisons between Central and South America. Further, we assessed local scale spatial genetic structure (SGS, d ≤ 8 km) in Panama and developed an agent-based model parameterized with data from F. insipida to estimate minimum pollination distances, which determine the contribution of pollen dispersal on SGS. The local scale data for F. insipida was compared to SGS data collected for an additional free-standing fig, F. yoponensis (subgenus Pharmacosycea), and two species of strangler figs, F. citrifolia and F. obtusifolia (subgenus Urostigma) sampled in Panama. All four species displayed significant SGS (mean Sp = 0.014 ± 0.012). Model simulations indicated that most pollination events likely occur at distances > > 1 km, largely ruling out spatially limited pollen dispersal as the determinant of SGS in F. insipida and, by extension, the other fig species. Our results are consistent with the view that Ficus develops fine-scale SGS primarily as a result of localized seed dispersal and/or clumped seedling establishment despite extensive long-distance pollen dispersal. We discuss several ecological and life history factors that could have species- or subgenus-specific impacts on the genetic structure of Neotropical figs.

Polymorphic microsatellite markers for a wind-dispersed tropical tree species, Triplaris cumingiana (Polygonaceae).

PREMISE OF THE STUDY: Novel microsatellite markers were characterized in the wind-dispersed and dioecious neotropical tree Triplaris cumingiana (Polygonaceae) for use in understanding the ecological processes and genetic impacts of pollen- and seed-mediated gene flow in tropical forests. • METHODS AND RESULTS: Sixty-two microsatellite primer pairs were screened, from which 12 markers showing five or more alleles per locus (range 5-17) were tested on 47 individuals. Observed and expected heterozygosities averaged 0.692 and 0.731, respectively. Polymorphism information content was between 0.417 and 0.874. Linkage disequilibrium was observed in one of the 66 pairwise comparisons between loci. Two loci showed deviation from Hardy-Weinberg equilibrium. An additional 14 markers exhibiting lower polymorphism were characterized on a smaller number of individuals. • CONCLUSIONS: These microsatellite markers have high levels of polymorphism and reproducibility and will be useful in studying gene flow and population structure in T. cumingiana.

The effects of read length, quality and quantity on microsatellite discovery and primer development: from Illumina to PacBio.

The advent of next-generation sequencing (NGS) technologies has transformed the way microsatellites are isolated for ecological and evolutionary investigations. Recent attempts to employ NGS for microsatellite discovery have used the 454, Illumina, and Ion Torrent platforms, but other methods including single-molecule real-time DNA sequencing (Pacific Biosciences or PacBio) remain viable alternatives. We outline a workflow from sequence quality control to microsatellite marker validation in three plant species using PacBio circular consensus sequencing (CCS). We then evaluate the performance of PacBio CCS in comparison with other NGS platforms for microsatellite isolation, through simulations that focus on variations in read length, read quantity and sequencing error rate. Although quality control of CCS reads reduced microsatellite yield by around 50%, hundreds of microsatellite loci that are expected to have improved conversion efficiency to functional markers were retrieved for each species. The simulations quantitatively validate the advantages of long reads and emphasize the detrimental effects of sequencing errors on NGS-enabled microsatellite development. In view of the continuing improvement in read length on NGS platforms, sequence quality and the corresponding strategies of quality control will become the primary factors to consider for effective microsatellite isolation. Among current options, PacBio CCS may be optimal for rapid, small-scale microsatellite development due to its flexibility in scaling sequencing effort, while platforms such as Illumina MiSeq will provide cost-efficient solutions for multispecies microsatellite projects.

Fine-scale niche structure of Neotropical forests reflects a legacy of the Great American Biotic Interchange.

The tendency of species to retain their ancestral niches may link processes that determine community assembly with biogeographic histories that span geological time scales. Biogeographic history is likely to have had a particularly strong impact on Neotropical forests because of the influence of the Great American Biotic Interchange, which followed emergence of a land connection between North and South America ~3 Ma. Here we examine the community structure, ancestral niches and ancestral distributions of the related, hyperdiverse woody plant genera Psychotria and Palicourea (Rubiaceae) in Panama. We find that 49% of the variation in hydraulic traits, a strong determinant of community structure, is explained by species' origins in climatically distinct biogeographic regions. Niche evolution models for a regional sample of 152 species indicate that ancestral climatic niches are associated with species' habitat distributions, and hence local community structure and composition, even millions of years after dispersal into new geographic regions.

Exon-primed intron-crossing (EPIC) markers for evolutionary studies of Ficus and other taxa in the fig family (Moraceae).

PREMISE OF THE STUDY: The genus Ficus (fig trees) comprises ca. 750 species of trees, vines, and stranglers found in tropical forests throughout the world. Fig trees are keystone species in many tropical forests, and their relationship with host-specific wasp pollinators has received much attention, although many questions remain unresolved regarding the levels of host specificity, cospeciation, and the role of hybridization in fig and wasp speciation. We developed exon-primed intron-crossing (EPIC) markers to obtain phylogenetic resolution needed to address these questions. • METHODS AND RESULTS: Expressed sequence tags (ESTs) from F. elastica were compared to Arabidopsis and Populus genomes to locate introns and to design primers in flanking exons. Primer pairs for 80 EPIC markers were tested in samples from divergent clades within Ficus and the outgroup Poulsenia (Moraceae). • CONCLUSIONS: Thirty-one EPIC markers were successfully sequenced across Ficus, and 29 of the markers also amplified in Poulsenia, indicating broad transferability within Moraceae. All of the EPIC markers were polymorphic and showed levels of polymorphism similar to that of the widely used internal transcribed spacer (ITS).