Prezygotic barriers effectively limit hybridization in a rapid evolutionary radiation.

Hybridization is increasingly recognized as an important evolutionary process across the tree of life. In many clades, phylogenomic approaches have permitted unparalleled insight into the extent and frequency of hybridization. However, we continue to lack a deep understanding of the factors that limit and shape patterns of hybridization, especially in evolutionary radiations. In this study, we characterized patterns of introgression across Costus (Costaceae), a young evolutionary radiation of tropical understory plants that maintain widespread interfertility despite exhibiting strong prezygotic reproductive isolation. We analyzed a phylogenomic dataset of 756 genes from 54 Costus species using multiple complementary approaches - D-statistics, gene-tree-based tests, and phylogenetic network analyses - to detect and characterize introgression events throughout the evolutionary history of the radiation. Our results identified a moderate number of introgression events, including a particularly ancient, well-supported event spanning one of the deepest divergences in the clade. Most introgression events occurred between taxa or ancestral lineages that shared the same pollination syndrome (bee-pollinated or hummingbird-pollinated). These findings suggest that prezygotic barriers, including pollinator specialization, have been key to the balance between introgression and reproductive isolation in Costus.

Genome assemblies and comparison of two Neotropical spiral gingers: Costus pulverulentus and C. lasius.

The spiral gingers (Costus L.) are a pantropical genus of herbaceous perennial monocots; the Neotropical clade of Costus radiated rapidly in the past few million years into over 60 species. The Neotropical spiral gingers have a rich history of evolutionary and ecological research that can motivate and inform modern genetic investigations. Here, we present the first 2 chromosome-level genome assemblies in the genus, for C. pulverulentus and C. lasius, and briefly compare their synteny. We assembled the C. pulverulentus genome from a combination of short-read data, Chicago and Dovetail Hi-C chromatin-proximity sequencing, and alignment with a linkage map. We annotated the genome by mapping a C. pulverulentus transcriptome and querying mapped transcripts against a protein database. We assembled the C. lasius genome with Pacific Biosciences HiFi long reads and alignment to the C. pulverulentus genome. These 2 assemblies are the first published genomes for non-cultivated tropical plants. These genomes solidify the spiral gingers as a model system and will facilitate research on the poorly understood genetic basis of tropical plant diversification.

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.

A consensus phylogenomic approach highlights paleopolyploid and rapid radiation in the history of Ericales.

PREMISE: Large genomic data sets offer the promise of resolving historically recalcitrant species relationships. However, different methodologies can yield conflicting results, especially when clades have experienced ancient, rapid diversification. Here, we analyzed the ancient radiation of Ericales and explored sources of uncertainty related to species tree inference, conflicting gene tree signal, and the inferred placement of gene and genome duplications. METHODS: We used a hierarchical clustering approach, with tree-based homology and orthology detection, to generate six filtered phylogenomic matrices consisting of data from 97 transcriptomes and genomes. Support for species relationships was inferred from multiple lines of evidence including shared gene duplications, gene tree conflict, gene-wise edge-based analyses, concatenation, and coalescent-based methods, and is summarized in a consensus framework. RESULTS: Our consensus approach supported a topology largely concordant with previous studies, but suggests that the data are not capable of resolving several ancient relationships because of lack of informative characters, sensitivity to methodology, and extensive gene tree conflict correlated with paleopolyploidy. We found evidence of a whole-genome duplication before the radiation of all or most ericalean families, and demonstrate that tree topology and heterogeneous evolutionary rates affect the inferred placement of genome duplications. CONCLUSIONS: We provide several hypotheses regarding the history of Ericales, and confidently resolve most nodes, but demonstrate that a series of ancient divergences are unresolvable with these data. Whether paleopolyploidy is a major source of the observed phylogenetic conflict warrants further investigation.

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.

Bayesian and likelihood phylogenetic reconstructions of morphological traits are not discordant when taking uncertainty into consideration: a comment on Puttick et al.

Puttick et al. (2017 Proc. R. Soc. B284, 20162290 (doi:10.1098/rspb.2016.2290)) performed a simulation study to compare accuracy among methods of inferring phylogeny from discrete morphological characters. They report that a Bayesian implementation of the Mk model (Lewis 2001 Syst. Biol.50, 913-925 (doi:10.1080/106351501753462876)) was most accurate (but with low resolution), while a maximum-likelihood (ML) implementation of the same model was least accurate. They conclude by strongly advocating that Bayesian implementations of the Mk model should be the default method of analysis for such data. While we appreciate the authors' attempt to investigate the accuracy of alternative methods of analysis, their conclusion is based on an inappropriate comparison of the ML point estimate, which does not consider confidence, with the Bayesian consensus, which incorporates estimation credibility into the summary tree. Using simulation, we demonstrate that ML and Bayesian estimates are concordant when confidence and credibility are comparably reflected in summary trees, a result expected from statistical theory. We therefore disagree with the conclusions of Puttick et al. and consider their prescription of any default method to be poorly founded. Instead, we recommend caution and thoughtful consideration of the model or method being applied to a morphological dataset.

Conflicting phylogenomic signals reveal a pattern of reticulate evolution in a recent high-Andean diversification (Asteraceae: Astereae: Diplostephium).

High-throughput sequencing is helping biologists to overcome the difficulties of inferring the phylogenies of recently diverged taxa. The present study analyzes the phylogenetic signal of genomic regions with different inheritance patterns using genome skimming and ddRAD-seq in a species-rich Andean genus (Diplostephium) and its allies. We analyzed the complete nuclear ribosomal cistron, the complete chloroplast genome, a partial mitochondrial genome, and a nuclear-ddRAD matrix separately with phylogenetic methods. We applied several approaches to understand the causes of incongruence among datasets, including simulations and the detection of introgression using the D-statistic (ABBA-BABA test). We found significant incongruence among the nuclear, chloroplast, and mitochondrial phylogenies. The strong signal of hybridization found by simulations and the D-statistic among genera and inside the main clades of Diplostephium indicate reticulate evolution as a main cause of phylogenetic incongruence. Our results add evidence for a major role of reticulate evolution in events of rapid diversification. Hybridization and introgression confound chloroplast and mitochondrial phylogenies in relation to the species tree as a result of the uniparental inheritance of these genomic regions. Practical implications regarding the prevalence of hybridization are discussed in relation to the phylogenetic method.

Allopatric speciation is more prevalent than parapatric ecological divergence in a recent high-Andean diversification (Linochilus: Asteraceae).

Elucidating how species accumulate in diversity hotspots is an ongoing debate in evolutionary biology. The páramo, in the Northern Andes, has remarkably high indices of plant diversity, endemicity, and diversification rates. A hypothesis for explaining such indices is that allopatric speciation is high in the páramo given its island-like distribution. An alternative hypothesis is that the altitudinal gradient of the Andean topography provides a variety of niches that drive vertical parapatric ecological speciation. A formal test for evaluating the relative roles of allopatric and parapatric ecological speciation is lacking. The main aim of our study is to test which kind of speciation is more common in an endemic páramo genus. We developed a framework incorporating phylogenetics, species' distributions, and a morpho-ecological trait (leaf area) to compare sister species and infer whether allopatric or parapatric ecological divergence caused their speciation. We applied our framework to the species-rich genus Linochilus (63 spp.) and found that the majority of recent speciation events in it (12 events, 80%) have been driven by allopatric speciation, while a smaller fraction (one event, 6.7%) is attributed to parapatric ecological speciation; two pairs of sister species produced inconclusive results (13.3%). We conclude that páramo autochthonous (in-situ) diversification has been primarily driven by allopatric speciation.

Patterns of speciation are similar across mountainous and lowland regions for a Neotropical plant radiation (Costaceae: Costus).

High species richness and endemism in tropical mountains are recognized as major contributors to the latitudinal diversity gradient. The processes underlying mountain speciation, however, are largely untested. The prevalence of steep ecogeographic gradients and the geographic isolation of populations by topographic features are predicted to promote speciation in mountains. We evaluate these processes in a species-rich Neotropical genus of understory herbs that range from the lowlands to montane forests and have higher species richness in topographically complex regions. We ask whether climatic niche divergence, geographic isolation, and pollination shifts differ between mountain-influenced and lowland Amazonian sister pairs inferred from a 756-gene phylogeny. Neotropical Costus ancestors diverged in Central America during a period of mountain formation in the last 3 million years with later colonization of Amazonia. Although climatic divergence, geographic isolation, and pollination shifts are prevalent in general, these factors do not differ between mountain-influenced and Amazonian sister pairs. Despite higher climatic niche and species diversity in the mountains, speciation modes in Costus appear similar across regions. Thus, greater species richness in tropical mountains may reflect differences in colonization history, diversification rates, or the prevalence of rapidly evolving plant life forms, rather than differences in speciation mode.

Characterizing gene tree conflict in plastome-inferred phylogenies.

Evolutionary relationships among plants have been inferred primarily using chloroplast data. To date, no study has comprehensively examined the plastome for gene tree conflict. Using a broad sampling of angiosperm plastomes, we characterize gene tree conflict among plastid genes at various time scales and explore correlates to conflict (e.g., evolutionary rate, gene length, molecule type). We uncover notable gene tree conflict against a backdrop of largely uninformative genes. We find alignment length and tree length are strong predictors of concordance, and that nucleotides outperform amino acids. Of the most commonly used markers, matK, greatly outperforms rbcL; however, the rarely used gene rpoC2 is the top-performing gene in every analysis. We find that rpoC2 reconstructs angiosperm phylogeny as well as the entire concatenated set of protein-coding chloroplast genes. Our results suggest that longer genes are superior for phylogeny reconstruction. The alleviation of some conflict through the use of nucleotides suggests that stochastic and systematic error is likely the root of most of the observed conflict, but further research on biological conflict within plastome is warranted given documented cases of heteroplasmic recombination. We suggest that researchers should filter genes for topological concordance when performing downstream comparative analyses on phylogenetic data, even when using chloroplast genomes.

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.