Increased resolution in the face of conflict: phylogenomics of the Neotropical bellflowers (Campanulaceae: Lobelioideae), a rapid plant radiation

Background and Aims- The centropogonid clade (Lobelioideae: Campanulaceae) is an Andean-centered rapid radiation characterized by repeated convergent evolution of morphological traits, including fruit type and pollination syndromes. While previous studies have resolved relationships of lineages with fleshy fruits into subclades, relationships among capsular species remain unresolved. This lack of resolution has impeded reclassification of non-monophyletic genera, whose current taxonomy relies heavily on traits that have undergone convergent evolution. Methods- Targeted sequence capture using a probeset recently developed for the centropogonid clade was used to obtain phylogenomic data from DNA extracted from both silica-dried and herbarium leaf tissue. These data were used to infer relationships among species using concatenated and partitioned species tree methods, and to quantify gene tree discordance. Key Results- While silica-dried leaf tissue resulted in more and longer sequence data, the inclusion of herbarium samples improved phylogenetic reconstruction. Relationships among baccate lineages are similar previous studies, though differ within and among capsular lineages. We improve phylogenetic resolution of Siphocampylus, which forms ten groups of closely related species which we informally name. Two subclades of Siphocampylus and two individual species are rogue taxa whose placement differs widely across analyses. Gene tree discordance (including cytonuclear discordance) is rampant. Conclusions- The first phylogenomic study of the centropogonid clade considerably improves our understanding of relationships in this rapid radiation. Differences across analyses and the possibility of additional lineage discoveries still hamper a solid and stable reclassification. Rapid morphological innovation corresponds with a high degree of phylogenomic complexity, including cytonuclear discordance, nuclear gene tree conflict, and well-supported differences between analyses based on different nuclear loci. Taken together, these results point to a potential role of hemiplasy underlying repeated convergent evolution. This hallmark of rapid radiations is likely present in many other species-rich Andean plant radiations.

How to Tackle Phylogenetic Discordance in Recent and Rapidly Radiating Groups? Developing a Workflow Using Loricaria (Asteraceae) as an Example

A major challenge in phylogenetics and -genomics is to resolve young rapidly radiating groups. The fast succession of species increases the probability of incomplete lineage sorting (ILS), and different topologies of the gene trees are expected, leading to gene tree discordance, i.e., not all gene trees represent the species tree. Phylogenetic discordance is common in phylogenomic datasets, and apart from ILS, additional sources include hybridization, whole-genome duplication, and methodological artifacts. Despite a high degree of gene tree discordance, species trees are often well supported and the sources of discordance are not further addressed in phylogenomic studies, which can eventually lead to incorrect phylogenetic hypotheses, especially in rapidly radiating groups. We chose the high-Andean Asteraceae genus Loricaria to shed light on the potential sources of phylogenetic discordance and generated a phylogenetic hypothesis. By accounting for paralogy during gene tree inference, we generated a species tree based on hundreds of nuclear loci, using Hyb-Seq, and a plastome phylogeny obtained from off-target reads during target enrichment. We observed a high degree of gene tree discordance, which we found implausible at first sight, because the genus did not show evidence of hybridization in previous studies. We used various phylogenomic analyses (trees and networks) as well as the D-statistics to test for ILS and hybridization, which we developed into a workflow on how to tackle phylogenetic discordance in recent radiations. We found strong evidence for ILS and hybridization within the genus Loricaria. Low genetic differentiation was evident between species located in different Andean cordilleras, which could be indicative of substantial introgression between populations, promoted during Pleistocene glaciations, when alpine habitats shifted creating opportunities for secondary contact and hybridization.

Sage Insights Into the Phylogeny of Salvia: Dealing With Sources of Discordance Within and Across Genomes

Next-generation sequencing technologies have facilitated new phylogenomic approaches to help clarify previously intractable relationships while simultaneously highlighting the pervasive nature of incongruence within and among genomes that can complicate definitive taxonomic conclusions. Salvia L., with ∼1,000 species, makes up nearly 15% of the species diversity in the mint family and has attracted great interest from biologists across subdisciplines. Despite the great progress that has been achieved in discerning the placement of Salvia within Lamiaceae and in clarifying its infrageneric relationships through plastid, nuclear ribosomal, and nuclear single-copy genes, the incomplete resolution has left open major questions regarding the phylogenetic relationships among and within the subgenera, as well as to what extent the infrageneric relationships differ across genomes. We expanded a previously published anchored hybrid enrichment dataset of 35 exemplars of Salvia to 179 terminals. We also reconstructed nearly complete plastomes for these samples from off-target reads. We used these data to examine the concordance and discordance among the nuclear loci and between the nuclear and plastid genomes in detail, elucidating both broad-scale and species-level relationships within Salvia. We found that despite the widespread gene tree discordance, nuclear phylogenies reconstructed using concatenated, coalescent, and network-based approaches recover a common backbone topology. Moreover, all subgenera, except for Audibertia, are strongly supported as monophyletic in all analyses. The plastome genealogy is largely resolved and is congruent with the nuclear backbone. However, multiple analyses suggest that incomplete lineage sorting does not fully explain the gene tree discordance. Instead, horizontal gene flow has been important in both the deep and more recent history of Salvia. Our results provide a robust species tree of Salvia across phylogenetic scales and genomes. Future comparative analyses in the genus will need to account for the impacts of hybridization/introgression and incomplete lineage sorting in topology and divergence time estimation.

A Time-calibrated Firefly (Coleoptera: Lampyridae) Phylogeny: Using Genomic Data for Divergence Time Estimation

Fireflies (Coleoptera: Lampyridae) consist of over 2,000 described extant species. A well-resolved phylogeny of fireflies is important for the study of their bioluminescence, evolution, and conservation. We used a recently published anchored hybrid enrichment dataset (AHE; 436 loci for 88 Lampyridae species and 10 outgroup species) and state-of-the-art statistical methods (the fossilized birth-death-range process implemented in a Bayesian framework) to estimate a time-calibrated phylogeny of Lampyridae. Unfortunately, estimating calibrated phylogenies using AHE and the latest and most robust time-calibration strategies is not possible because of computational constraints. As a solution, we subset the full dataset and applied three different strategies: using the most complete loci, the most homogeneous loci, and the loci with the highest accuracy to infer the well established Photinus clade. The estimated topology using the three data subsets agreed on almost all major clades and only showed minor discordance with less supported nodes. The estimated divergence times overlapped for all nodes that are shared between the topologies. Thus, divergence time estimation is robust as long as the topology inference is robust and any well selected data subset suffices. Additionally, we observed an unexpected amount of gene tree discordance between the 436 AHE loci. Our assessment of model adequacy showed that standard phylogenetic substitution models are not adequate for any of the 436 AHE loci which is likely to bias phylogenetic inferences. We performed a simulation study to explore the impact of (a) incomplete lineage sorting, (b) uniformly distributed and systematic missing data, and (c) systematic bias in the position of highly variable and conserved sites. For our simulated data, we observed less gene tree variation and hence the empirically observed amount of gene tree discordance for the AHE dataset is unexpected.

Phylogenomic analyses using genomes and transcriptomes do not resolve relationships among major clades in Phrymaceae

Premise of the study: Phylogenomic datasets using genomes and transcriptomes provide rich opportunities beyond resolving bifurcating phylogenetic relationships. Monkeyflower (Phrymaceae) is a model system for evolutionary ecology. However, it lacks a well-supported phylogeny for a stable taxonomy and for macroevolutionary comparisons. Methods: We sampled 24 genomes and transcriptomes in Phrymaceae and closely related families, including eight newly sequenced transcriptomes. We reconstructed the phylogeny using IQ-TREE and ASTRAL, evaluated gene tree discordance using PhyParts, Quartet Sampling, and cloudogram, and carried out phylogenetic network analyses using PhyloNet and HyDe. We searched for whole genome duplication (WGD) events using chromosome numbers, synonymous distance, and gene duplication events. Key results: Most gene trees support the monophyly of Phrymaceae and each of its tribes. Most gene trees also support the tribe Mimuleae being sister to Phrymeae + Diplaceae + Leucocarpeae, with extensive gene tree discordance among the latter three. Despite the discordance, polyphyly of Mimulus s.l. is strongly supported, and no particular reticulation event among the Phrymaceae tribes is well supported. Reticulation likely occurred among Erythranthe bicolor and close relatives. No ancient WGD event was detected in Phrymaceae. Instead, small-scale duplications are among potential drivers of macroevolutionary diversification of Phrymaceae. Conclusions: We show that analysis of reticulate evolution is sensitive to taxon sampling and methods used. We also demonstrate that genome-scale data do not always fully "resolve" phylogenetic relationships. They present rich opportunities to investigate reticulate evolution, and gene and genome evolution involved in lineage diversification and adaptation.

On the hybrid origin of the C2Salsola divaricata agg. (Amaranthaceae) from C3 and C4 parental lineages

C2 photosynthesis is characterized by recapturing photorespiratory CO2 by RuBisCO in Kranz-like cells and is therefore physiologically intermediate between C3 and C4 photosynthesis. C2 is either interpreted as an evolutionary precursor of C4 or as the result of hybridization between a C3 and C4 lineage. We compared the expression of photosynthetic traits among populations of the Salsola divaricata agg. (C2) from humid subtropical to arid habitats on the coasts of the Canary Islands and Morocco, and subjected them to salt and drought treatments. We screened for enhanced C4-like expression of traits related to habitat or treatment. We estimated species trees with a transcriptome dataset of Salsoleae and explored patterns of gene tree discordance. With phylogenetic networks and hybridization analyses we tested for hybrid origin of the Salsola divaricata agg. We observed independent variation of photosynthetic traits within and among populations and no clear evidence for selection towards C4-like trait expression in more stressful habitats or treatments. We found reticulation and gene tree incongruencies in the Salsoleae supporting a putative hybrid origin of the Salsola divaricata agg. C2 photosynthesis in the Salsola divaricata agg. combines traits inherited from its C3 and C4 parental lineages and seems well adapted to a wide climatic amplitude.

Phylogenomic data reveal hard polytomies across the backbone of the large genus Solanum (Solanaceae)

Increased volumes of phylogenomic data have revealed incongruent topologies in gene trees, both between and within genomes across many organisms. Some of these incongruences indicate polytomies that may remain impossible to resolve. Here, widespread gene-tree discordance is uncovered along the backbone of Solanum, one of the largest flowering plant genera that includes the cultivated potato, tomato, and eggplant, as well as 24 minor crop plants. First, a densely sampled species-level phylogeny of Solanum is built using unpublished and publicly available Sanger sequences comprising 60% of all accepted species (742 spp.) and nine regions (ITS, waxy, and seven plastid markers). The robustness of the Sanger-based topology is tested by examining a plastome dataset with 140 species and a nuclear target-capture dataset with 39 species of Solanum. Clear incongruences between species trees generated from the supermatrix, plastome, and nuclear target-capture datasets are revealed. Discordance within the plastome and target-capture dataset are found at different evolutionary depths in three different areas along the backbone of these phylogenetic trees, with polytomy tests suggesting that most of these nodes have short branches and should be collapsed. We argue that incomplete lineage sorting due to rapid diversification is the most likely cause behind these polytomies, and that embracing the uncertainty that underlies them is crucial to depict the evolution of large and rapidly radiating lineages.

Dissecting incongruence between concatenation- and quartet-based approaches in phylogenomic data

Topological conflict or incongruence is widespread in phylogenomic data. Concatenation- and coalescent-based approaches often result in incongruent topologies, but the causes of this conflict can be difficult to characterize. We examined incongruence stemming from conflict between likelihood-based signal (quantified by the difference in gene-wise log likelihood score or ΔGLS) and quartet-based topological signal (quantified by the difference in gene-wise quartet score or ΔGQS) for every gene in three phylogenomic studies in animals, fungi, and plants, which were chosen because their concatenation-based IQ-TREE (T1) and quartet-based ASTRAL (T2) phylogenies are known to produce eight conflicting internal branches (bipartitions). By comparing the types of phylogenetic signal for all genes in these three data matrices, we found that 30% - 36% of genes in each data matrix are inconsistent, that is, each of these genes has higher log likelihood score for T1 versus T2 (i.e., ΔGLS >0) whereas its T1 topology has lower quartet score than its T2 topology (i.e., ΔGQS <0) or vice versa. Comparison of inconsistent and consistent genes using a variety of metrics (e.g., evolutionary rate, gene tree topology, distribution of branch lengths, hidden paralogy, and gene tree discordance) showed that inconsistent genes are more likely to recover neither T1 nor T2 and have higher levels of gene tree discordance than consistent genes. Simulation analyses demonstrate that removal of inconsistent genes from datasets with low levels of incomplete lineage sorting (ILS) and low and medium levels of gene tree estimation error (GTEE) reduced incongruence and increased accuracy. In contrast, removal of inconsistent genes from datasets with medium and high ILS levels and high GTEE levels eliminated or extensively reduced incongruence, but the resulting congruent species phylogenies were not always topologically identical to the true species trees.

Widespread introgression across a phylogeny of 155 Drosophila genomes

Genome-scale sequence data has invigorated the study of hybridization and introgression, particularly in animals. However, outside of a few notable cases, we lack systematic tests for introgression at a larger phylogenetic scale across entire clades. Here we leverage 155 genome assemblies, from 149 species, to generate a fossil-calibrated phylogeny and conduct multilocus tests for introgression across 9 monophyletic radiations within the genus Drosophila. Using complementary phylogenomic approaches, we identify widespread introgression across the evolutionary history of Drosophila. Mapping gene-tree discordance onto the phylogeny revealed that both ancient and recent introgression has occurred, with introgression at the base of species radiations being particularly common. Our results provide the first evidence of introgression occurring across the evolutionary history of Drosophila and highlight the need to continue to study the evolutionary consequences of hybridization and introgression in this genus and across the Tree of Life.