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.

Genomic Characterization of Parengyodontium torokii sp. nov., a Biofilm-Forming Fungus Isolated from Mars 2020 Assembly Facility

A fungal strain (FJII-L10-SW-P1) was isolated from the Mars 2020 spacecraft assembly facility and exhibited biofilm formation on spacecraft-qualified Teflon surfaces. The reconstruction of a six-loci gene tree (ITS, LSU, SSU, RPB1 and RPB2, and TEF1) using multi-locus sequence typing (MLST) analyses of the strain FJII-L10-SW-P1 supported a close relationship to other known Parengyodontium album subclade 3 isolates while being phylogenetically distinct from subclade 1 strains. The zig-zag rachides morphology of the conidiogenous cells and spindle-shaped conidia were the distinct morphological characteristics of the P. album subclade 3 strains. The MLST data and morphological analysis supported the conclusion that the P. album subclade 3 strains could be classified as a new species of the genus Parengyodontium and placed in the family Cordycipitaceae. The name Parengyodontium torokii sp. nov. is proposed to accommodate the strain, with FJII-L10-SW-P1 as the holotype. The genome of the FJII-L10-SW-P1 strain was sequenced, annotated, and the secondary metabolite clusters were identified. Genes predicted to be responsible for biofilm formation and adhesion to surfaces were identified. Homology-based assignment of gene ontologies to the predicted proteome of P. torokii revealed the presence of gene clusters responsible for synthesizing several metabolic compounds, including a cytochalasin that was also verified using traditional metabolomic analysis.

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.

Identification and expression of odorant binding proteins in the egg-parasitoid Trissolcus basalis (Wollaston) (Hymenoptera, Scelionidae, Telenominae)

Trissolcus basalis (Wollaston) (Hymenoptera, Scelionidae) is an egg-parasitoid of the southern green stink bug, Nezara viridula (Linneaus) (Hemiptera, Pentatomidae). Many behaviors associated with female T. basalis host-finding and acceptance are mediated by chemosensory pathways, for which olfactory, gustatory and ionotropic receptors have been previously identified. Odorant binding proteins (OBPs) are small, globular proteins, one of the functions of which is the transport of odorant ligands through the aqueous lymph of chemosensory sensilla to these receptors. We identified 18 classical OBP sequences in the T. basalis genome and transcriptomes sharing an average 26.8% pairwise identity. Gene tree analyses suggest very limited lineage-specific expansion and identify potential orthologs among other scelionids and Hymenoptera. Transcriptome mapping and qPCR comparison of expression levels in antennae and bodies of both sexes determine that at least five TbOBPs are preferentially expressed in the female antennae. These are, therefore, prime candidates for further study to determine their role in detecting host-produced semiochemicals.

A target capture approach for phylogenomic analyses at multiple evolutionary timescales in rosewoods (Dalbergia spp.) and the legume family (Fabaceae)

Understanding the genetic changes associated with the evolution of biological diversity is of fundamental interest to molecular ecologists. The assessment of genetic variation at hundreds or thousands of unlinked genetic loci forms a sound basis to address questions ranging from micro- to macro-evolutionary timescales, and is now possible thanks to advances in sequencing technology. Major difficulties are associated with i) the lack of genomic resources for many taxa, especially from tropical biodiversity hotspots, ii) scaling the numbers of individuals analyzed and loci sequenced, and iii) building tools for reproducible bioinformatic analyses of such datasets. To address these challenges, we developed a set of target capture probes for phylogenomic studies of the highly diverse, pantropically distributed and economically significant rosewoods (Dalbergia spp.), explored the performance of an overlapping probe set for target capture across the legume family (Fabaceae), and built a general-purpose bioinformatics pipeline. Phylogenomic analyses of Dalbergia species from Madagascar yielded highly resolved and well supported hypotheses of evolutionary relationships. Population genomic analyses identified differences between closely related species and revealed the existence of a potentially new species, suggesting that the diversity of Malagasy Dalbergia species has been underestimated. Analyses at the family level corroborated previous findings by the recovery of monophyletic subfamilies and many well-known clades, as well as high levels of gene tree discordance, especially near the root of the family. The new genomic and bioinformatics resources will hopefully advance systematics and ecological genetics research in legumes, and promote conservation of the highly diverse and endangered Dalbergia rosewoods.

A target capture approach for phylogenomic analyses at multiple evolutionary timescales in rosewoods (Dalbergia spp.) and the legume family (Fabaceae)

Understanding the genetic changes associated with the evolution of biological diversity is of fundamental interest to molecular ecologists. The assessment of genetic variation at hundreds or thousands of unlinked genetic loci forms a sound basis to address questions ranging from micro- to macro-evolutionary timescales, and is now possible thanks to advances in sequencing technology. Major difficulties are associated with i) the lack of genomic resources for many taxa, especially from tropical biodiversity hotspots, ii) scaling the numbers of individuals analyzed and loci sequenced, and iii) building tools for reproducible bioinformatic analyses of such datasets. To address these challenges, we developed a set of target capture probes for phylogenomic studies of the highly diverse, pantropically distributed and economically significant rosewoods (Dalbergia spp.), explored the performance of an overlapping probe set for target capture across the legume family (Fabaceae), and built a general-purpose bioinformatics pipeline. Phylogenomic analyses of Dalbergia species from Madagascar yielded highly resolved and well supported hypotheses of evolutionary relationships. Population genomic analyses identified differences between closely related species and revealed the existence of a potentially new species, suggesting that the diversity of Malagasy Dalbergia species has been underestimated. Analyses at the family level corroborated previous findings by the recovery of monophyletic subfamilies and many well-known clades, as well as high levels of gene tree discordance, especially near the root of the family. The new genomic and bioinformatics resources will hopefully advance systematics and ecological genetics research in legumes, and promote conservation of the highly diverse and endangered Dalbergia rosewoods.

Taxonomic revision of Romaleosyrphus Bigot (Diptera, Syrphidae), including descriptions of seven new species

The genus Romaleosyrphus Bigot is reviewed, including the description of seven new species (R. argosi Moran, sp. nov., R. bigoti Moran, sp. nov., R. drysus Moran, sp. nov., R. nephelaeus Moran & Thompson, sp. nov., R. soletluna Moran & Thompson, sp. nov., R. vockerothi Moran & Thompson, sp. nov. and R. woodi Moran, sp. nov.). Romaleosyrphus arctophiloides (Giglio-Tos), comb. nov. is transferred to Romaleosyrphus. Romaleosyrphusstat. rev. is redefined to represent the monophyletic unit of species within Criorhinina which possess holoptic males, a proximal ventral half of vein C with setae, a broad intersection of vein R1 with vein C, the distal part of R4+5 beyond M1 longer than cross-vein h and appressed pile on the abdomen. Descriptions, habitus and genitalia photographs, distributions, and an illustrated key for all nine Romaleosyrphus are presented. DNA barcode data are provided for eight of the species with a cytochrome c oxidase subunit I gene tree presented and discussed.

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.