Tackling Rapid Radiations With Targeted Sequencing

In phylogenetic studies across angiosperms, at various taxonomic levels, polytomies have persisted despite efforts to resolve them by increasing sampling of taxa and loci. The large amount of genomic data now available and statistical tools to analyze them provide unprecedented power for phylogenetic inference. Targeted sequencing has emerged as a strong tool for estimating species trees in the face of rapid radiations, lineage sorting, and introgression. Evolutionary relationships in Cyperaceae have been studied mostly using Sanger sequencing until recently. Despite ample taxon sampling, relationships in many genera remain poorly understood, hampered by diversification rates that outpace mutation rates in the loci used. The C4 Cyperus clade of the genus Cyperus has been particularly difficult to resolve. Previous studies based on a limited set of markers resolved relationships among Cyperus species using the C3 photosynthetic pathway, but not among C4 Cyperus clade taxa. We test the ability of two targeted sequencing kits to resolve relationships in the C4 Cyperus clade, the universal Angiosperms-353 kit and a Cyperaceae-specific kit. Sequences of the targeted loci were recovered from data generated with both kits and used to investigate overlap in data between kits and relative efficiency of the general and custom approaches. The power to resolve shallow-level relationships was tested using a summary species tree method and a concatenated maximum likelihood approach. High resolution and support are obtained using both approaches, but high levels of missing data disproportionately impact the latter. Targeted sequencing provides new insights into the evolution of morphology in the C4 Cyperus clade, demonstrating for example that the former segregate genus Alinula is polyphyletic despite its seeming morphological integrity. An unexpected result is that the Cyperus margaritaceus-Cyperus niveus complex comprises a clade separate from and sister to the core C4 Cyperus clade. Our results demonstrate that data generated with a family-specific kit do not necessarily have more power than those obtained with a universal kit, but that data generated with different targeted sequencing kits can often be merged for downstream analyses. Moreover, our study contributes to the growing consensus that targeted sequencing data are a powerful tool in resolving rapid radiationsEspaña Ministry of Economy and Competitiveness (project CGL2016- 77401-P

Tackling rapid radiations with targeted sequencing

In phylogenetic studies across angiosperms, at various taxonomic levels, polytomies have persisted despite efforts to resolve them by increasing sampling of taxa and loci. The large amount of genomic data now available and statistical tools to analyze them provide unprecedented power for phylogenetic inference. Targeted sequencing has emerged as a strong tool for estimating species trees in the face of rapid radiations, lineage sorting, and introgression. Evolutionary relationships in Cyperaceae have been studied mostly using Sanger sequencing until recently. Despite ample taxon sampling, relationships in many genera remain poorly understood, hampered by diversification rates that outpace mutation rates in the loci used. The C4 Cyperus clade of the genus Cyperus has been particularly difficult to resolve. Previous studies based on a limited set of markers resolved relationships among Cyperus species using the C3 photosynthetic pathway, but not among C4 Cyperus clade taxa. We test the ability of two targeted sequencing kits to resolve relationships in the C4 Cyperus clade, the universal Angiosperms-353 kit and a Cyperaceae-specific kit. Sequences of the targeted loci were recovered from data generated with both kits and used to investigate overlap in data between kits and relative efficiency of the general and custom approaches. The power to resolve shallow-level relationships was tested using a summary species tree method and a concatenated maximum likelihood approach. High resolution and support are obtained using both approaches, but high levels of missing data disproportionately impact the latter. Targeted sequencing provides new insights into the evolution of morphology in the C4 Cyperus clade, demonstrating for example that the former segregate genus Alinula is polyphyletic despite its seeming morphological integrity. An unexpected result is that the Cyperus margaritaceus-Cyperus niveus complex comprises a clade separate from and sister to the core C4 Cyperus clade. Our results demonstrate that data generated with a family-specific kit do not necessarily have more power than those obtained with a universal kit, but that data generated with different targeted sequencing kits can often be merged for downstream analyses. Moreover, our study contributes to the growing consensus that targeted sequencing data are a powerful tool in resolving rapid radiations

Targeted sequencing supports morphology and embryo features in resolving the classification of Cyperaceae tribe Fuireneae s.l.

Molecular phylogenetic studies based on Sanger sequences have shown that Cyperaceae tribe Fuireneae s.l. is paraphyletic. However, taxonomic sampling in these studies has been poor, topologies have been inconsistent, and support for the backbone of trees has been weak. Moreover, uncertainty still surrounds the morphological limits of Schoenoplectiella, a genus of mainly small, amphicarpic annuals that was recently segregated from Schoenoplectus. Consequently, despite ample evidence from molecular analyses that Fuireneae s.l. might consist of two to four tribal lineages, no taxonomic changes have yet been made. Here, we use the Angiosperms353 enrichment panel for targeted sequencing in order to: (1) clarify the relationships of Fuireneae s.l. with the related tribes Abildgaardieae, Eleocharideae and Cypereae; (2) define the limits of Fuireneae s.s., and (3) test the monophyly of Fuireneae s.l. genera with emphasis on Schoenoplectus and Schoenoplectiella. Using more than a third of Fuireneae s.l. diversity, our phylogenomic analyses strongly support six genera and four major Fuireneae s.l. clades that we recognise as tribes: Bolboschoeneae stat.nov., Fuireneae s.s., Schoenoplecteae, and Pseudoschoeneae tr.nov. These results are consistent with morphological, micromorphological (nutlet epidermal cell shape), and embryo differences detected for each tribe. At the generic level, most sub‐Saharan African perennials currently treated in Schoenoplectus are transferred to Schoenoplectiella. Our targeted sequencing results show that these species are nested in Schoenoplectiella, and their treatment here is consistent with micromorphological and embryo characters shared by all Schoenoplectiella species. Keys to recognised tribes and genera are provided

A universal probe set for targeted sequencing of 353 nuclear genes from any flowering plant designed using k-medoids clustering

Sequencing of target-enriched libraries is an efficient and cost-effective method for obtaining DNA sequence data from hundreds of nuclear loci for phylogeny reconstruction. Much of the cost of developing targeted sequencing approaches is associated with the generation of preliminary data needed for the identification of orthologous loci for probe design. In plants, identifying orthologous loci has proven difficult due to a large number of whole-genome duplication events, especially in the angiosperms (flowering plants).We used multiple sequence alignments from over 600 angiosperms for 353 putatively single-copy protein-coding genes identified by the One Thousand Plant Transcriptomes Initiative to design a set of targeted sequencing probes for phylogenetic studies of any angiosperm group. To maximize the phylogenetic potential of the probes, while minimizing the cost of production, we introduce a k-medoids clustering approach to identify the minimum number of sequences necessary to represent each coding sequence in the final probe set. Using this method, 5–15 representative sequences were selected per orthologous locus, representing the sequence diversity of angiosperms more efficiently than if probes were designed using available sequenced genomes alone. To test our approximately 80,000 probes, we hybridized libraries from 42 species spanning all higher-order groups of angiosperms, with a focus on taxa not present in the sequence alignments used to design the probes. Out of a possible 353 coding sequences, we recovered an average of 283 per species and at least 100 in all species. Differences among taxa in sequence recovery could not be explained by relatedness to the representative taxa selected for probe design, suggesting that there is no phylogenetic bias in the probe set. Our probe set, which targeted 260 kbp of coding sequence, achieved a median recovery of 137 kbp per taxon in coding regions, a maximum recovery of 250 kbp, and an additional median of 212 kbp per taxon in flanking non-coding regions across all species. These results suggest that the Angiosperms353 probe set described here is effective for any group of flowering plants and would be useful for phylogenetic studies from the species level to higher-order groups, including the entire angiosperm clade itself

Investigating antimalarial drug interactions of emetine dihydrochloride hydrate using CalcuSyn-based interactivity calculations

The widespread introduction of artemisinin-based combination therapy has contributed to recent reductions in malaria mortality. Combination therapies have a range of advantages, including synergism, toxicity reduction, and delaying the onset of resistance acquisition. Unfortunately, antimalarial combination therapy is limited by the depleting repertoire of effective drugs with distinct target pathways. To fast-track antimalarial drug discovery, we have previously employed drug-repositioning to identify the anti-amoebic drug, emetine dihydrochloride hydrate, as a potential candidate for repositioned use against malaria. Despite its 1000-fold increase in in vitro antimalarial potency (ED50 47 nM) compared with its anti-amoebic potency (ED50 26±32 uM), practical use of the compound has been limited by dose-dependent toxicity (emesis and cardiotoxicity). Identification of a synergistic partner drug would present an opportunity for dose-reduction, thus increasing the therapeutic window. The lack of reliable and standardised methodology to enable the in vitro definition of synergistic potential for antimalarials is a major drawback. Here we use isobologram and combination-index data generated by CalcuSyn software analyses (Biosoft v2.1) to define drug interactivity in an objective, automated manner. The method, based on the median effect principle proposed by Chou and Talalay, was initially validated for antimalarial application using the known synergistic combination (atovaquone-proguanil). The combination was used to further understand the relationship between SYBR Green viability and cytocidal versus cytostatic effects of drugs at higher levels of inhibition. We report here the use of the optimised Chou Talalay method to define synergistic antimalarial drug interactivity between emetine dihydrochloride hydrate and atovaquone. The novel findings present a potential route to harness the nanomolar antimalarial efficacy of this affordable natural product

A new classification of Cyperaceae (Poales) supported by phylogenomic data

Cyperaceae (sedges) are the third largest monocot family and are of considerable economic and ecological importance. Sedges represent an ideal model family to study evolutionary biology because of their species richness, global distribution, large discrepancies in lineage diversity, broad range of ecological preferences, and adaptations including multiple origins of C4 photosynthesis and holocentric chromosomes. Goetghebeur’s seminal work on Cyperaceae published in 1998 provided the most recent complete classification at tribal and generic level, based on a morphological study of Cyperaceae inflorescence, spikelet, flower and embryo characters plus anatomical and other information. Since then, several family‐level molecular phylogenetic studies using Sanger sequence data have been published. Here, more than 20 years after the last comprehensive classification of the family, we present the first family‐wide phylogenomic study of Cyperaceae based on targeted sequencing using the Angiosperms353 probe kit sampling 311 accessions. Additionally, 62 accessions available from GenBank were mined for overlapping reads and included in the phylogenomic analyses. Informed by this backbone phylogeny, a new classification for the family at the tribal, subtribal and generic levels is proposed. The majority of previously recognized suprageneric groups are supported, and for the first time we establish support for tribe Cryptangieae as a clade including the genus Koyamaea. We provide a taxonomic treatment including identification keys and diagnoses for the 2 subfamilies, 24 tribes and 10 subtribes and basic information on the 95 genera. The classification includes five new subtribes in tribe Schoeneae: Anthelepidinae, Caustiinae, Gymnoschoeninae, Lepidospermatinae and Oreobolinae. This article is protected by copyright. All rights reserved