The past decade has seen a major breakthrough in our ability to easily and inexpensively sequence genome‐scale data from diverse lineages. The development of high‐throughput sequencing and long‐read technologies has ushered in the era of phylogenomics, where hundreds to thousands of nuclear genes and whole organellar genomes are routinely used to reconstruct evolutionary relationships. As a result, understanding which options are best suited for a particular set of questions can be difficult, especially for those just starting in the field. Here, we review the most recent advances in plant phylogenomic methods and make recommendations for project‐dependent best practices and considerations. We focus on the costs and benefits of different approaches in regard to the information they provide researchers and the questions they can address. We also highlight unique challenges and opportunities in plant systems, such as polyploidy, reticulate evolution, and the use of herbarium materials, identifying optimal methodologies for each. Finally, we draw attention to lingering challenges in the field of plant phylogenomics, such as reusability of data sets, and look at some up‐and‐coming technologies that may help propel the field even further.

Advances in high-throughput sequencing (HTS) have allowed researchers to obtain large amounts of biological sequence information at speeds and costs unimaginable only a decade ago. Phylogenetics, and the study of evolution in general, is quickly migrating towards using HTS to generate larger and more complex molecular datasets. In this paper, we present a method that utilizes microfluidic PCR and HTS to generate large amounts of sequence data suitable for phylogenetic analyses. The approach uses the Fluidigm Access Array System (Fluidigm, San Francisco, CA, USA) and two sets of PCR primers to simultaneously amplify 48 target regions across 48 samples, incorporating sample-specific barcodes and HTS adapters (2,304 unique amplicons per Access Array). The final product is a pooled set of amplicons ready to be sequenced, and thus, there is no need to construct separate, costly genomic libraries for each sample. Further, we present a bioinformatics pipeline to process the raw HTS reads to either generate consensus sequences (with or without ambiguities) for every locus in every sample or—more importantly—recover the separate alleles from heterozygous target regions in each sample. This is important because it adds allelic information that is well suited for coalescent-based phylogenetic analyses that are becoming very common in conservation and evolutionary biology. To test our approach and bioinformatics pipeline, we sequenced 576 samples across 96 target regions belonging to the South American clade of the genus Bartsia L. in the plant family Orobanchaceae. After sequencing cleanup and alignment, the experiment resulted in ~25,300bp across 486 samples for a set of 48 primer pairs targeting the plastome, and ~13,500bp for 363 samples for a set of primers targeting regions in the nuclear genome. Finally, we constructed a combined concatenated matrix from all 96 primer combinations, resulting in a combined aligned length of ~40,500bp for 349 samples.

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This work advances understanding of Vitis evolutionary relationships and provides the foundation for ongoing germplasm enhancement. It supports conservation and breeding efforts by contributing to a growing genetic framework for identifying novel genetic variation and for incorporating new, unsampled populations into the germplasm repository system.

• Premise of the study: We present an alternative approach for molecular systematic studies that combines long PCR and next-generation sequencing. Our approach can be used to generate templates from any DNA source for next-generation sequencing. Here we test our approach by amplifying complete chloroplast genomes, and we present a set of 58 potentially universal primers for angiosperms to do so. Additionally, this approach is likely to be particularly useful for nuclear and mitochondrial regions.• Methods and Results: Chloroplast genomes of 30 species across angiosperms were amplified to test our approach. Amplification success varied depending on whether PCR conditions were optimized for a given taxon. To further test our approach, some amplicons were sequenced on an Illumina HiSeq 2000.• Conclusions: Although here we tested this approach by sequencing plastomes, long PCR amplicons could be generated using DNA from any genome, expanding the possibilities of this approach for molecular systematic studies.