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-centred 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 probe-set 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 longer assembled sequence data, the inclusion of herbarium samples improved taxonomic representation. Relationships among baccate lineages are similar to those inferred in previous studies, although they differ for lineages within and among capsular clades. We improve the 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: This 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. Together, these results point to a potential role of hemiplasy underlying repeated convergent evolution. This hallmark of rapid radiations is probably present in many other species-rich Andean plant radiations.

Utility of targeted sequence capture for phylogenomics in rapid, recent angiosperm radiations: Neotropical Burmeistera bellflowers as a case study.

Targeted sequence capture is a promising approach for large-scale phylogenomics. However, rapid evolutionary radiations pose significant challenges for phylogenetic inference (e.g. incomplete lineages sorting (ILS), phylogenetic noise), and the ability of targeted nuclear loci to resolve species trees despite such issues remains poorly studied. We test the utility of targeted sequence capture for inferring phylogenetic relationships in rapid, recent angiosperm radiations, focusing on Burmeistera bellflowers (Campanulaceae), which diversified into ~130 species over less than 3 million years. We compared phylogenies estimated from supercontig (exons plus flanking sequences), exon-only, and flanking-only datasets with 506-546 loci (~4.7 million bases) for 46 Burmeistera species/lineages and 10 outgroup taxa. Nuclear loci resolved backbone nodes and many congruent internal relationships with high support in concatenation and coalescent-based species tree analyses, and inferences were largely robust to effects of missing taxa and base composition biases. Nevertheless, species trees were incongruent between datasets, and gene trees exhibited remarkably high levels of conflict (~4-60% congruence, ~40-99% conflict) not simply driven by poor gene tree resolution. Higher gene tree heterogeneity at shorter branches suggests an important role of ILS, as expected for rapid radiations. Phylogenetic informativeness analyses also suggest this incongruence has resulted from low resolving power at short internal branches, consistent with ILS, and homoplasy at deeper nodes, with exons exhibiting much greater risk of incorrect topologies due to homoplasy than other datasets. Our findings suggest that targeted sequence capture is feasible for resolving rapid, recent angiosperm radiations, and that results based on supercontig alignments containing nuclear exons and flanking sequences have higher phylogenetic utility and accuracy than either alone. We use our results to make practical recommendations for future target capture-based studies of Burmeistera and other rapid angiosperm radiations, including that such studies should analyze supercontigs to maximize the phylogenetic information content of loci.

Practical considerations for plant phylogenomics.

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.

Incongruence in molecular species delimitation schemes: What to do when adding more data is difficult.

Using multiple, independent approaches to molecular species delimitation is advocated to accommodate limitations and assumptions of a single approach. Incongruence in delimitation schemes is a potential by-product of employing multiple methods on the same data, and little attention has been paid to its reconciliation. Instead, a particular scheme is prioritized, and/or molecular delimitations are coupled with additional, independent lines of evidence that mitigate incongruence. We advocate that incongruence within a line of evidence should be accounted for before comparing across lines of evidence that can themselves be incongruent. Additionally, it is not uncommon for empiricists working in nonmodel systems to be data-limited, generating some concern for the adequacy of available data to address the question of interest. With conservation and management decisions often hinging on the status of species, it seems prudent to understand the capabilities of approaches we use given the data we have. Here, we apply two molecular species delimitation approaches, spedeSTEM and BPP, to the Castilleja ambigua (Orobanchaceae) species complex, a relatively young plant lineage in western North America. Upon finding incongruence in our delimitation, we employed a post hoc simulation study to examine the power of these approaches to delimit species. Given the data we collected, we find that spedeSTEM lacks the power to delimit while BPP is capable, thus allowing us to address incongruence before proceeding in delimitation. We suggest post hoc simulation studies like this compliment empirical delimitation and serve as a means of exploring conflict within a line of evidence and dealing with it appropriately.

High-throughput sequencing data clarify evolutionary relationships among North American Vitis species and improve identification in USDA Vitis germplasm collections.

PREMISE OF THE STUDY: Grapes are one of the most economically important berry crops worldwide, with the vast majority of production derived from the domesticated Eurasian species Vitis vinifera. Expansion of production into new areas, development of new cultivars, and concerns about adapting grapevines for changing climates necessitate the use of wild grapevine species in breeding programs. Diversity within Vitis has long been a topic of study; however, questions remain regarding relationships between species. Furthermore, the identity of some living accessions is unclear. METHODS: This study generated 11,020 single nucleotide polymorphism (SNP) markers for more than 300 accessions in the USDA-ARS grape germplasm repository using genotyping-by-sequencing. Resulting data sets were used to reconstruct evolutionary relationships among several North American and Eurasian Vitis species, and to suggest taxonomic labels for previously unidentified and misidentified germplasm accessions based on genetic distance. KEY RESULTS: Maximum likelihood analyses of SNP data support the monophyly of Vitis, subg. Vitis, a Eurasian subg. Vitis clade, and a North American subg. Vitis clade. Data delineate species groups within North America. In addition, analysis of genetic distance suggested taxonomic identities for 20 previously unidentified Vitis accessions and for 28 putatively misidentified accessions. CONCLUSIONS: 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.

Primers for Castilleja and their utility across Orobanchaceae: I. Chloroplast primers.

PREMISE OF THE STUDY: Chloroplast primers were developed from genomic data for the taxonomically challenging genus Castilleja. We further tested the broader utility of these primers across Orobanchaceae, identifying a core set of chloroplast primers amplifying across the clade. METHODS AND RESULTS: Using a combination of three low-coverage Castilleja genomes and sequence data from 12 Castilleja plastomes, 76 primer combinations were specifically designed and tested for Castilleja. The primers targeted the most variable portions of the plastome and were validated for their applicability across the clade. Of these, 38 primer combinations were subsequently evaluated in silico and then validated across other major clades in Orobanchaceae. CONCLUSIONS: These results demonstrate the utility of these primers, not only across Castilleja, but for other clades in Orobanchaceae-particularly hemiparasitic lineages-and will contribute to future phylogenetic studies of this important clade of parasitic plants.

Phylogenetic relationships of Burmeistera (Campanulaceae: Lobelioideae): Combining whole plastome with targeted loci data in a recent radiation.

The field of molecular systematics has benefited greatly with the advent of high-throughput sequencing (HTS), making large genomic datasets commonplace. However, a large number of targeted Sanger sequences produced by many studies over the last two decades are publicly available and should not be overlooked. In this study, we elucidate the phylogenetic relationships of the plant genus Burmeistera (Campanulaceae: Lobelioideae), while investigating how to best combine targeted Sanger loci with HTS data. We sequence, annotate, and analyze complete to nearly complete plastomes for a subset of the genus. We then combine these data with a much larger taxonomic dataset for which only Sanger sequences are available, making this the most comprehensively sampled study in the genus to date. We show that using a phylogeny inferred from the species with plastome data as a topological constraint for the larger dataset increases the resolution of our data and produces a more robust evolutionary hypothesis for the group. We then use the resulting phylogeny to study the evolution of morphological traits thought to be important in Burmeistera, and assess their usefulness in the current taxonomic classification of the genus. The main morphological character used to delimit subgeneric sections, the presence or absence of hairs on the apex of the two ventral anthers, shows a complex evolutionary history with many changes in the tree, suggesting that this character should not be used for taxonomic classification. Although it is too soon to propose a new subgeneric classification for Burmeistera, our results highlight some morphological traits shared by whole clades that could potentially be used in future taxonomic work.

A Phylogenomic Approach Based on PCR Target Enrichment and High Throughput Sequencing: Resolving the Diversity within the South American Species of Bartsia L. (Orobanchaceae).

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 ~25,300 bp across 486 samples for a set of 48 primer pairs targeting the plastome, and ~13,500 bp 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 ~40,500 bp for 349 samples.

Shifts in diversification rates linked to biogeographic movement into new areas: An example of a recent radiation in the Andes.

PREMISE OF THE STUDY: Clade-specific bursts in diversification are often associated with the evolution of key innovations. However, in groups with no obvious morphological innovations, observed upticks in diversification rates have also been attributed to the colonization of a new geographic environment. In this study, we explore the systematics, diversification dynamics, and historical biogeography of the plant clade Rhinantheae in the Orobanchaceae, with a special focus on the Andean clade of the genus Bartsia. METHODS: We sampled taxa from across Rhinantheae, including a representative sample of Andean Bartsia species. Using standard phylogenetic methods, we reconstructed evolutionary relationships, inferred divergence times among the clades of Rhinantheae, elucidated their biogeographic history, and investigated diversification dynamics. KEY RESULTS: We confirmed that the South American Bartsia species form a highly supported monophyletic group. The median crown age of Rhinantheae was determined to be ca. 30 Myr, and Europe played an important role in the biogeographic history of the lineages. South America was first reconstructed in the biogeographic analyses around 9 Myr ago, and with a median age of 2.59 Myr, this clade shows a significant uptick in diversification. CONCLUSIONS: Increased net diversification of the South American clade corresponds to biogeographic movement into the New World. This movement happened at a time when the Andes were reaching the necessary elevation to host an alpine environment. Although a specific route could not be identified with certainty, we provide plausible hypotheses to how the group colonized the New World.

Oligocene niche shift, Miocene diversification - cold tolerance and accelerated speciation rates in the St. John's Worts (Hypericum, Hypericaceae).

BACKGROUND: Our aim is to understand the evolution of species-rich plant groups that shifted from tropical into cold/temperate biomes. It is well known that climate affects evolutionary processes, such as how fast species diversify, species range shifts, and species distributions. Many plant lineages may have gone extinct in the Northern Hemisphere due to Late Eocene climate cooling, while some tropical lineages may have adapted to temperate conditions and radiated; the hyper-diverse and geographically widespread genus Hypericum is one of these. RESULTS: To investigate the effect of macroecological niche shifts on evolutionary success we combine historical biogeography with analyses of diversification dynamics and climatic niche shifts in a phylogenetic framework. Hypericum evolved cold tolerance c. 30 million years ago, and successfully colonized all ice-free continents, where today ~500 species exist. The other members of Hypericaceae stayed in their tropical habitats and evolved into ~120 species. We identified a 15-20 million year lag between the initial change in temperature preference in Hypericum and subsequent diversification rate shifts in the Miocene. CONCLUSIONS: Contrary to the dramatic niche shift early in the evolution of Hypericum most extant species occur in temperate climates including high elevations in the tropics. These cold/temperate niches are a distinctive characteristic of Hypericum. We conclude that the initial release from an evolutionary constraint (from tropical to temperate climates) is an important novelty in Hypericum. However, the initial shift in the adaptive landscape into colder climates appears to be a precondition, and may not be directly related to increased diversification rates. Instead, subsequent events of mountain formation and further climate cooling may better explain distribution patterns and species-richness in Hypericum. These findings exemplify important macroevolutionary patterns of plant diversification during large-scale global climate change.

A long PCR-based approach for DNA enrichment prior to next-generation sequencing for systematic studies.

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.