Community-level phylogenetic diversity does not differ between rare and common lineages across tallgrass prairies in the northern Great Plains.

Niche differentiation has served as one explanation for species coexistence, and phylogenetic relatedness provides a means to approximate how ecologically similar species are to each other. To explore the contribution of rare species to community phylogenetic diversity, we sampled 21 plant communities across the Prairie Coteau ecoregion, an area of high conservation concern. We used breakpoint analysis through the iterative addition of less abundant species to the phylogenetic tree for each community to assess the contribution of rare species to community phylogenetic diversity. We also quantify the phylogenetic signal of abundance using Blomberg's K statistic and calculated the phylogenetic similarity between rare and common species using a phylogenetic beta-diversity metric (D nn). To estimate the phylogenetic structuring of these prairie communities, we calculated two common metrics that capture evolutionary relatedness at different scales (MPD and MNTD). Additionally, we examine the correlation between Faith's PD, MPD, and MNTD and species richness. We found rare species do not generally contribute higher levels of phylogenetic diversity than common species. Eight communities had significant breakpoints, with only four communities having an increasing trend for the rarest species. The phylogenetic signal for abundance was low but significant in only four communities, and communities had lower phylogenetic diversity than expected from the regional species pool. Finally, the strength of the correlation between species richness and phylogenetic diversity was mixed. Our results indicate niche differentiation does not explain the persistence of rare species in tallgrass prairies, as they were more closely related than expected from random, suggesting high functional redundancy between rare and common species. This is promising for the long-term resilience of this ecosystem, but only insofar as enough species remain in the system. With ongoing biodiversity loss, it is essential that we understand the role rare species play in their communities.

Digitized collections elucidate invasion history and patterns of awn polymorphism in Microstegium vimineum.

PREMISE: Digitized collections can help illuminate the mechanisms behind the establishment and spread of invasive plants. These databases provide a record of traits in space and time that allows for investigation of abiotic and biotic factors that influence invasive species. METHODS: Over 1100 digitized herbarium records were examined to investigate the invasion history and trait variation of Microstegium vimineum. Presence-absence of awns was investigated to quantify geographic patterns of this polymorphic trait, which serves several functions in grasses, including diaspore burial and dispersal to germination sites. Floret traits were further quantified, and genomic analyses of contemporary samples were conducted to investigate the history of M. vimineum's introduction and spread into North America. RESULTS: Herbarium records revealed similar patterns of awn polymorphism in native and invaded ranges of M. vimineum, with awned forms predominating at higher latitudes and awnless forms at lower latitudes. Herbarium records and genomic data suggested initial introduction and spread of the awnless form in the southeastern United States, followed by a putative secondary invasion and spread of the awned form from eastern Pennsylvania. Awned forms have longer florets, and floret size varies significantly with latitude. There is evidence of a transition zone with short-awned specimens at mid-latitudes. Genomic analyses revealed two distinct clusters corresponding to awnless and awned forms, with evidence of admixture. CONCLUSIONS: Our results demonstrate the power of herbarium data to elucidate the invasion history of a problematic weed in North America and, together with genomic data, reveal a possible key trait in introduction success: presence or absence of an awn.

Examining the utility of DNA barcodes for the identification of tallgrass prairie flora.

PREMISE: The tallgrass prairies of North America are one of the most threatened ecosystems in the world, making efficient species identification essential for understanding and managing diversity. Here, we assess DNA barcoding with high-throughput sequencing as a method for rapid plant species identification. METHODS: Using herbarium collections representing the tallgrass prairie flora of Oak Lake Field Station, South Dakota, USA, we amplified and examined four common nuclear and plastid barcode regions (ITS, matK, psbA-trnH, and rbcL), individually and in combination, to test their success in identifying samples to family, genus, and species levels using BLAST searches of three databases of varying size. RESULTS: Concatenated barcodes increased performance, although none were significantly different than single-region barcodes. The plastid region psbA-trnH performed significantly more poorly than the others, while barcodes containing ITS performed best. Database size significantly affected identification success at all three taxonomic levels. Confident species-level identification ranged from 8-44% for the global database, 13-56% for the regional database, and 21-80% for the sampled species database, depending on the barcode used. DISCUSSION: Barcoding was generally successful in identifying tallgrass prairie genera and families, but was of limited use in species-level identifications. Database size was an important factor in successful plant identification. We discuss future directions and considerations for improving the performance of DNA barcoding in tallgrass prairies.

Fluidigm2PURC: Automated processing and haplotype inference for double-barcoded PCR amplicons.

PREMISE OF THE STUDY: Targeted enrichment strategies for phylogenomic inference are a time- and cost-efficient way to collect DNA sequence data for large numbers of individuals at multiple, independent loci. Automated and reproducible processing of these data is a crucial step for researchers conducting phylogenetic studies. METHODS AND RESULTS: We present Fluidigm2PURC, an open source Python utility for processing paired-end Illumina data from double-barcoded PCR amplicons. In combination with the program PURC (Pipeline for Untangling Reticulate Complexes), our scripts process raw FASTQ files for analysis with PURC and use its output to infer haplotypes for diploids, polyploids, and samples with unknown ploidy. We demonstrate the use of the pipeline with an example data set from the genus Thalictrum (Ranunculaceae). CONCLUSIONS: Fluidigm2PURC is freely available for Unix-like operating systems on GitHub (https://github.com/pblischak/fluidigm2purc) and for all operating systems through Docker (https://hub.docker.com/r/pblischak/fluidigm2purc).

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.

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.

Primers for Castilleja and their utility across Orobanchaceae: II. Single-copy nuclear loci.

PREMISE OF THE STUDY: We developed primers targeting nuclear loci in Castilleja with the goal of reconstructing the evolutionary history of this challenging clade. These primers were tested across other major clades in Orobanchaceae to assess their broader utility. METHODS AND RESULTS: We assembled low-coverage genomes for three taxa in Castilleja and developed primer combinations for the single-copy conserved ortholog set (COSII) and the pentatricopeptide repeat (PPR) gene family. These primer combinations were designed to take advantage of the Fluidigm microfluidic PCR platform and are well suited for high-throughput sequencing applications. Eighty-seven primers were designed for Castilleja, and 27 were found to have broader utility in Orobanchaceae. CONCLUSIONS: These results demonstrate the utility of these primers, not only across Castilleja, but for other lineages within Orobanchaceae as well. This expanded molecular toolkit will be an asset to future phylogenetic studies in Castilleja and throughout Orobanchaceae.

Phylogenetic relationships and character evolution analysis of Saxifragales using a supermatrix approach.

PREMISE OF THE STUDY: We sought novel evolutionary insights for the highly diverse Saxifragales by constructing a large phylogenetic tree encompassing 36.8% of the species-level biodiversity. • METHODS: We built a phylogenetic tree for 909 species of Saxifragales and used this hypothesis to examine character evolution for annual or perennial habit, woody or herbaceous habit, ovary position, petal number, carpel number, and stamen to petal ratio. We employed likelihood approaches to investigate the effect of habit and life history on speciation and extinction within this clade. • KEY RESULTS: Two major shifts occurred from a woody ancestor to the herbaceous habit, with multiple secondary changes from herbaceous to woody. Transitions among superior, subinferior, and inferior ovaries appear equiprobable. A major increase in petal number is correlated with a large increase in carpel number; these increases have co-occurred multiple times in Crassulaceae. Perennial or woody lineages have higher rates of speciation than annual or herbaceous ones, but higher probabilities of extinction offset these differences. Hence, net diversification rates are highest for annual, herbaceous lineages and lowest for woody perennials. The shift from annuality to perenniality in herbaceous taxa is frequent. Conversely, woody perennial lineages to woody annual transitions are infrequent; if they occur, the woody annual state is left immediately. • CONCLUSIONS: The large tree provides new insights into character evolution that are not obvious with smaller trees. Our results indicate that in some cases the evolution of angiosperms might be conditioned by constraints that have been so far overlooked.

Angiosperm phylogeny: 17 genes, 640 taxa.

PREMISE OF THE STUDY: Recent analyses employing up to five genes have provided numerous insights into angiosperm phylogeny, but many relationships have remained unresolved or poorly supported. In the hope of improving our understanding of angiosperm phylogeny, we expanded sampling of taxa and genes beyond previous analyses. METHODS: We conducted two primary analyses based on 640 species representing 330 families. The first included 25260 aligned base pairs (bp) from 17 genes (representing all three plant genomes, i.e., nucleus, plastid, and mitochondrion). The second included 19846 aligned bp from 13 genes (representing only the nucleus and plastid). KEY RESULTS: Many important questions of deep-level relationships in the nonmonocot angiosperms have now been resolved with strong support. Amborellaceae, Nymphaeales, and Austrobaileyales are successive sisters to the remaining angiosperms (Mesangiospermae), which are resolved into Chloranthales + Magnoliidae as sister to Monocotyledoneae + [Ceratophyllaceae + Eudicotyledoneae]. Eudicotyledoneae contains a basal grade subtending Gunneridae. Within Gunneridae, Gunnerales are sister to the remainder (Pentapetalae), which comprises (1) Superrosidae, consisting of Rosidae (including Vitaceae) and Saxifragales; and (2) Superasteridae, comprising Berberidopsidales, Santalales, Caryophyllales, Asteridae, and, based on this study, Dilleniaceae (although other recent analyses disagree with this placement). Within the major subclades of Pentapetalae, most deep-level relationships are resolved with strong support. CONCLUSIONS: Our analyses confirm that with large amounts of sequence data, most deep-level relationships within the angiosperms can be resolved. We anticipate that this well-resolved angiosperm tree will be of broad utility for many areas of biology, including physiology, ecology, paleobiology, and genomics.

Rosid radiation and the rapid rise of angiosperm-dominated forests.

The rosid clade (70,000 species) contains more than one-fourth of all angiosperm species and includes most lineages of extant temperate and tropical forest trees. Despite progress in elucidating relationships within the angiosperms, rosids remain the largest poorly resolved major clade; deep relationships within the rosids are particularly enigmatic. Based on parsimony and maximum likelihood (ML) analyses of separate and combined 12-gene (10 plastid genes, 2 nuclear; >18,000 bp) and plastid inverted repeat (IR; 24 genes and intervening spacers; >25,000 bp) datasets for >100 rosid species, we provide a greatly improved understanding of rosid phylogeny. Vitaceae are sister to all other rosids, which in turn form 2 large clades, each with a ML bootstrap value of 100%: (i) eurosids I (Fabidae) include the nitrogen-fixing clade, Celastrales, Huaceae, Zygophyllales, Malpighiales, and Oxalidales; and (ii) eurosids II (Malvidae) include Tapisciaceae, Brassicales, Malvales, Sapindales, Geraniales, Myrtales, Crossosomatales, and Picramniaceae. The rosid clade diversified rapidly into these major lineages, possibly over a period of <15 million years, and perhaps in as little as 4 to 5 million years. The timing of the inferred rapid radiation of rosids [108 to 91 million years ago (Mya) and 107-83 Mya for Fabidae and Malvidae, respectively] corresponds with the rapid rise of angiosperm-dominated forests and the concomitant diversification of other clades that inhabit these forests, including amphibians, ants, placental mammals, and ferns.

Floral variation and floral genetics in basal angiosperms.

Recent advances in phylogeny reconstruction and floral genetics set the stage for new investigations of the origin and diversification of the flower. We review the current state of angiosperm phylogeny, with an emphasis on basal lineages. With the surprising inclusion of Hydatellaceae with Nymphaeales, recent studies support the topology of Amborella sister to all other extant angiosperms, with Nymphaeales and then Austrobaileyales as subsequent sisters to all remaining angiosperms. Notable modifications from most recent analyses are the sister relationships of Chloranthaceae with the magnoliids and of Ceratophyllaceae with eudicots. We review "trends" in floral morphology and contrast historical, intuitive interpretations with explicit character-state reconstructions using molecular-based trees, focusing on (1) the size, number, and organization of floral organs; (2) the evolution of the perianth; (3) floral symmetry; and (4) floral synorganization. We provide summaries of those genes known to affect floral features that contribute to much of floral diversity. Although most floral genes have not been investigated outside of a few model systems, sufficient information is emerging to identify candidate genes for testing specific hypotheses in nonmodel plants. We conclude with a set of evo-devo case studies in which floral genetics have been linked to variation in floral morphology.

Floral gigantism in Rafflesiaceae.

Species of Rafflesiaceae possess the world's largest flowers (up to 1 meter in diameter), yet their precise evolutionary relationships have been elusive, hindering our understanding of the evolution of their extraordinary reproductive morphology. We present results of phylogenetic analyses of mitochondrial, nuclear, and plastid data showing that Rafflesiaceae are derived from within Euphorbiaceae, the spurge family. Most euphorbs produce minute flowers, suggesting that the enormous flowers of Rafflesiaceae evolved from ancestors with tiny flowers. Given the inferred phylogeny, we estimate that there was a circa 79-fold increase in flower diameter on the stem lineage of Rafflesiaceae, making this one of the most dramatic cases of size evolution reported for eukaryotes.