Sean W. Graham

A DNA barcode for land plants

DNA barcoding involves sequencing a standard region of DNA as a tool for species identification. However, there has been no agreement on which region(s) should be used for barcoding land plants. To provide a community recommendation on a standard plant barcode, we have compared the performance of 7 leading candidate plastid DNA regions (atpF–atpH spacer, matK gene, rbcL gene, rpoB gene, rpoC1 gene, psbK–psbI spacer, and trnH–psbA spacer). Based on assessments of recoverability, sequence quality, and levels of species discrimination, we recommend the 2-locus combination of rbcL+matK as the plant barcode. This core 2-locus barcode will provide a universal framework for the routine use of DNA sequence data to identify specimens and contribute toward the discovery of overlooked species of land plants.

Hydatellaceae identified as a new branch near the base of the angiosperm phylogenetic tree

Although the relationship of angiosperms to other seed plants remains controversial, great progress has been made in identifying the earliest extant splits in flowering-plant phylogeny, with the discovery that the New Caledonian shrub Amborella trichopoda, the water lilies (Nymphaeales), and the woody Austrobaileyales constitute a basal grade of lines that diverged before the main radiation in the clade. By focusing attention on these ancient lines, this finding has re-written our understanding of angiosperm structural and reproductive biology, physiology, ecology and taxonomy. The discovery of a new basal lineage would lead to further re-evaluation of the initial angiosperm radiation, but would also be unexpected, as nearly all of the ∼460 flowering-plant families have been surveyed in molecular studies. Here we show that Hydatellaceae, a small family of dwarf aquatics that were formerly interpreted as monocots, are instead a highly modified and previously unrecognized ancient lineage of angiosperms. Molecular phylogenetic analyses of multiple plastid genes and associated noncoding regions from the two genera of Hydatellaceae identify this overlooked family as the sister group of Nymphaeales. This surprising result is further corroborated by evidence from the nuclear gene phytochrome C (PHYC), and by numerous morphological characters. This indicates that water lilies are part of a larger lineage that evolved more extreme and diverse modifications for life in an aquatic habitat than previously recognized.

Choosing and using a plant DNA barcode.

The main aim of DNA barcoding is to establish a shared community resource of DNA sequences that can be used for organismal identification and taxonomic clarification. This approach was successfully pioneered in animals using a portion of the cytochrome oxidase 1 (CO1) mitochondrial gene. In plants, establishing a standardized DNA barcoding system has been more challenging. In this paper, we review the process of selecting and refining a plant barcode; evaluate the factors which influence the discriminatory power of the approach; describe some early applications of plant barcoding and summarise major emerging projects; and outline tool development that will be necessary for plant DNA barcoding to advance.

Phylotranscriptomic analysis of the origin and early diversification of land plants

Significance Early branching events in the diversification of land plants and closely related algal lineages remain fundamental and unresolved questions in plant evolutionary biology. Accurate reconstructions of these relationships are critical for testing hypotheses of character evolution: for example, the origins of the embryo, vascular tissue, seeds, and flowers. We investigated relationships among streptophyte algae and land plants using the largest set of nuclear genes that has been applied to this problem to date. Hypothesized relationships were rigorously tested through a series of analyses to assess systematic errors in phylogenetic inference caused by sampling artifacts and model misspecification. Results support some generally accepted phylogenetic hypotheses, while rejecting others. This work provides a new framework for studies of land plant evolution. Reconstructing the origin and evolution of land plants and their algal relatives is a fundamental problem in plant phylogenetics, and is essential for understanding how critical adaptations arose, including the embryo, vascular tissue, seeds, and flowers. Despite advances in molecular systematics, some hypotheses of relationships remain weakly resolved. Inferring deep phylogenies with bouts of rapid diversification can be problematic; however, genome-scale data should significantly increase the number of informative characters for analyses. Recent phylogenomic reconstructions focused on the major divergences of plants have resulted in promising but inconsistent results. One limitation is sparse taxon sampling, likely resulting from the difficulty and cost of data generation. To address this limitation, transcriptome data for 92 streptophyte taxa were generated and analyzed along with 11 published plant genome sequences. Phylogenetic reconstructions were conducted using up to 852 nuclear genes and 1,701,170 aligned sites. Sixty-nine analyses were performed to test the robustness of phylogenetic inferences to permutations of the data matrix or to phylogenetic method, including supermatrix, supertree, and coalescent-based approaches, maximum-likelihood and Bayesian methods, partitioned and unpartitioned analyses, and amino acid versus DNA alignments. Among other results, we find robust support for a sister-group relationship between land plants and one group of streptophyte green algae, the Zygnematophyceae. Strong and robust support for a clade comprising liverworts and mosses is inconsistent with a widely accepted view of early land plant evolution, and suggests that phylogenetic hypotheses used to understand the evolution of fundamental plant traits should be reevaluated.

Multiple Multilocus DNA Barcodes from the Plastid Genome Discriminate Plant Species Equally Well

A universal barcode system for land plants would be a valuable resource, with potential utility in fields as diverse as ecology, floristics, law enforcement and industry. However, the application of plant barcoding has been constrained by a lack of consensus regarding the most variable and technically practical DNA region(s). We compared eight candidate plant barcoding regions from the plastome and one from the mitochondrial genome for how well they discriminated the monophyly of 92 species in 32 diverse genera of land plants (N = 251 samples). The plastid markers comprise portions of five coding (rpoB, rpoC1, rbcL, matK and 23S rDNA) and three non-coding (trnH-psbA, atpF–atpH, and psbK–psbI) loci. Our survey included several taxonomically complex groups, and in all cases we examined multiple populations and species. The regions differed in their ability to discriminate species, and in ease of retrieval, in terms of amplification and sequencing success. Single locus resolution ranged from 7% (23S rDNA) to 59% (trnH-psbA) of species with well-supported monophyly. Sequence recovery rates were related primarily to amplification success (85–100% for plastid loci), with matK requiring the greatest effort to achieve reasonable recovery (88% using 10 primer pairs). Several loci (matK, psbK–psbI, trnH-psbA) were problematic for generating fully bidirectional sequences. Setting aside technical issues related to amplification and sequencing, combining the more variable plastid markers provided clear benefits for resolving species, although with diminishing returns, as all combinations assessed using four to seven regions had only marginally different success rates (69–71%; values that were approached by several two- and three-region combinations). This performance plateau may indicate fundamental upper limits on the precision of species discrimination that is possible with DNA barcoding systems that include moderate numbers of plastid markers. Resolution to the contentious debate on plant barcoding should therefore involve increased attention to practical issues related to the ease of sequence recovery, global alignability, and marker redundancy in multilocus plant DNA barcoding systems.

MICROSTRUCTURAL CHANGES IN NONCODING CHLOROPLAST DNA: INTERPRETATION, EVOLUTION, AND UTILITY OF INDELS AND INVERSIONS IN BASAL ANGIOSPERM PHYLOGENETIC INFERENCE

Microstructural changes in several very slowly evolving chloroplast introns and intergenic spacers were characterized across a broad range of angiosperms, including most of the major basal lineages. Insertion/deletion events (indels) in the surveyed noncoding regions of the large inverted repeat (IR) region were shown to be rarer than nucleotide substitutions and thus constitute one of the slowest and least homoplastic types of data available to plant systematists. In our study we scored 180 indels in noncoding regions, of which 36 were parsimony informative within the angiosperms. Because they are relatively few in number, their general utility is currently limited. However, they provide support for specific major taxa, including the angiosperms as a whole, the water lilies, and Illiciaceae and relatives. Support for the basalmost angiosperm split is largely inconclusive, but a single indel supported a basal placement of the water lilies, not Amborella. We estimate that roughly double or triple the current amount (ca. 2.2 kb) of noncoding IR DNA would be required to obtain indel support for most of the deepest branches at the base of the angiosperms. A variety of molecular processes appear to be responsible for the observed indels. Indels are more frequently associated with tandem repeat sequences than not. Insertions are significantly more frequently associated with tandem repeats than are deletions. The latter finding may be, in part, a function of an ascertainment bias for insertions versus deletions. Single‐base indels were the most common size class, but there was an unexplained deficit of some other small indel size classes. Coding indels can be problematical, particularly when they overlap among taxa in an alignment. We favor one simple scheme for coding overlapping indels but argue that no existing scheme for coding overlapping indels for phylogenetic analysis, or dealing with them in alignment, is ideal. Several small inversions were observed. These included the most homoplastic microstructural character in the current study. Each inversion was associated with short flanking inverted repeats.

Data access for the 1,000 Plants (1KP) project

The 1,000 plants (1KP) project is an international multi-disciplinary consortium that has generated transcriptome data from over 1,000 plant species, with exemplars for all of the major lineages across the Viridiplantae (green plants) clade. Here, we describe how to access the data used in a phylogenomics analysis of the first 85 species, and how to visualize our gene and species trees. Users can develop computational pipelines to analyse these data, in conjunction with data of their own that they can upload. Computationally estimated protein-protein interactions and biochemical pathways can be visualized at another site. Finally, we comment on our future plans and how they fit within this scalable system for the dissemination, visualization, and analysis of large multi-species data sets.

Are plant species inherently harder to discriminate than animal species using DNA barcoding markers

The ability to discriminate between species using barcoding loci has proved more difficult in plants than animals, raising the possibility that plant species boundaries are less well defined. Here, we review a selection of published barcoding data sets to compare species discrimination in plants vs. animals. Although the use of different genetic markers, analytical methods and depths of taxon sampling may complicate comparisons, our results using common metrics demonstrate that the number of species supported as monophyletic using barcoding markers is higher in animals (> 90%) than plants (~70%), even after controlling for the amount of parsimony‐informative information per species. This suggests that more than a simple lack of variability limits species discrimination in plants. Both animal and plant species pairs have variable size gaps between intra‐ and interspecific genetic distances, but animal species tend to have larger gaps than plants, even in relatively densely sampled genera. An analysis of 12 plant genera suggests that hybridization contributes significantly to variation in genetic discontinuity in plants. Barcoding success may be improved in some plant groups by careful choice of markers and appropriate sampling; however, overall fine‐scale species discrimination in plants relative to animals may be inherently more difficult because of greater levels of gene‐tree paraphyly.

Assembling the Tree of the Monocotyledons: Plastome Sequence Phylogeny and Evolution of Poales1

Abstract The order Poales comprises a substantial portion of plant life (7% of all angiosperms and 33% of monocots) and includes taxa of enormous economic and ecological significance. Molecular and morphological studies over the past two decades, however, leave uncertain many relationships within Poales and among allied commelinid orders. Here we present the results of an initial project by the Monocot AToL (Angiosperm Tree of Life) team on phylogeny and evolution in Poales, using sequence data for 81 plastid genes (exceeding 101 aligned kb) from 83 species of angiosperms. We recovered highly concordant relationships using maximum likelihood (ML) and maximum parsimony (MP), with 98.2% mean ML bootstrap support across monocots. For the first time, ML resolves ties among Poales and other commelinid orders with moderate to strong support. Analyses provide strong support for Bromeliaceae being sister to the rest of Poales; Typhaceae, Rapateaceae, and cyperids (sedges, rushes, and their allies) emerge next along the phylogenetic spine. Graminids (grasses and their allies) and restiids (Restionaceae and its allies) are well supported as sister taxa. MP identifies a xyrid clade (Eriocaulaceae, Mayacaceae, Xyridaceae) sister to cyperids, but ML (with much stronger support) places them as a grade with respect to restiids + graminids. The conflict in resolution between these analyses likely reflects long-branch attraction and highly elevated substitution rates in some Poales. All other familial relationships within the order are strongly supported by both MP and ML analyses. Character-state mapping implies that ancestral Poales lived in sunny, fire-prone, at least seasonally damp/wet, and possibly nutrient-poor sites, and were animal pollinated. Five subsequent shifts to wind pollination—in Typhaceae, cyperids, restiids, Ecdeiocoleaceae, and the vast PACCMAD-BEP clade of grasses—are significantly correlated with shifts to open habitats and small, inconspicuous, unisexual, and nectar-free flowers. Prime ecological movers driving the repeated evolution of wind pollination in Poales appear to include open habitats combined with the high local dominance of conspecific taxa, with the latter resulting from large-scale disturbances, combined with tall plant stature, vigorous vegetative spread, and positive ecological feedback. Reproductive assurance in the absence of reliable animal visitation probably favored wind pollination in annuals and short-statured perennials of Centrolepidaceae in ephemerally wet depressions and windswept alpine sites.

Reconstruction of the evolution of reproductive characters in Pontederiaceae using phylogenetic evidence from chloroplast DNA restriction-site variation

We reconstructed the phylogenetic history of Pontederiaceae using chloroplast DNA restriction‐site variation from approximately two‐thirds of the species in this family of aquatic monocotyledons. The molecular phylogeny was used to evaluate hypotheses concerning the evolution of reproductive characters associated with the breeding system. The family has four main genera, two of which (Eichhornia and Pontederia) have tristylous, predominantly outcrossing species, while two (Monochoria and Heteranthera) have enantiostylous taxa. Self‐incompatibility is restricted to some but not all tristylous species. In Eichhornia and Pontederia, predominantly selfing species with small monomorphic flowers (homostyly) have been hypothesized to result from the multiple breakdown of tristyly. Restriction‐site variation provided a well supported phylogeny of ingroup taxa, enabling the mapping of reproductive characters onto trees. Two contrasting optimization schemes were assessed, differing in the relative weights assigned to shifts in character states. The reconstructed sequence of floral character‐state change was used to assess competing hypotheses concerning the origin and breakdown of tristyly, and the relationships between tristylous and enantiostylous syndromes. Our results indicate that the class of optimization scheme used was the most critical factor in reconstructing character evolution. Despite some topological uncertainties and difficulty in reconstructing the primitive floral form in the family, several broad conclusions were possible when an unordered, unequally‐weighted optimization scheme was used: (1) tristyly originated either once or twice, while the occurrence of enantiostyly in Monochoria and Heteranthera was always found to have independent origins; (2) tristyly has repeatedly broken down leading to selfing, homostylous taxa; and (3) self‐incompatibility probably arose after the origin of floral trimorphism, a sequence of events that conflicts with some evolutionary models.