Dennis W. Stevenson

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.

A comparison of algorithms for the identification of specimens using DNA barcodes: examples from gymnosperms

In order to use DNA sequences for specimen identification (e.g., barcoding, fingerprinting) an algorithm to compare query sequences with a reference database is needed. Precision and accuracy of query sequence identification was estimated for hierarchical clustering (parsimony and neighbor joining), similarity methods (BLAST, BLAT and megaBLAST), combined clustering/similarity methods (BLAST/parsimony and BLAST/neighbor joining), diagnostic methods (DNA–BAR and DOME ID), and a new method (ATIM). We offer two novel alignment‐free algorithmic solutions (DOME ID and ATIM) to identify query sequences for the purposes of DNA barcoding. Publicly available gymnosperm nrITS 2 and plastid matK sequences were used as test data sets. On the test data sets, almost all of the methods were able to accurately identify sequences to genus; however, no method was able to accurately identify query sequences to species at a frequency that would be considered useful for routine specimen identification (42–71% unambiguously correct). Clustering methods performed the worst (perhaps due to alignment issues). Similarity methods, ATIM, DNA–BAR, and DOME ID all performed at approximately the same level. Given the relative precision of the algorithms (median = 67% unambiguous), the low accuracy of species‐level identification observed could be ascribed to the lack of correspondence between patterns of allelic similarity and species delimitations. Application of DNA barcoding to sequences of CITES listed cycads (Cycadopsida) provides an example of the potential application of DNA barcoding to enforcement of conservation laws.

A reevaluation of seed plant phylogeny

Seed plant phylogeny is evaluated using a data set of 46 terminals (taxa) and 103 morphological and anatomical characters. Cladistic analyses using the criterion of parsimony were performed on the complete data set as well as on subsets of the data, e.g., excluding fossils and/or combining various complex taxa into single terminals. The results support the placement of the cycads as the sister group of a monophyletic group that includes several fossil «seed ferns» as well as extant Ginkgo, conifers, gnetopsids, and angiosperms. When fossils were included, Bennettitales (cycadeoids) were part of an «anthophyte» clade that included gnetopsids and angiosperms. Pentoxylon was a sister taxon to the core anthophyte clade, in some, but not all, of the most parsimonious trees

DNA barcoding in the cycadales: testing the potential of proposed barcoding markers for species identification of cycads.

Barcodes are short segments of DNA that can be used to uniquely identify an unknown specimen to species, particularly when diagnostic morphological features are absent. These sequences could offer a new forensic tool in plant and animal conservation—especially for endangered species such as members of the Cycadales. Ideally, barcodes could be used to positively identify illegally obtained material even in cases where diagnostic features have been purposefully removed or to release confiscated organisms into the proper breeding population. In order to be useful, a DNA barcode sequence must not only easily PCR amplify with universal or near-universal reaction conditions and primers, but also contain enough variation to generate unique identifiers at either the species or population levels. Chloroplast regions suggested by the Plant Working Group of the Consortium for the Barcode of Life (CBoL), and two alternatives, the chloroplast psbA-trnH intergenic spacer and the nuclear ribosomal internal transcribed spacer (nrITS), were tested for their utility in generating unique identifiers for members of the Cycadales. Ease of amplification and sequence generation with universal primers and reaction conditions was determined for each of the seven proposed markers. While none of the proposed markers provided unique identifiers for all species tested, nrITS showed the most promise in terms of variability, although sequencing difficulties remain a drawback. We suggest a workflow for DNA barcoding, including database generation and management, which will ultimately be necessary if we are to succeed in establishing a universal DNA barcode for plants.

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.

A genome triplication associated with early diversification of the core eudicots.

BackgroundAlthough it is agreed that a major polyploidy event, gamma, occurred within the eudicots, the phylogenetic placement of the event remains unclear.ResultsTo determine when this polyploidization occurred relative to speciation events in angiosperm history, we employed a phylogenomic approach to investigate the timing of gene set duplications located on syntenic gamma blocks. We populated 769 putative gene families with large sets of homologs obtained from public transcriptomes of basal angiosperms, magnoliids, asterids, and more than 91.8 gigabases of new next-generation transcriptome sequences of non-grass monocots and basal eudicots. The overwhelming majority (95%) of well-resolved gamma duplications was placed before the separation of rosids and asterids and after the split of monocots and eudicots, providing strong evidence that the gamma polyploidy event occurred early in eudicot evolution. Further, the majority of gene duplications was placed after the divergence of the Ranunculales and core eudicots, indicating that the gamma appears to be restricted to core eudicots. Molecular dating estimates indicate that the duplication events were intensely concentrated around 117 million years ago.ConclusionsThe rapid radiation of core eudicot lineages that gave rise to nearly 75% of angiosperm species appears to have occurred coincidentally or shortly following the gamma triplication event. Reconciliation of gene trees with a species phylogeny can elucidate the timing of major events in genome evolution, even when genome sequences are only available for a subset of species represented in the gene trees. Comprehensive transcriptome datasets are valuable complements to genome sequences for high-resolution phylogenomic analysis.

A Phylogeny of the Monocots, as Inferred from rbcL and atpA Sequence Variation, and a Comparison of Methods for Calculating Jackknife and Bootstrap Values

Abstract A phylogenetic analysis of the monocots was conducted on the basis of nucleotide sequence variation in two genes (atpA, encoded in the mitochondrial genome, and rbcL, encoded in the plastid genome). The taxon sample of 218 angiosperm terminals included 177 monocots and 41 dicots. Among the major results of the analysis are the resolution of a clade comprising four magnoliid lineages (Canellales, Piperales, Magnoliales, and Laurales) as sister of the monocots, with the deepest branch within the monocots between a clade consisting of Araceae, Tofieldiaceae, Acorus, and Alismatales, and a clade that includes all other monocots. Nartheciaceae are placed as the sister of Pandanales, and Corsiaceae as the sister of Liliales. The Triuridaceae, represented by three genera, including Lacandonia, are resolved as monophyletic and placed in a range of positions, generally within Pandanales. Dasypogonaceae and Arecaceae diverge sequentially from a clade that includes all other commelinid taxa, and within the latter group Poales s. lat. are sister of a clade in which Zingiberales and Commelinales are sisters. Within Poales s. lat., Trithuria (Hydatellaceae) and Mayaca appear to be closely related to some or all elements of Xyridaceae. A comparison was conducted of jackknife and bootstrap values, as computed using strict-consensus (SC) and frequency-within-replicates (FWR) approaches. Jackknife values tend to be higher than bootstrap values, and for each of these methods support values obtained with the FWR approach tend to exceed those obtained with the SC approach.

Shortcuts in systematics? A commentary on DNA-based taxonomy

The primary aims of taxonomy are to name, circumscribe, describe and classify species. The first goal is convention but the remainder are science. The International Codes of Nomenclature are legislative instruments and nomenclature is simply a mechanism to ensure that a species name is legitimately attached to a type specimen, regardless of scientific status. The type of a species does not serve, as Tautz et al. ([1xDNA points the way ahead in taxonomy. Tautz, D. et al. Nature. 2002; 418: 479CrossRef | PubMedSee all References, 2xA plea for DNA taxonomy. Tautz, D. et al. Trends Ecol. Evol. 2003; 18Abstract | Full Text | Full Text PDF | Scopus (441)See all References], but see [3xThe status of taxonomic literature. Minelli, A. Trends Ecol. Evol. 2003; 18Abstract | Full Text | Full Text PDF | Scopus (22)See all References][3]) assert, as ‘the central reference for comparisons’. The crucial link between names and scientific investigation is species circumscription followed by description. The Codes require Linnaean binomials: a genus name and a species epithet. The rules are totally silent about what constitutes a species; rather this is a key goal of biological investigation. Circumscriptions of genera and species evolve as science progresses.The Linnaean binomial system is not ‘inherently unstable’ but is used to interpret the underlying science. The problem that ‘a name that has been used for a long time thus can suddenly disappear’ (or reappear, for that matter) is a nuisance for everyone. However, if name changes are considered a serious problem, then the conservation and rejection criteria available in the Codes can be invoked.Neither the Botanical nor the Zoological Code has a fixed authoritarian supervisory body; rather, they have committees chosen by a democratic process that oversee changes. The Codes work by consensus and are designed to be open and universally applicable [4xTaxonomy needs evolution, not revolution. Knapp, S. et al. Nature. 2002; 419: 559CrossRef | PubMedSee all References][4]. Tautz et al. ([2xA plea for DNA taxonomy. Tautz, D. et al. Trends Ecol. Evol. 2003; 18Abstract | Full Text | Full Text PDF | Scopus (441)See all References][2], and see [3xThe status of taxonomic literature. Minelli, A. Trends Ecol. Evol. 2003; 18Abstract | Full Text | Full Text PDF | Scopus (22)See all References][3]) advocate universal, centralized, apparently obligatory registration, a concept emphatically rejected by the International Botanical Congress in 1999. Taxonomists from developing nations led the move against centralization, fearful that the wealthier nations were attempting to monopolize information. In our view, registration would eventually strangle systematics, as debate will be discouraged.Most current taxonomy is pursued using low-cost technology. Mandatory introduction of DNA sequences into taxonomy seems to us a retrograde step. In most instances, a quick survey of morphology will serve the same purpose and, although morphology has its problems, DNA has as many pitfalls. A sufficiently different sequence might warrant the description of a new species, as will a sufficiently different morphology. An expensive and centralized DNA-based taxonomy would only add to the North–South divide in taxonomy, and might exclude the many taxonomists who have limited access to sequencing technology.Acknowledging that there need not (or cannot) be universal agreement about which region of the genome to sequence, Tautz et al. suggest using house-keeping genes, especially the ribosomal genes (at least in animals). Although abundant, ribosomal sequences might be an inappropriate choice in the long run, because they have profound alignment problems and are subject to different degrees of concerted evolution.Matching existing Linnaean names with DNA sequences is fraught with hazards. Although it is tempting to create a new starting date for priority for the Botanical and Zoological Codes [5xChallenges for taxonomy. Godfray, H.C.J. Nature. 2002; 417: 17–19CrossRef | PubMed | Scopus (246)See all References][5], it seems futile to replace existing types (from which DNA in some cases cannot be extracted) with neotypes. As indicated by the authors [1xDNA points the way ahead in taxonomy. Tautz, D. et al. Nature. 2002; 418: 479CrossRef | PubMedSee all References][1], expert taxonomists are in short supply and many important groups are neglected. It takes little imagination to envisage the problems broad-scale designation of neotypes would cause.Tautz et al. argue that existing DNA data bases represent bad taxonomy, as ‘there is no guarantee that the correct species names were assigned by the submitter of the sequence, because there are no established taxonomic standards under which such submissions have to be done’. The circumscription of a species is an opinion [6xSee all References][6]. One might ask therefore who is going to decide on ‘taxonomic standards’. Circumscription changes with increasing knowledge; thats the science. Many sequences are deposited in DNA data bases, but, if a specimen has been misidentified, only inspection of the voucher can solve this problem. It is naïve to think that ‘phylogenetic analysis of query sequences, will readily place any sequences from new species’, as if the problems of homoplasy, alignment, and even phylogenetic methods would not add to the unreliability of information in existing data bases.The role of collections in systematics is vital. Collections represent a comparative model of diversity, and therefore, as the authors [1xDNA points the way ahead in taxonomy. Tautz, D. et al. Nature. 2002; 418: 479CrossRef | PubMedSee all References][1] point out, specimens should be retained as intact as possible. Destructive sampling of type specimens for any characters has always been a severe problem and such procedures are accepted only as a last resort. To destroy a sample to extract its DNA might be inadvisable in the light of future needs.With regard to taxonomic and phylogenetic studies, current practise attempts to create higher level classifications that do not conflict directly with cladograms [7xSee all References][7]. Today, phylogenetic methods applied to DNA data are a strong focus of systematics research, and it is difficult to believe that the pendulum might swing in another direction. However, methods do change and the fixed opinions of today might seem redundant in the future.So what is the upshot? Indeed, what has the molecular revolution really achieved for taxonomy? The main advance is that it has provided access to copious data for cladistic analysis and, in our view, has provided new data sets and a new class of characters that can be extremely useful in broad-scale comparisons of everything from bacteria to mammals and plants. Often one is forced to use one, or a few, carefully selected specimens as representatives of a taxon, but experience has repeatedly shown that this can be a major mistake. Deliberately using a single specimen as a representative of the taxon will only create havoc in taxonomy, a fact long realized by taxonomists working with other types of data. Individual bases and DNA sequences are simply characters, tiny fragments of the lifecycle. It seems perverse to us to advocate using a DNA sequence as a mandatory identification tag for a species, even as a first approximation. Therefore, this plea for a DNA-based taxonomy is little more than a cri de coeur for bioinformatics and increased reliance on sequence data. We agree that there is a very strong need for efficient, although perhaps not centralized, repositories for DNA samples. How and where such samples should be curated, and how the curators should be funded, is an open question. We also agree that it would be a good idea to include DNA sequences in the diagnoses of taxa. However, we feel that a DNA-based taxonomy along the suggested lines would catastrophically retard taxonomic activity, and it would certainly not relieve the need for many more taxonomists, especially in the tropics, for the good of taxonomys many dependent user groups.

Data decisiveness, data quality, and incongruence in phylogenetic analysis: an example from the monocotyledons using mitochondrial atp A sequences.

We examined three parallel data sets with respect to qualities relevant to phylogenetic analysis of 20 exemplar monocotyledons and related dicotyledons. The three data sets represent restriction-site variation in the inverted repeat region of the chloroplast genome, and nucleotide sequence variation in the chloroplast-encoded gene rbcL and in the mitochondrion-encoded gene atpA, the latter of which encodes the alpha-subunit of mitochondrial ATP synthase. The plant mitochondrial genome has been little used in plant systematics, in part because nucleotide sequence evolution in enzyme-encoding genes of this genome is relatively slow. The three data sets were examined in separate and combined analyses, with a focus on patterns of congruence, homoplasy, and data decisiveness. Data decisiveness (described by P. Goloboff) is a measure of robustness of support for most parsimonious trees by a data set in terms of the degree to which those trees are shorter than the average length of all possible trees. Because indecisive data sets require relatively fewer additional steps than decisive ones to be optimized on nonparsimonious trees, they will have a lesser tendency to be incongruent with other data sets. One consequence of this relationship between decisiveness and character incongruence is that if incongruence is used as a criterion of noncombinability, decisive data sets, which provide robust support for relationships, are more likely to be assessed as noncombinable with other data sets than are indecisive data sets, which provide weak support for relationships. For the sampling of taxa in this study, the atpA data set has about half as many cladistically informative nucleotides as the rbcL data set per site examined, and is less homoplastic and more decisive. The rbcL data set, which is the least decisive of the three, exhibits the lowest levels of character incongruence. Whatever the molecular evolutionary cause of this phenomenon, it seems likely that the poorer performance of rbcL than atpA, in terms of data decisiveness, is due to both its higher overall level of homoplasy and the fact that it is performing especially poorly at nonsynonymous sites.

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.