Does complete plastid genome sequencing improve species discrimination and phylogenetic resolution in Araucaria?

Obtaining accurate phylogenies and effective species discrimination using a small standardized set of plastid genes is challenging in evolutionarily young lineages. Complete plastid genome sequencing offers an increasingly easy-to-access source of characters that helps address this. The usefulness of this approach, however, depends on the extent to which plastid haplotypes track morphological species boundaries. We have tested the power of complete plastid genomes to discriminate among multiple accessions of 11 of 13 New Caledonian Araucaria species, an evolutionarily young lineage where the standard DNA barcoding approach has so far failed and phylogenetic relationships have remained elusive. Additionally, 11 nuclear gene regions were Sanger sequenced for all accessions to ascertain the success of species discrimination using a moderate number of nuclear genes. Overall, fewer than half of the New Caledonian Araucaria species with multiple accessions were monophyletic in the plastid or nuclear trees. However, the plastid data retrieved a phylogeny with a higher resolution compared to any previously published tree of this clade and supported the monophyly of about twice as many species and nodes compared to the nuclear data set. Modest gains in discrimination thus are possible, but using complete plastid genomes or a small number of nuclear genes in DNA barcoding may not substantially raise species discriminatory power in many evolutionarily young lineages. The big challenge therefore remains to develop techniques that allow routine access to large numbers of nuclear markers scaleable to thousands of individuals from phylogenetically disparate sample sets.

Genotyping-by-sequencing for Populus population genomics: an assessment of genome sampling patterns and filtering approaches.

Continuing advances in nucleotide sequencing technology are inspiring a suite of genomic approaches in studies of natural populations. Researchers are faced with data management and analytical scales that are increasing by orders of magnitude. With such dramatic advances comes a need to understand biases and error rates, which can be propagated and magnified in large-scale data acquisition and processing. Here we assess genomic sampling biases and the effects of various population-level data filtering strategies in a genotyping-by-sequencing (GBS) protocol. We focus on data from two species of Populus, because this genus has a relatively small genome and is emerging as a target for population genomic studies. We estimate the proportions and patterns of genomic sampling by examining the Populus trichocarpa genome (Nisqually-1), and demonstrate a pronounced bias towards coding regions when using the methylation-sensitive ApeKI restriction enzyme in this species. Using population-level data from a closely related species (P. tremuloides), we also investigate various approaches for filtering GBS data to retain high-depth, informative SNPs that can be used for population genetic analyses. We find a data filter that includes the designation of ambiguous alleles resulted in metrics of population structure and Hardy-Weinberg equilibrium that were most consistent with previous studies of the same populations based on other genetic markers. Analyses of the filtered data (27,910 SNPs) also resulted in patterns of heterozygosity and population structure similar to a previous study using microsatellites. Our application demonstrates that technically and analytically simple approaches can readily be developed for population genomics of natural populations.

Biphasic capnogram in a single-lung transplant recipient: a case report.

Capnography is a graphic representation of the carbon dioxide concentration in the airways plotted against time. We report an unusual biphasic-shaped capnogram in a single-lung transplant recipient. In keeping with the only 2 prior reports of which we are aware, we ascribe the unusual biphasic pattern to differential emptying of the native and transplanted lungs. This experience reminds respiratory clinicians of the value of capnometry as a monitoring tool and hopefully will heighten awareness regarding the diagnostic value of usual and unusual capnographic patterns.

Widespread triploidy in Western North American aspen (Populus tremuloides).

We document high rates of triploidy in aspen (Populus tremuloides) across the western USA (up to 69% of genets), and ask whether the incidence of triploidy across the species range corresponds with latitude, glacial history (as has been documented in other species), climate, or regional variance in clone size. Using a combination of microsatellite genotyping, flow cytometry, and cytology, we demonstrate that triploidy is highest in unglaciated, drought-prone regions of North America, where the largest clone sizes have been reported for this species. While we cannot completely rule out a low incidence of undetected aneuploidy, tetraploidy or duplicated loci, our evidence suggests that these phenomena are unlikely to be significant contributors to our observed patterns. We suggest that the distribution of triploid aspen is due to a positive synergy between triploidy and ecological factors driving clonality. Although triploids are expected to have low fertility, they are hypothesized to be an evolutionary link to sexual tetraploidy. Thus, interactions between clonality and polyploidy may be a broadly important component of geographic speciation patterns in perennial plants. Further, cytotypes are expected to show physiological and structural differences which may influence susceptibility to ecological factors such as drought, and we suggest that cytotype may be a significant and previously overlooked factor in recent patterns of high aspen mortality in the southwestern portion of the species range. Finally, triploidy should be carefully considered as a source of variance in genomic and ecological studies of aspen, particularly in western U.S. landscapes.

Hemisphere-scale differences in conifer evolutionary dynamics.

Fundamental differences in the distribution of oceans and landmasses in the Northern and Southern Hemispheres potentially impact patterns of biological diversity in the two areas. The evolutionary history of conifers provides an opportunity to explore these dynamics, because the majority of extant conifer species belong to lineages that have been broadly confined to the Northern or Southern Hemisphere during the Cenozoic. Incorporating genetic information with a critical review of fossil evidence, we developed an age-calibrated phylogeny sampling ∼80% of living conifer species. Most extant conifer species diverged recently during the Neogene within clades that generally were established during the later Mesozoic, but lineages that diversified mainly in the Southern Hemisphere show a significantly older distribution of divergence ages than their counterparts in the Northern Hemisphere. Our tree topology and divergence times also are best fit by diversification models in which Northern Hemisphere conifer lineages have higher rates of species turnover than Southern Hemisphere lineages. The abundance of recent divergences in northern clades may reflect complex patterns of migration and range shifts during climatic cycles over the later Neogene leading to elevated rates of speciation and extinction, whereas the scattered persistence of mild, wetter habitats in the Southern Hemisphere may have favored the survival of older lineages.

Utility of a large, multigene plastid data set in inferring higher-order relationships in ferns and relatives (monilophytes).

PREMISE OF THE STUDY: The monilophytes (ferns and relatives)-the third largest group of land plants-exhibit a diverse array of vegetative and reproductive morphologies. Investigations into their early ecological and life-history diversification require accurate, well-corroborated phylogenetic estimates. We examined the utility of a large plastid-based data set in inferring backbone relationships for monilophytes. • METHODS: We recovered 17 plastid genes for exemplar taxa using published and new primers. We compared results from maximum-likelihood and parsimony analyses, assessed the effects of removing rapidly evolving characters, and examined the extent to which our data corroborate or contradict the results of other studies, or resolve current ambiguities. • KEY RESULTS: Considering multifamily clades, we found bootstrap support comparable to or better than that in published studies that used fewer genes from fewer or more taxa. We firmly establish filmy ferns (Hymenophyllales) as the sister group of all leptosporangiates except Osmundaceae, resolving the second deepest split in leptosporangiate-fern phylogeny. A clade comprising Ophioglossaceae and Psilotaceae is currently accepted as the sister group of other monilophytes, but we recover Equisetum in this position. We also recover marattioid and leptosporangiate ferns as sister groups. Maximum-likelihood rate-class estimates are somewhat skewed when a long-branch lineage (Selaginella) is included, negatively affecting bootstrap support for early branches. • CONCLUSIONS: Our findings support the utility of this gene set in corroborating relationships found in previous studies, improving support, and resolving uncertainties in monilophyte phylogeny. Despite these advances, our results also underline the need for continued work on resolving the very earliest splits in monilophyte phylogeny.

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 approximately 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.

Repeated evolution of net venation and fleshy fruits among monocots in shaded habitats confirms a priori predictions: evidence from an ndhF phylogeny.

We present a well-resolved, highly inclusive phylogeny for monocots, based on ndhF sequence variation, and use it to test a priori hypotheses that net venation and vertebrate-dispersed fleshy fruits should undergo concerted convergence, representing independent but often concurrent adaptations to shaded conditions. Our data demonstrate that net venation arose at least 26 times and was lost eight times over the past 90 million years; fleshy fruits arose at least 21 times and disappeared 11 times. Both traits show a highly significant pattern of concerted convergence (p<10(-9)), arising 16 times and disappearing four times in tandem. This phenomenon appears driven by even stronger tendencies for both traits to evolve in shade and be lost in open habitats (p<10(-13)-10(-29)). These patterns are among the strongest ever demonstrated for evolutionary convergence in individual traits and the predictability of evolution, and the strongest evidence yet uncovered for concerted convergence. The rate of adaptive shifts per taxon has declined exponentially over the past 90 million years, as expected when large-scale radiations fill adaptive zones.

Inference of higher-order relationships in the cycads from a large chloroplast data set.

We investigated higher-order relationships in the cycads, an ancient group of seed-bearing plants, by examining a large portion of the chloroplast genome from seven species chosen to exemplify our current understanding of taxonomic diversity in the order. The regions considered span approximately 13.5 kb of unaligned data per taxon, and comprise a diverse range of coding sequences, introns and intergenic spacers dispersed throughout the plastid genome. Our results provide substantial support for most of the inferred backbone of cycad phylogeny, and weak evidence that the sister-group of the cycads among living seed plants is Ginkgo biloba. Cycas (representing Cycadaceae) is the sister-group of the remaining cycads; Dioon is part of the next most basal split. Two of the three commonly recognized families of cycads (Zamiaceae and Stangeriaceae) are not monophyletic; Stangeria is embedded within Zamiaceae, close to Zamia and Ceratozamia, and not closely allied to the other genus of Stangeriaceae, Bowenia. In contrast to the other seed plants, cycad chloroplast genomes share two features with Ginkgo: a reduced rate of evolution and an elevated transition:transversion ratio. We demonstrate that the latter aspect of their molecular evolution is unlikely to have affected inference of cycad relationships in the context of seed-plant wide analyses.