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Plant genomes vary over several orders of magnitude in size, even among closely related species, yet the origin, genesis and significance of this variation are not clear. Because DNA content varies over a sevenfold range among diploid species in the cotton genus (Gossypium) and its allies, this group offers opportunities for exploring patterns and mechanisms of genome size evolution. For example, the question has been raised whether plant genomes have a 'one-way ticket to genomic obesity', as a consequence of retroelement accumulation. Few empirical studies directly address this possibility, although it is consistent with recent insights gleaned from evolutionary genomic investigations. We used a phylogenetic approach to evaluate the directionality of genome size evolution among Gossypium species and their relatives in the cotton tribe (Gossypieae, Malvaceae). Our results suggest that both DNA content increase and decrease have occurred repeatedly during evolution. In contrast to a model of unidirectional genome size change, the frequency of inferred genome size contraction exceeded that of expansion. In conjunction with other evidence, this finding highlights the dynamic nature of plant genome size evolution, and suggests that poorly understood genomic contraction mechanisms operate on a more extensive scale that previously recognized. Moreover, the research sets the stage for fine-scale analysis of the evolutionary dynamics and directionality of change for the full spectrum of genomic constituents.

Premise of the study: We investigated whether wood metabolite profiles from direct analysis in real time (time-of-flight) mass spectrometry (DART-TOFMS) could be used to determine the geographic origin of Douglas-fir wood cores originating from two regions in western Oregon, USA. Methods: Three annual ring mass spectra were obtained from 188 adult Douglas-fir trees, and these were analyzed using random forest models to determine whether samples could be classified to geographic origin, growth year, or growth year and geographic origin. Specific wood molecules that contributed to geographic discrimination were identified. Results: Douglas-fir mass spectra could be differentiated into two geographic classes with an accuracy between 70% and 76%. Classification models could not accurately classify sample mass spectra based on growth year. Thirty-two molecules were identified as key for classifying western Oregon Douglas-fir wood cores to geographic origin. Discussion: DART-TOFMS is capable of detecting minute...

The cultivated strawberry, Fragaria ×ananassa, is one of the youngest domesticated plants. Its 18th century origin via hybridization in Europe between the North American F. virginiana and the South American F. chiloensis was documented by the botanist Antoine Nicolas Duchesne. His 1766 "Natural History of Strawberries" is an extraordinary work that integrates fundamental discoveries on the biology, ecology, and phylogeny of Fragaria with applied information on cultivation and ethnobotanical uses, serving as an inspiration for current research in the genus. Fragaria species exhibit the full range of sexual systems in the gynodioecy pathway from hermaphroditism to dioecy (and back again), as well as variation in self-compatibility, and evidence of sex chromosomes with female heterogamety. The genus is also characterized by interspecific hybridization and polyploidy, with a natural range of ploidy levels from diploids to decaploids. This biological diversity, combined with the availability of genomic resources and the ease of growing and experimenting with the plants, makes Fragaria a very attractive system for ecological and evolutionary genomics. The goal of this review is to introduce Fragaria as a model genus and to provide a roadmap for future integrative research. These research directions will deepen our understanding of the ecological and evolutionary context that shaped the ancestors of the cultivated strawberry, not only providing information that can be applied to efforts to shape the future of this important fruit crop but also our understanding of key transitions in plant evolution.

Premise of the study: Hyb-Seq, the combination of target enrichment and genome skimming, allows simultaneous data collection for low-copy nuclear genes and high-copy genomic targets for plant systematics and evolution studies. Methods and Results: Genome and transcriptome assemblies for milkweed (Asclepias syriaca) were used to design enrichment probes for 3385 exons from 768 genes (>1.6 Mbp) followed by Illumina sequencing of enriched libraries. Hyb-Seq of 12 individuals (10 Asclepias species and two related genera) resulted in at least partial assembly of 92.6% of exons and 99.7% of genes and an average assembly length >2 Mbp. Importantly, complete plastomes and nuclear ribosomal DNA cistrons were assembled using off-target reads. Phylogenomic analyses demonstrated signal conflict between genomes. Conclusions: The Hyb-Seq approach enables targeted sequencing of thousands of low-copy nuclear exons and flanking regions, as well as genome skimming of high-copy repeats and organellar genomes, to efficiently produce genome-scale data sets for phylogenomics.

Recent work has demonstrated that allopolyploid speciation in plants may be associated with non-Mendelian genomic changes in the early generations following polyploid synthesis. To address the question of whether rapid genomic changes also occur in allopolyploid cotton (Gossypium) species, amplified fragment length polymorphism (AFLP) analysis was performed to evaluate nine sets of newly synthesized allotetraploid and allohexaploid plants, their parents, and the selfed progeny from colchicine-doubled synthetics. Using both methylation-sensitive and methylation- insensitive enzymes, the extent of fragment additivity in newly combined genomes was ascertained for a total of ap- proximately 22 000 genomic loci. Fragment additivity was observed in nearly all cases, with the few exceptions most likely reflecting parental heterozygosity or experimental error. In addition, genomic Southern analysis on six sets of synthetic allopolyploids probed with five retrotransposons also revealed complete additivity. Because no alterations were observed using methylation-sensitive isoschizomers, epigenetic changes following polyploid synthesis were also mini - mal. These indications of genomic additivity and epigenetic stasis during allopolyploid formation provide a contrast to recent evidence from several model plant allopolyploids, most notably wheat and Brassica, where rapid and unex- plained genomic changes have been reported. In addition, the data contrast with evidence from repetitive DNAs in Gossypium, some of which are subject to non-Mendelian molecular evolutionary phenomena in extant polyploids. These contrasts indicate polyploid speciation in plants is accompanied by a diverse array of molecular evolutionary phenomena, which will vary among both genomic constituents and taxa. Resume : De recents travaux ont demontre que la speciation via l'allopolyploidisation chez les plantes s'accompagne parfois de changements genomiques non-mendeliens au cours des premieres generations. Afin de determiner si des changements genomiques rapides surviennent chez des especes allopolyploides du cotonnier (Gossypium), une analyse AFLP (polymorphisme de longueur des fragments amplifies) a ete faite pour evaluer neuf jeux d'hybrides synthetiques allotetraploides ou allohexaploides, leurs parents et la progeniture obtenue par autofecondation suite au doublement chromosomique induit par la colchicine. A l'aide d'enzymes sensibles ou insensibles a la methylation, le degre d'additivite des fragments a ete evalue chez les genomes nouvellement combines pour environ 22 000 locus genomi- ques. L'additivite des fragments a ete observee dans presque tous les cas et les quelques rares exceptions refletaient vraisemblablement une heterozygotie parentale ou une erreur experimentale. De plus, des analyses Southern sur six jeux d'allopolyploides examines a l'aide de sondes constituees de retrotransposons ont egalement revele une complete additivite. Comme aucune alteration n'a ete observee a l'aide des isoschizomeres sensibles a la methylation, les chan - gements epigenetiques decoulant de la synthese des polyploides sont egalement peu nombreux. Ces evidences d'additivite genomique et de stabilite epigenetique lors de l'allopolyploidisation contrastent avec les resultats recents chez plusieurs especes modeles vegetales, notamment le ble et Brassica. Chez ces dernieres, des changements rapides et inexpliques ont ete rapportes. De plus, les donnees presentees contrastent avec celles obtenues avec des ADN repetes chez le Gossypium dont certains sont sujets a des phenomenes d'evolution moleculaire non-mendelienne chez les poly- ploides existants. Ces differences montrent que la speciation via allopolyploidisation chez les plantes s'accompagne de divers phenomenes evolutifs moleculaires, lesquels peuvent varier parmi les composantes genomiques et les taxons. Mots cles : polyploidie, evolution genomique, cotonnier, Gossypium, AFLP. (Traduit par la Redaction) Liu et al. 330

One of the most striking examples of plant hairs is the single-celled epidermal seed trichome of cultivated cotton. The developmental morphology of these commercial "fibers" has been well-characterized in Gossypium hirsutum, but little is known about the pattern and tempo of fiber development in wild Gossypium species, all of which have short, agronomically inferior fiber. To identify developmental differences that account for variation in fiber length, and to place these differences in a phylogenetic context, we conducted SEM studies of ovules at and near the time of flowering, and generated growth curves for cultivated and wild diploid and tetraploid species. Trichome initiation was found to be similar in all taxa, with few notable differences in trichome density or early growth. Developmental profiles of the fibers of most wild species are similar, with fiber elongation terminating at about two weeks post-anthesis. In contrast, growth is extended to three weeks in the A- and F-genome diploids. This prolonged elongation period is diagnosed as a key evolutionary event in the origin of long fiber. A second evolutionary innovation is that absolute growth rate is higher in species with long fibers. Domestication of species is associated with a further prolongation of elongation at both the diploid and allopolyploid levels, suggesting the effects of parallel artificial selection. Comparative analysis of fiber growth curves lends developmental support to previous quantitative genetic suggestions that genes for fiber "improvement" in tetraploid cotton were contributed by the agronomically inferior D-genome diploid parent.

We needed a reliable way to identify species and confirm potential interspecific and intergeneric hybrids in a landscape level study of gene flow from transgenic glyphosate-resistant Agrostis stolonifera (Poaceae) to compatible relatives. We developed 12 new polymorphic chloroplast microsatellite markers to aid in identifying species recipient of transgenic pollen both within the Agrostis complex and the related genera Polypogon.

During allopolyploid formation, divergent sets of chromosomes are combined in a common nucleus. Accordingly, loci that are single-copy in progenitor diploid genomes become duplicated, homoeologous loci in allopolyploids. Although homoeologous loci have been identified using classical genetic and linkage mapping approaches, there are few examples of the isolation and molecular characterization of homoeologous loci from allopolyploids. Here we describe two methods for the isolation of orthologous duplicated loci and demonstrate the feasibility of the techniques using allotetraploid cotton. The methods utilize restriction-digested, size-fractionated genomic DNA as a template for either plasmid cloning or PCR amplification. This fractionation procedure, when combined with Southern hybridization and mapping information, can separate homoeologues from each other and from more divergent cross-hybridizing sequences (paralogues). Each homoeologue can be then be isolated from a pool of size-fractionated genomic DNA that is enriched for target loci. While these methods were specifically designed for isolating homoeologous loci from allotetraploids, they should be applicable to a broad spectrum of diploid and polyploid plants and be useful for studying both ancient and recent gene duplications. In addition, the procedures enrich for orthologous sequences, which can be used for phylogenetic analysis. Thus, a general approach is detailed for the isolation of nuclear genes for phylogeny reconstruction.Key words: polyploidy, homoeology, orthology, Gossypium.

Phylogenetic resolution is often low within groups of recently diverged taxa due to a paucity of phylogenetically informative characters. We tested the relative utility of seven noncoding cpDNA regions and a pair of homoeologous nuclear genes for resolving recent divergences, using tetraploid cottons ( Gossypium) as a model system. The five tetraploid species of Gossypium are a monophyletic assemblage derived from an allopolyploidization event that probably occurred within the last 0.5‐2 million years. Previous analysis of cpDNA restriction site data provided only partial resolution within this clade despite a large number of enzymes employed. We sequenced three cpDNA introns (rpl16, rpoC1, ndhA) and four cpDNA spacers (accD-psaI, trnL-trnF, trnT-trnL, atpB-rbcL) for a total of over 7 kb of sequence per taxon, yet obtained only four informative nucleotide substitutions (0.05%) resulting in incomplete phylogenetic resolution. In addition, we sequenced a 1.65-kb region of a homoeologous pair of nuclear-encoded alcohol dehydrogenase (Adh) genes. In contrast with the cpDNA sequence data, the Adh homoeologues yielded 25 informative characters (0.76%) and provided a robust and completely resolved topology that is concordant with previous cladistic and phenetic analyses. The enhanced resolution obtained using the nuclear genes reflects an approximately three- to sixfold increase in nucleotide substitution rate relative to the plastome spacers and introns.

Emergent diseases are an ever-increasing threat to forests and forest ecosystems and necessitate the development of research tools for species that often may have few pre-existing resources. We sequenced the mRNA expressed by the sudden oak death pathogen Phytophthora ramorum and its most susceptible forest host, tanoak, within the same tissue at two time points after inoculation, and in uninfected tanoak controls. Using the P. ramorum genome to differentiate host and pathogen transcripts, we detected more than 850 P. ramorum transcripts at 5 days post-inoculation and a concurrent upregulation of host genes usually associated with pathogenicity. At 1 day, in contrast, we did not detect pathogen expression or significant enrichment of functional categories of host transcripts relative to controls, highlighting the importance of sequencing depth for in planta studies of host-pathogen interactions. This study highlights processes in molecular host-pathogen interactions in forest trees and provides a first reference transcriptome for tanoak, allowing the preliminary identification of disease-related genes in this study and facilitating future work for this and other members of the family Fagaceae.

Based on residue concentrations in honey bees (Apis mellifera L.), we mapped the spatial distribution of fluoride across much of the upper Snake River Plain in southeastern Idaho. Our results suggest that facilities at the Idaho National Engineering Laboratory (INEL) intermittently release this chemical. These emissions affect localized areas on the INEL and probably contribute little fluoride to the surrounding region. Phosphate ore processing near Pocatello, ID, is a recurrent fluoride source and influences a wide area, possibly even the INEL. These results are reviewed with respect to anthropogenic and natural sources of fluoride. We also compare bee fluoride concentrations to those reported for lichens, vegetation, and soils in southern Idaho.

5S RNA genes and their nontranscribed spacers are tandemly repeated in plant genomes at one or more chromosomal loci. To facilitate an understanding of the forces that govern 5S rDNA evolution, copy-number estimation and DNA sequencing were conducted for a phylogenetically well-characterized set of 16 diploid species of cotton (Gossypium) and 4 species representing allopolyploid derivatives of the diploids. Copy number varies over twentyfold in the genus, from approximately 1,000 to 20,000 copies/2C genome. When superimposed on the organismal phylogeny, these data reveal examples of both array expansion and contraction. Across species, a mean of 12% of nucleotide positions are polymorphic within individual arrays, for both gene and spacer sequences. This shows, in conjunction with phylogenetic evidence for ancestral polymorphisms that survive speciation events, that intralocus concerted evolutionary forces are relatively weak and that the rate of interrepeat homogenization is approximately equal to the rate of speciation. Evidence presented also shows that duplicated 5S rDNA arrays in allopolyploids have retained their subgenomic identity since polyploid formation, thereby indicating that interlocus concerted evolution has not been an important factor in the evolution of these arrays. A descriptive model, one which incorporates the opposing forces of mutation and homogenization within a selective framework, is outlined to account for the empirical data presented. Weak homogenizing forces allow equivalent levels of sequence polymorphism to accumulate in the 5S gene and spacer sequences, but fixation of mutations is nearly prohibited in the 5S gene. As a consequence, fixed interspecific differences are statistically underrepresented for 5S genes. This result explains the apparent paradox that despite similar levels of gene and spacer diversity, phylogenetic analysis of spacer sequences yields highly resolved trees, whereas analyses based on 5S gene sequences do not.

Aspen (Populus tremuloides) is a temperate North American tree species with a geographical distribution more extensive than any other tree species on the continent. Because it is economically important for pulp and paper industries and ecologically important for its role as a foundation species in forest ecosystems, the decline of aspen in large portions of its range is of serious concern. The availability and annotation of the black cottonwood (Populus trichocarpa) genome enables a range of high throughput sequencing approaches that can be used to understand rangewide patterns of genetic variation, adaptation, and responses to environmental challenges in other Populus species, including aspen. Gene expression studies are particularly useful for understanding the molecular basis of ecological responses, but are limited by the availability of transcriptome data. We explored the aspen transcriptome through the use of high-throughput sequencing with two main goals: (1) characterization of the expressed portion of the P. tremuloides genome in leaves and (2) assessment of variation in gene expression among genets collected from distinct latitudes but reared in a common garden. We also report a large single nucleotide polymorphism dataset that provides the groundwork for future studies of aspen evolution and ecology, and we identify a set of differentially expressed genes across individuals and population boundaries for the leaf transcriptome of P. tremuloides.

Large population sizes and global distributions generally associate with high mitochondrial DNA control region (CR) diversity. The sperm whale (Physeter macrocephalus) is an exception, showing low CR diversity relative to other cetaceans; however, diversity levels throughout the remainder of the sperm whale mitogenome are unknown. We sequenced 20 mitogenomes from 17 sperm whales representative of worldwide diversity using Next Generation Sequencing (NGS) technologies (Illumina GAIIx, Roche 454 GS Junior). Resequencing of three individuals with both NGS platforms and partial Sanger sequencing showed low discrepancy rates (454-Illumina: 0.0071%; Sanger-Illumina: 0.0034%; and Sanger-454: 0.0023%) confirming suitability of both NGS platforms for investigating low mitogenomic diversity. Using the 17 sperm whale mitogenomes in a phylogenetic reconstruction with 41 other species, including 11 new dolphin mitogenomes, we tested two hypotheses for the low CR diversity. First, the hypothesis that CR-specific constraints have reduced diversity solely in the CR was rejected as diversity was low throughout the mitogenome, not just in the CR (overall diversity π = 0.096%; protein-coding 3rd codon = 0.22%; CR = 0.35%), and CR phylogenetic signal was congruent with protein-coding regions. Second, the hypothesis that slow substitution rates reduced diversity throughout the sperm whale mitogenome was rejected as sperm whales had significantly higher rates of CR evolution and no evidence of slow coding region evolution relative to other cetaceans. The estimated time to most recent common ancestor for sperm whale mitogenomes was 72,800 to 137,400 years ago (95% highest probability density interval), consistent with previous hypotheses of a bottleneck or selective sweep as likely causes of low mitogenome diversity.

The cultivated strawberry is one of the youngest domesticated plants, developed in France in the 1700s from chance hybridization between two western hemisphere octoploid species. However, little is known about the evolution of the species that gave rise to this important fruit crop. Phylogenetic analysis of chloroplast genome sequences of 21 Fragaria species and subspecies resolves the western North American diploid F. vesca subsp. bracteata as sister to the clade of octoploid/decaploid species. No extant tetraploids or hexaploids are directly involved in the maternal ancestry of the octoploids. There is strong geographic segregation of chloroplast haplotypes in subsp . bracteata , and the gynodioecious Pacific Coast populations are implicated as both the maternal lineage and the source of male-sterility in the octoploid strawberries. Analysis of sexual system evolution in Fragaria provides evidence that the loss of male and female function can follow polyploidization, but does not seem to be associated with loss of self-incompatibility following genome doubling. Character-state mapping provided insight into sexual system evolution and its association with loss of self-incompatibility and genome doubling/merger. Fragaria attained its circumboreal and amphitropical distribution within the past one to four million years and the rise of the octoploid clade is dated at 0.372–2.05 million years ago.

 Premise of the study: Just as Sanger sequencing did more than 20 years ago, next-generation sequencing (NGS) is poised to revolutionize plant systematics. By combining multiplexing approaches with NGS throughput, systematists may no longer need to choose between more taxa or more characters. Here we describe a genome skimming (shallow sequencing) approach for plant systematics.  Methods: Through simulations, we evaluated optimal sequencing depth and performance of single-end and paired-end short read sequences for assembly of nuclear ribosomal DNA (rDNA) and plastomes and addressed the effect of divergence on reference-guided plastome assembly. We also used simulations to identify potential phylogenetic markers from low-copy nuclear loci at different sequencing depths. We demonstrated the utility of genome skimming through phylogenetic analysis of the Sonoran Desert clade (SDC) of Asclepias (Apocynaceae).  Key results: Paired-end reads performed better than single-end reads. Minimum sequencing depths for high quality rDNA and plastome assemblies were 40 × and 30 × , respectively. Divergence from the reference signifi cantly affected plastome assembly, but relatively similar references are available for most seed plants. Deeper rDNA sequencing is necessary to characterize intragenomic polymorphism. The low-copy fraction of the nuclear genome was readily surveyed, even at low sequencing depths. Nearly 160 000 bp of sequence from three organelles provided evidence of phylogenetic incongruence in the SDC.  Conclusions: Adoption of NGS will facilitate progress in plant systematics, as whole plastome and rDNA cistrons, partial mitochondrial genomes, and low-copy nuclear markers can now be effi ciently obtained for molecular phylogenetics studies.

 Premise of the study: Microsatellite primers were developed for the Pacifi c Northwest conifer, Chamaecyparis lawsoniana (Cupressaceae), to enhance effi ciencies in disease-resistance breeding and germplasm screening for gene conservation of this rare species.  Methods and Results: Using multiplexed massively parallel Illumina sequencing, we identifi ed over 300 000 microsatellitecontaining sequences from 2 million paired-end microreads. After stringent fi ltering and primer evaluation, we selected 11 primer pairs and used these to screen variation in four populations of C. lawsoniana. Loci show between three and 10 repeats per locus, with an average of eight. Screening of these markers in the North American relative Callitropsis nootkatensis demonstrated limited marker transferability, but these markers could have utility in Asian species of Chamaecyparis .  Conclusions: These microsatellite primers show high polymorphism and should provide a high level of individual discrimination for paternity analysis in defi ned pedigrees, and routine screening of wild variation in Chamaecyparis lawsoniana .

Background Big sagebrush (Artemisia tridentata) is one of the most widely distributed and ecologically important shrub species in western North America. This species serves as a critical habitat and food resource for many animals and invertebrates. Habitat loss due to a combination of disturbances followed by establishment of invasive plant species is a serious threat to big sagebrush ecosystem sustainability. Lack of genomic data has limited our understanding of the evolutionary history and ecological adaptation in this species. Here, we report on the sequencing of expressed sequence tags (ESTs) and detection of single nucleotide polymorphism (SNP) and simple sequence repeat (SSR) markers in subspecies of big sagebrush. Results cDNA of A. tridentata sspp. tridentata and vaseyana were normalized and sequenced using the 454 GS FLX Titanium pyrosequencing technology. Assembly of the reads resulted in 20,357 contig consensus sequences in ssp. tridentata and 20,250 contigs in ssp. vaseyana. A BLASTx search against the non-redundant (NR) protein database using 29,541 consensus sequences obtained from a combined assembly resulted in 21,436 sequences with significant blast alignments (≤ 1e-15). A total of 20,952 SNPs and 119 polymorphic SSRs were detected between the two subspecies. SNPs were validated through various methods including sequence capture. Validation of SNPs in different individuals uncovered a high level of nucleotide variation in EST sequences. EST sequences of a third, tetraploid subspecies (ssp. wyomingensis) obtained by Illumina sequencing were mapped to the consensus sequences of the combined 454 EST assembly. Approximately one-third of the SNPs between sspp. tridentata and vaseyana identified in the combined assembly were also polymorphic within the two geographically distant ssp. wyomingensis samples. Conclusion We have produced a large EST dataset for Artemisia tridentata, which contains a large sample of the big sagebrush leaf transcriptome. SNP mapping among the three subspecies suggest the origin of ssp. wyomingensis via mixed ancestry. A large number of SNP and SSR markers provide the foundation for future research to address questions in big sagebrush evolution, ecological genetics, and conservation using genomic approaches.

Conservation and management of natural populations requires accurate and inexpensive genotyping methods. Traditional microsatellite, or simple sequence repeat (SSR), marker analysis remains a popular genotyping method because of the comparatively low cost of marker development, ease of analysis and high power of genotype discrimination. With the availability of massively parallel sequencing (MPS), it is now possible to sequence microsatellite-enriched genomic libraries in multiplex pools. To test this approach, we prepared seven microsatellite-enriched, barcoded genomic libraries from diverse taxa (two conifer trees, five birds) and sequenced these on one lane of the Illumina Genome Analyzer using paired-end 80-bp reads. In this experiment, we screened 6.1 million sequences and identified 356 958 unique microreads that contained di- or trinucleotide microsatellites. Examination of four species shows that our conversion rate from raw sequences to polymorphic markers compares favourably to Sanger- and 454-based methods. The advantage of multiplexed MPS is that the staggering capacity of modern microread sequencing is spread across many libraries; this reduces sample preparation and sequencing costs to less than $400 (USD) per species. This price is sufficiently low that microsatellite libraries could be prepared and sequenced for all 1373 organisms listed as ‘threatened’ and ‘endangered’ in the United States for under $0.5 M (USD).

Previous molecular phylogenetic studies have failed to resolve the branching order among the major cotton (Gossypium) lineages, and it has been unclear whether this reflects actual history (rapid radiation) or sampling properties of the genes evaluated. In this paper, we reconsider the phylogenetic relationships of diploid cotton genome groups using DNA sequences from 11 single-copy nuclear loci (10 293 base pairs [bp]), nuclear ribosomal DNA (695 bp), and four chloroplast loci (7370 bp). Results from individual loci and combined nuclear and chloroplast DNA partitions reveal that the cotton genome groups radiated in rapid succession following the formation of the genus. Maximum likelihood analysis of nuclear synonymous sites shows that this radiation occurred within a time span equivalent to 17% of the time since the separation of Gossypium from its nearest extant relatives in the genera Kokia and Gossypioides. Chloroplast and nuclear phylogenies differ significantly with respect to resolution of the basal divergence in the genus and to interrelationships among African cottons. This incongruence is due to limited character evolution in cpDNA and either previously unsuspected hybridization or unreliable phylogenetic performance of the cpDNA characters. This study highlights the necessity of using multiple, independent data sets for resolving phylogenetic relationships of rapidly diverged lineages.

Sequence data from nrITS and cpDNA have failed to fully resolve phylogenetic relationships among Pinus species. Four low-copy nuclear genes, developed from the screening of 73 mapped conifer anchor loci, were sequenced from 12 species representing all subsections. Individual loci do not uniformly support either the nrITS or cpDNA hypotheses and in some cases produce unique topologies. Combined analysis of low-copy nuclear loci produces a well-supported subsectional topology of two subgenera, each divided into two sections, congruent with prior hypotheses of deep divergence in Pinus. The placements of P. nelsonii, P. krempfii, and P. contorta have been of continued systematic interest. Results strongly support the placement of P. nelsonii as sister to the remaining members of sect. Parrya, suggest a moderately well-supported and consistent position of P. krempfii as sister to the remaining members of sect. Quinquefoliae, and are ambiguous about the placement of P. contorta. A successful phylogenetic strategy in Pinus will require many low-copy nuclear loci that include a high proportion of silent sites and derive from independent linkage groups. The locus screening and evaluation strategy presented here can be broadly applied to facilitate the development of phylogenetic markers from the increasing number of available genomic resources.

Hybridization and polyploidization are exceedingly important processes because both infl uence the ecological envelope and evolutionary trajectory of land plants. These processes are frequently invoked for Castilleja (Indian paintbrushes) as contributors to morphological and genetic novelty and as complicating factors in species delimitations. Here, we provide a detailed analysis of morphological and genetic evidence for hybridization in a well-characterized hybrid swarm involving three broadly sympatric species ( C. miniata , C. rhexiifolia , C. sulphurea ) in western Colorado. Field-classifi ed hybrids are present at high frequencies at these sites and show morphological intermediacy to and segregate for chloroplast DNA haplotypes with C. rhexiifolia and C. sulphurea . Contrarily, DNA content and AFLP variation show that fi eld-classifi ed hybrids are not recent hybrids but a distinctive fourth taxon. Actual hybrids (plants showing admixture ≥ 10% for two genotypic groups) comprised 13% of our sample, with most admixture involving C. rhexiifolia , C. sulphurea , and the unknown taxon. The identity of the fi eld-classifi ed “ hybrids ” remains unknown; they either represent a stabilized hybrid species or a species with uncharacteristically high diversity for color alleles. This study highlights the importance of examining concordance and discordance between morphology, cytology, and genetic criteria to understand the complex evolutionary history of diverse groups such as Castilleja .

Background Milkweeds (Asclepias L.) have been extensively investigated in diverse areas of evolutionary biology and ecology; however, there are few genetic resources available to facilitate and compliment these studies. This study explored how low coverage genome sequencing of the common milkweed (Asclepias syriaca L.) could be useful in characterizing the genome of a plant without prior genomic information and for development of genomic resources as a step toward further developing A. syriaca as a model in ecology and evolution. Results A 0.5× genome of A. syriaca was produced using Illumina sequencing. A virtually complete chloroplast genome of 158,598 bp was assembled, revealing few repeats and loss of three genes: accD, clpP, and ycf1. A nearly complete rDNA cistron (18S-5.8S-26S; 7,541 bp) and 5S rDNA (120 bp) sequence were obtained. Assessment of polymorphism revealed that the rDNA cistron and 5S rDNA had 0.3% and 26.7% polymorphic sites, respectively. A partial mitochondrial genome sequence (130,764 bp), with identical gene content to tobacco, was also assembled. An initial characterization of repeat content indicated that Ty1/copia-like retroelements are the most common repeat type in the milkweed genome. At least one A. syriaca microread hit 88% of Catharanthus roseus (Apocynaceae) unigenes (median coverage of 0.29×) and 66% of single copy orthologs (COSII) in asterids (median coverage of 0.14×). From this partial characterization of the A. syriaca genome, markers for population genetics (microsatellites) and phylogenetics (low-copy nuclear genes) studies were developed. Conclusions The results highlight the promise of next generation sequencing for development of genomic resources for any organism. Low coverage genome sequencing allows characterization of the high copy fraction of the genome and exploration of the low copy fraction of the genome, which facilitate the development of molecular tools for further study of a target species and its relatives. This study represents a first step in the development of a community resource for further study of plant-insect co-evolution, anti-herbivore defense, floral developmental genetics, reproductive biology, chemical evolution, population genetics, and comparative genomics using milkweeds, and A. syriaca in particular, as ecological and evolutionary models.

Loblolly pine (LP; Pinus taeda L.) is the most economically important tree in the U.S. and a cornerstone species in southeastern forests. However, genomics research on LP and other conifers has lagged behind studies on flowering plants due, in part, to the large size of conifer genomes. As a means to accelerate conifer genome research, we constructed a BAC library for the LP genotype 7-56. The LP BAC library consists of 1,824,768 individually-archived clones making it the largest single BAC library constructed to date, has a mean insert size of 96 kb, and affords 7.6X coverage of the 21.7 Gb LP genome. To demonstrate the efficacy of the library in gene isolation, we screened macroarrays with overgos designed from a pine EST anchored on LP chromosome 10. A positive BAC was sequenced and found to contain the expected full-length target gene, several gene-like regions, and both known and novel repeats. Macroarray analysis using the retrotransposon IFG-7 (the most abundant repeat in the sequenced BAC) as a probe indicates that IFG-7 is found in roughly 210,557 copies and constitutes about 5.8% or 1.26 Gb of LP nuclear DNA; this DNA quantity is eight times the Arabidopsis genome. In addition to its use in genome characterization and gene isolation as demonstrated herein, the BAC library should hasten whole genome sequencing of LP via next-generation sequencing strategies/technologies and facilitate improvement of trees through molecular breeding and genetic engineering. The library and associated products are distributed by the Clemson University Genomics Institute (www.genome.clemson.edu).

Background Science-based wildlife management relies on genetic information to infer population connectivity and identify conservation units. The most commonly used genetic marker for characterizing animal biodiversity and identifying maternal lineages is the mitochondrial genome. Mitochondrial genotyping figures prominently in conservation and management plans, with much of the attention focused on the non-coding displacement ("D") loop. We used massively parallel multiplexed sequencing to sequence complete mitochondrial genomes from 40 fishers, a threatened carnivore that possesses low mitogenomic diversity. This allowed us to test a key assumption of conservation genetics, specifically, that the D-loop accurately reflects genealogical relationships and variation of the larger mitochondrial genome. Results Overall mitogenomic divergence in fishers is exceedingly low, with 66 segregating sites and an average pairwise distance between genomes of 0.00088 across their aligned length (16,290 bp). Estimates of variation and genealogical relationships from the displacement (D) loop region (299 bp) are contradicted by the complete mitochondrial genome, as well as the protein coding fraction of the mitochondrial genome. The sources of this contradiction trace primarily to the near-absence of mutations marking the D-loop region of one of the most divergent lineages, and secondarily to independent (recurrent) mutations at two nucleotide position in the D-loop amplicon. Conclusions Our study has two important implications. First, inferred genealogical reconstructions based on the fisher D-loop region contradict inferences based on the entire mitogenome to the point that the populations of greatest conservation concern cannot be accurately resolved. Whole-genome analysis identifies Californian haplotypes from the northern-most populations as highly distinctive, with a significant excess of amino acid changes that may be indicative of molecular adaptation; D-loop sequences fail to identify this unique mitochondrial lineage. Second, the impact of recurrent mutation appears most acute in closely related haplotypes, due to the low level of evolutionary signal (unique mutations that mark lineages) relative to evolutionary noise (recurrent, shared mutation in unrelated haplotypes). For wildlife managers, this means that the populations of greatest conservation concern may be at the highest risk of being misidentified by D-loop haplotyping. This message is timely because it highlights the new opportunities for basing conservation decisions on more accurate genetic information.

Background Molecular evolutionary studies share the common goal of elucidating historical relationships, and the common challenge of adequately sampling taxa and characters. Particularly at low taxonomic levels, recent divergence, rapid radiations, and conservative genome evolution yield limited sequence variation, and dense taxon sampling is often desirable. Recent advances in massively parallel sequencing make it possible to rapidly obtain large amounts of sequence data, and multiplexing makes extensive sampling of megabase sequences feasible. Is it possible to efficiently apply massively parallel sequencing to increase phylogenetic resolution at low taxonomic levels? Results We reconstruct the infrageneric phylogeny of Pinus from 37 nearly-complete chloroplast genomes (average 109 kilobases each of an approximately 120 kilobase genome) generated using multiplexed massively parallel sequencing. 30/33 ingroup nodes resolved with ≥ 95% bootstrap support; this is a substantial improvement relative to prior studies, and shows massively parallel sequencing-based strategies can produce sufficient high quality sequence to reach support levels originally proposed for the phylogenetic bootstrap. Resampling simulations show that at least the entire plastome is necessary to fully resolve Pinus, particularly in rapidly radiating clades. Meta-analysis of 99 published infrageneric phylogenies shows that whole plastome analysis should provide similar gains across a range of plant genera. A disproportionate amount of phylogenetic information resides in two loci (ycf1, ycf2), highlighting their unusual evolutionary properties. Conclusion Plastome sequencing is now an efficient option for increasing phylogenetic resolution at lower taxonomic levels in plant phylogenetic and population genetic analyses. With continuing improvements in sequencing capacity, the strategies herein should revolutionize efforts requiring dense taxon and character sampling, such as phylogeographic analyses and species-level DNA barcoding.

We present an overview of the evolution and diversity in Gossypium (the cotton genus). This framework facilitates insight into fundamental aspects of plant biology, provides the necessary underpinnings for effective utilization of cotton genetic resources, and guides exploration of the genomic basis of morphological diversity in the genus. More than 50 species of Gossypium are distributed in arid to semi-arid regions of the tropics and subtropics. Included are four species that independently have been domesticated for their fiber, two each in Africa-Asia and the Americas. Gossypium species exhibit extraordinary morphological variation, ranging from trailing herbaceous perennials to ∼15 m trees with a diverse array of reproductive and vegetative characteristics. A parallel level of cytogenetic and genomic diversity has arisen during the global radiation of the genus, leading to the evolution of eight groups of diploid (n = 13) species (genome groups A through G, and K). Data implicate an origin for Gossypium about 5–10 million years ago and a rapid early diversification of the major genome groups. Allopolyploid cottons appear to have arisen within the last 1–2 million years, as a consequence of trans-oceanic dispersal of an A-genome taxon to the New World followed by hybridization with an indigenous D-genome diploid. Subsequent to formation, allopolyploids radiated into three modern lineages, two of which contain the commercially important species G. hirsutum and G. barbadense.

Reverse transcriptase has been purified from feline immunodeficiency virus (FIV) by DEAE-cellulose and phosphocellulose chromatography. The purified enzyme consists of a single protein with a Mr of 67,000. When proteolysis is not minimized during purification, a fragment of Mr 54,000 is also observed. This is similar to the reverse transcriptase from human immunodeficiency virus type 1 (HIV), which consists of a polypeptide of Mr 66,000; when proteolysis is not minimized during purification, a fragment of Mr 51,000 is also observed. In direct comparisons, the FIV reverse transcriptase is very similar to the HIV reverse transcriptase in template specificity and requirements for Mg2+. In contrast to these similarities, the FIV and HIV reverse transcriptases are substantially different in primary sequence, as determined by peptide mapping.

Pinus lambertiana (sugar pine) is an economically and ecologically important conifer with a 1600-km latitudinal range extending from Oregon, USA, to northern Baja California, Mexico. Like all North American white pines (subsect. Strobus), sugar pine is highly susceptible to white pine blister rust, a disease caused by the fungus Cronartium ribicola. We conducted a chloroplast DNA (cpDNA) survey of Pinus subsect. Strobus with comprehensive geographical sampling of P. lambertiana. Sequence analysis of 12 sugar pine individuals revealed strong geographical differentiation for two chloroplast haplotypes. A diagnostic restriction site survey of an additional 72 individuals demarcated a narrow 150-km contact zone in northeastern California. In the contact zone, maternal (megagametophtye) and paternal (embryo) haplotypes were identified in 31 single seeds, demonstrating bidirectional pollen flow extending beyond the range of maternal haplotypes. The frequencies of the Cr1 allele for white pine blister rust major gene resistance, previously determined for 41 seed zones, differ significantly among seed zones that are fixed for the alternate haplotypes, or contain a mixture of both haplotypes. Interspecific phylogenetic analysis reveals that the northern sugar pine haplotype belongs to a clade that includes Pinus albicaulis (whitebark pine) and all of the East Asian white pines. Furthermore, there is little cpDNA divergence between northern sugar pine and whitebark pine (dS = 0.00058). These results are consistent with a Pleistocene migration of whitebark pine into North America and subsequent chloroplast introgression from whitebark pine to sugar pine. This study demonstrates the importance of placing phylogeographical results in a broader phylogenetic context.

Silent mutation rate estimates for Pinus vary 50-fold, ranging from angiosperm-like to among the slowest reported for plants. These differences either reflect extraordinary genomic processes or inconsistent fossil calibration, and they have important consequences for population and biogeographical inferences. Here we estimate mutation rates from 4 Pinus species that represent the major lineages using 11 nuclear and 4 chloroplast loci. Calibration was tested at the divergence of Pinus subgenera with the oldest leaf fossil from subg. Strobus (Eocene; 45 MYA) or a recently published subg. Strobus wood fossil (Cretaceous; 85 MYA). These calibrations place the origin of Pinus 190-102 MYA and give absolute silent rate estimates of 0.70-1.31 × 10 −9 and 0.22-0.42 × 10 −9 ·site −1 ·year −1 for the nuclear and chloroplast genomes, respectively. These rates are approximately 4- to 20-fold slower than angiosperms, but unlike many previous estimates, they are more consistent with the high per-generation deleterious mutation rates observed in pines. Chronograms from nuclear and chloroplast genomes show that the divergence of subgenera accounts for about half of the time since Pinus diverged from Picea, with subsequent radiations occurring more recently. By extending the sampling to encompass the phylogenetic diversity of Pinus, we predict that most extant subsections diverged during the Miocene. Moreover, subsect. Australes, Ponderosae, and Contortae, containing over 50 extant species, radiated within a 5 Myr time span starting as recently as 18 MYA. An Eocene divergence of pine subgenera (using leaf fossils) does not conflict with fossil-based estimates of the Pinus-Picea split, but a Cretaceous divergence using wood fossils accommodates Oligocene fossils that may represent modern subsections. Because homoplasy and polarity of character states have not been tested for fossil pine assignments, the choice of fossil and calibration node represents a significant source of uncertainty. Based on several lines of evidence (including agreement with ages inferred using calibrations outside of Pinus), we conclude that the 85 MYA calibration at the divergence of pine subgenera provides a reasonable lower bound and that further refinements in age and mutation rate estimates will require a synthetic examination of pine fossil history.

Phylogeographic and population genetic screens of chloroplast DNA (cpDNA) provide insights into seed-based gene flow in angiosperms, yet studies are frequently hampered by the low mutation rate of this genome. Detection methods for intraspecific variation can be either direct (DNA sequencing) or indirect (PCR–RFLP), although no single method incorporates the best features of both approaches. We show that screening universal chloroplast amp li cons for length polymorphism provides an accurate and efficient method for identifying cpDNA variation. By sequencing 4500 bp of cpDNA from 17 accessions of Purshia tridentata (bitterbrush), we detected 9 haplotypes, 8 of which were identifiable by unique multilocus length combinations resolvable by automated fragment analysis. In silico estimates of PCR–RFLP for these loci show that 5 haplotypes would be resolved by agarose electrophoresis. A survey of 4 intraspecific data sets from diverse angiosperms revealed that length variation in cpDNA amplicons is nearly ubiquitous, and 61 of 67 haplotypes identified by direct sequencing could be identified by screening length variation. Combined with automated fluorescent detection, length polymorphism screening of universal cpDNA regions offers a simple screen for intraspecific variation that can be used across angiosperms with minimal optimization, providing detection limits that rival direct sequencing at a fraction of the cost.Key words: cpDNA, intraspecific polymorphism, population genetics, phylogeography, indels.

11 pages, 2 figures, 3 tables.-- PMID: 12679546 [PubMed].-- Full-text paper available Open Access at journal site. Molecular evolutionary rate variation in Gossypium (cotton) was characterized using sequence data for 48 nuclear genes from both genomes of allotetraploid cotton, models of its diploid progenitors, and an outgroup. Substitution rates varied widely among the 48 genes, with silent and replacement substitution levels varying from 0.018 to 0.162 and from 0.000 to 0.073, respectively, in comparisons between orthologous Gossypium and outgroup sequences. However, about 90% of the genes had silent substitution rates spanning a more narrow threefold range. Because there was no evidence of rate heterogeneity among lineages for any gene and because rates were highly correlated in independent tests, evolutionary rate is inferred to be a property of each gene or its genetic milieu rather than the clade to which it belongs. Evidence from approximately 200,000 nucleotides (40,000 per genome) suggests that polyploidy in Gossypium led to a modest enhancement in rates of nucleotide substitution. Phylogenetic analysis for each gene yielded the topology expected from organismal history, indicating an absence of gene conversion or recombination among homoeologs subsequent to allopolyploid formation. Using the mean synonymous substitution rate calculated across the 48 genes, allopolyploid cotton is estimated to have formed circa 1.5 million years ago (MYA), after divergence of the diploid progenitors about 6.7 MYA. Financial support was provided by the National Science Foundation, the US-Israel Binational Science Foundation, the Plant Sciences Institute of Iowa State University, and the Spanish Ministry of Education, Culture and Sports.

PCR recombination describes a process of in vitro chimera formation from non-identical templates. The key requirement of this process is the inclusion of two partially homologous templates in one reaction, a condition met when amplifying any locus from polyploid organisms and members of multigene families from diploid organisms. Because polyploids possess two or more divergent genomes ("homoeologues") in a common nucleus, intergenic chimeras can form during the PCR amplification of any gene. Here we report a high frequency of PCR-induced recombination for four low-copy genes from allotetraploid cotton ( Gossypium hirsutum). Amplification products from these genes ( Myb3, Myb5, G1262 and CesA1) range in length from 860 to 4,050 bp. Intergenomic recombinants were formed frequently, accounting for 23 of the 74 (31.1%) amplicons evaluated, with the frequency of recombination in individual reactions ranging from 0% to approximately 89%. Inspection of the putative recombination zones failed to reveal sequence-specific attributes that promote recombination. The high levels of observed in vitro recombination indicate that the tacit assumption of exclusive amplification of target templates may often be violated, particularly from polyploid genomes. This conclusion has profound implications for population and evolutionary genetic studies, where unrecognized artifactually recombinant molecules may bias results or alter interpretations.

The cotton genus ( Gossypium ) includes approximately 50 species distributed in arid to semi-arid regions of the tropic and subtropics. Included are four species that have independently been domesticated for their fiber, two each in Africa–Asia and the Americas. Gossypium species exhibit extraordinary morphological variation, ranging from herbaceous perennials to small trees with a diverse array of reproductive and vegetative characteristics. A parallel level of cytogenetic and genomic diversity has arisen during the global radiation of the genus, leading to the evolution of eight groups of diploid ( n =13) species (genome groups A–G, and K). The evolutionary history of the genus included multiple episodes of trans-oceanic dispersal, invasion of new ecological niches, and a surprisingly high frequency of natural interspecific hybridization among lineages that are presently both geographically isolated and intersterile. Recent investigations have clarified many aspects of this history, including relationships within and among the eight genome groups, the domestication history of each of the four cultivated species, and the origin of the allopolyploid cottons. Data implicate an origin for Gossypium 5–15 million years ago (mya) and a rapid early diversification of the major genome groups. Allopolyploid cottons appear to have arisen within the last million years, as a consequence of trans-oceanic dispersal of an A-genome taxon to the New World followed by hybridization with an indigenous D-genome diploid. Subsequent to formation, allopolyploids radiated into three modern lineages, including those containing the commercially important species G. hirsutum and G. barbadense . Genome doubling has led to an array of molecular genetic interactions, including inter-locus concerted evolution, differential rates of genomic evolution, inter-genomic genetic transfer, and probable alterations in gene expression. The myriad underlying mechanisms are also suggested to have contributed to both ecological success and agronomic potential.

The 5S rRNA genes and their associated non-transcribed spacer (NTS) regions are present as repeat units arranged in tandem arrays in plant genomes. Length heterogeneity in 5S rDNA repeats was previously identified in Populus deltoides and was also observed in the present study. Primers were designed to amplify the 5S rDNA NTS variants from the P. deltoides genome. The PCR-amplified products from the two accessions of P. deltoides (G3 and G48) suggested the presence of length heterogeneity of 5S rDNA units within and among accessions, and the size of the spacers ranged from 385 to 434 bp. Sequence analysis of the non-transcribed spacer (NTS) revealed two distinct classes of 5S rDNA within both accessions: class 1, which contained GAA trinucleotide microsatellite repeats, and class 2, which lacked the repeats. The class 1 spacer shows length variation owing to the microsatellite, with two clones exhibiting 10 GAA repeat units and one clone exhibiting 16 such repeat units. However, distance analysis shows that class 1 spacer sequences are highly similar inter se, yielding nucleotide diversity (π) estimates that are less than 0.15% of those obtained for class 2 spacers (π = 0.0183 vs. 0.1433, respectively). The presence of microsatellite in the NTS region leading to variation in spacer length is reported and discussed for the first time in P. deltoides.Key words: 5S rDNA, Populus, repetitive DNA, microsatellite, sequence heterogeneity.

Of the many processes that generate gene duplications, polyploidy is unique in that entire genomes are duplicated. This process has been important in the evolution of many eukaryotic groups, and it occurs with high frequency in plants. Recent evidence suggests that polyploidization may be accompanied by rapid genomic changes, but the evolutionary fate of discrete loci recently doubled by polyploidy (homoeologues) has not been studied. Here we use locus-specific isolation techniques with comparative mapping to characterize the evolution of homoeologous loci in allopolyploid cotton ( Gossypium hirsutum ) and in species representing its diploid progenitors. We isolated and sequenced 16 loci from both genomes of the allopolyploid, from both progenitor diploid genomes and appropriate outgroups. Phylogenetic analysis of the resulting 73.5 kb of sequence data demonstrated that for all 16 loci (14.7 kb/genome), the topology expected from organismal history was recovered. In contrast to observations involving repetitive DNAs in cotton, there was no evidence of interaction among duplicated genes in the allopolyploid. Polyploidy was not accompanied by an obvious increase in mutations indicative of pseudogene formation. Additionally, differences in rates of divergence among homoeologues in polyploids and orthologues in diploids were indistinguishable across loci, with significant rate deviation restricted to two putative pseudogenes. Our results indicate that most duplicated genes in allopolyploid cotton evolve independently of each other and at the same rate as those of their diploid progenitors. These indications of genic stasis accompanying polyploidization provide a sharp contrast to recent examples of rapid genomic evolution in allopolyploids.

Background As it becomes increasingly possible to obtain DNA sequences of orthologous genes from diverse sets of taxa, species trees are frequently being inferred from multilocus data. However, the behavior of many methods for performing this inference has remained largely unexplored. Some methods have been proven to be consistent given certain evolutionary models, whereas others rely on criteria that, although appropriate for many parameter values, have peculiar zones of the parameter space in which they fail to converge on the correct estimate as data sets increase in size. Results Here, using North American pines, we empirically evaluate the behavior of 24 strategies for species tree inference using three alternative outgroups (72 strategies total). The data consist of 120 individuals sampled in eight ingroup species from subsection Strobus and three outgroup species from subsection Gerardianae, spanning ∼47 kilobases of sequence at 121 loci. Each “strategy” for inferring species trees consists of three features: a species tree construction method, a gene tree inference method, and a choice of outgroup. We use multivariate analysis techniques such as principal components analysis and hierarchical clustering to identify tree characteristics that are robustly observed across strategies, as well as to identify groups of strategies that produce trees with similar features. We find that strategies that construct species trees using only topological information cluster together and that strategies that use additional non-topological information (e.g., branch lengths) also cluster together. Strategies that utilize more than one individual within a species to infer gene trees tend to produce estimates of species trees that contain clades present in trees estimated by other strategies. Strategies that use the minimize-deep-coalescences criterion to construct species trees tend to produce species tree estimates that contain clades that are not present in trees estimated by the Concatenation, RTC, SMRT, STAR, and STEAC methods, and that in general are more balanced than those inferred by these other strategies. Conclusions When constructing a species tree from a multilocus set of sequences, our observations provide a basis for interpreting differences in species tree estimates obtained via different approaches that have a two-stage structure in common, one step for gene tree estimation and a second step for species tree estimation. The methods explored here employ a number of distinct features of the data, and our analysis suggests that recovery of the same results from multiple methods that tend to differ in their patterns of inference can be a valuable tool for obtaining reliable estimates.

Background Through next-generation sequencing, the amount of sequence data potentially available for phylogenetic analyses has increased exponentially in recent years. Simultaneously, the risk of incorporating ‘noisy’ data with misleading phylogenetic signal has also increased, and may disproportionately influence the topology of weakly supported nodes and lineages featuring rapid radiations and/or elevated rates of evolution. Results We investigated the influence of phylogenetic noise in large data sets by applying two fundamental strategies, variable site removal and long-branch exclusion, to the phylogenetic analysis of a full plastome alignment of 107 species of Pinus and six Pinaceae outgroups. While high overall phylogenetic resolution resulted from inclusion of all data, three historically recalcitrant nodes remained conflicted with previous analyses. Close investigation of these nodes revealed dramatically different responses to data removal. Whereas topological resolution and bootstrap support for two clades peaked with removal of highly variable sites, the third clade resolved most strongly when all sites were included. Similar trends were observed using long-branch exclusion, but patterns were neither as strong nor as clear. When compared to previous phylogenetic analyses of nuclear loci and morphological data, the most highly supported topologies seen in Pinus plastome analysis are congruent for the two clades gaining support from variable site removal and long-branch exclusion, but in conflict for the clade with highest support from the full data set. Conclusions These results suggest that removal of misleading signal in phylogenomic datasets can result not only in increased resolution for poorly supported nodes, but may serve as a tool for identifying erroneous yet highly supported topologies. For Pinus chloroplast genomes, removal of variable sites appears to be more effective than long-branch exclusion for clarifying phylogenetic hypotheses.

The replication of feline immunodeficiency virus (FIV) in cultured cells was inhibited by 2′,3′-dideoxyadenosine (ddA) and by 9-(2-phosphonylmethoxyethyI)adenine (PMEA) with IC 50 values of 0.98 and 0.95 μM, respectively. The effects of the presumed active forms of these inhibitors, ddATP and PMEA-diphosphate (PMEApp), upon the FIV reverse transcriptase (RT) were examined with two different template-primer systems. Both of these compounds were potent inhibitors of the FIV RT in reactions with primed φX-174 DNA, yielding K i values of 8.8 nM for ddATP and 5.0 nM for PMEApp. However, they were both poor inhibitors of the reaction with poly(rU)-oligo(dA); concentrations of ddATP or PMEApp greater than 10 μM were required to inhibit this reaction by 50%. Further analysis of the reaction with poly(rU)-oligo(dA) revealed that even in the absence of inhibitors the primers were extended by less than 20 nucleotides. In contrast, high molecular weight products were obtained in reactions with φX-174 DNA. These results suggest that the reaction of FIV RT with poly(rU)-oligo(dA) is not highly processive. The high degree of termination encountered during this reaction with poly(rU)-oligo(dA) may be responsible for the low inhibitory potential of ddATP and PMEApp.

Reverse transcription of retroviral genomes requires the action of an RNase H for template switching and primer generation. In this report, we compare enzymatic properties of the RNase H associated with the reverse transcriptase (RT) from feline immunodeficiency virus (FIV) and that from human immunodeficiency virus (HIV). Both enzymes displayed substrate preference for poly[3H](rG) . poly(dC) hybird over poly[3H](rA) . poly(dT) and cation preference for Mg2+ over Mn2+. Activity of the FIV RNase H upon poly(rG) . poly(dC) produced hydrolysis products from 1 to 6 nucleotides in length, similar to that reported for HIV. Dextran sulfates were effective inhibitors of both the FIV and HIV RNase H and RT activities. Nearly identical inhibition constants (0.12 nM) were obtained for all enzyme activities with dextran sulfate 500,000, while different inhibition constants were observed with dextran sulfate 8,000. Our results suggest that FIV and HIV RTs contain a conserved region that is sensitive to the larger dextran sulfate and that dextran sulfate 8,000 may interact at a different site or by a different mechanism.

Organellar DNA sequences are widely used in evolutionary and population genetic studies, however, the conservative nature of chloroplast gene and genome evolution often limits phylogenetic resolution and statistical power. To gain maximal access to the historical record contained within chloroplast genomes, we have adapted multiplex sequencing-by-synthesis (MSBS) to simultaneously sequence multiple genomes using the Illumina Genome Analyzer. We PCR-amplified approximately 120 kb plastomes from eight species (seven Pinus, one Picea) in 35 reactions. Pooled products were ligated to modified adapters that included 3 bp indexing tags and samples were multiplexed at four genomes per lane. Tagged microreads were assembled by de novo and reference-guided assembly methods, using previously published Pinus plastomes as surrogate references. Assemblies for these eight genomes are estimated at 88-94% complete, with an average sequence depth of 55x to 186x. Mononucleotide repeats interrupt contig assembly with increasing repeat length, and we estimate that the limit for their assembly is 16 bp. Comparisons to 37 kb of Sanger sequence show a validated error rate of 0.056%, and conspicuous errors are evident from the assembly process. This efficient sequencing approach yields high-quality draft genomes and should have immediate applicability to genomes with comparable complexity.

The sensitivities of reverse transcriptases (RTs) from feline immunodeficiency virus (FIV) and human immunodeficiency virus type 1 (HIV) were directly compared. The two enzymes had similar sensitivities to three analogs of dTTP, namely, 3'-azido-3'-deoxythymidine 5'-triphosphate, 2',3'-dideoxythymidine 5'-triphosphate, and 2',3'-dideoxy-2',3'-didehydrothymidine 5'-triphosphate. Each of these analogs demonstrated competitive inhibition of both enzymes. Ki values for inhibition of FIV RT by these three inhibitors were 3.3, 6.7, and 1.8 nM, respectively; Ki values for inhibition of the HIV enzyme were 6.5, 5.9, and 8.3 nM, respectively. Ratios of the Ki for the inhibitor to the Km for the substrate were also determined for each inhibitor, and no differences between the two enzymes greater than threefold were observed. Inhibition constants for 3'-amino-3'-deoxythymidine 5'-triphosphate and 3'-fluoro-3'-deoxythymidine 5'-triphosphate were determined for FIV RT, and these were similar to published values for HIV RT. The activities of three dideoxynucleoside 5'-triphosphates against FIV RT were determined; ddGTP was slightly more potent than ddTTP, whereas both were much more effective than ddCTP. The activity of a noncompetitive inhibitor, phosphonoformate, was also examined with the FIV enzyme; it was much more active with poly(rA)-oligo(dT) as the template-primer than with poly(rC)-oligo(dG) or poly(rI)-oligo(dC).

Molecular data have had a profound impact on the field of plant systematics, and the application of DNA-sequence data to phylogenetic problems is now routine. The majority of data used in plant molecular phylogenetic studies derives from chloroplast DNA and nuclear rDNA, while the use of low-copy nuclear genes has not been widely adopted. This is due, at least in part, to the greater difficulty of isolating and characterising low-copy nuclear genes relative to chloroplast and rDNA sequences that are readily amplified with universal primers. The higher level of sequence variation characteristic of low-copy nuclear genes, however, often compensates for the experimental effort required to obtain them. In this review, we briefly discuss the strengths and limitations of chloroplast and rDNA sequences, and then focus our attention on the use of low-copy nuclear sequences. Advantages of low-copy nuclear sequences include a higher rate of evolution than for organellar sequences, the potential to accumulate datasets from multiple unlinked loci, and bi-parental inheritance. Challenges intrinsic to the use of low-copy nuclear sequences include distinguishing orthologous loci from divergent paralogous loci in the same gene family, being mindful of the complications arising from concerted evolution or recombination among paralogous sequences, and the presence of intraspecific, intrapopulational and intraindividual polymorphism. Finally, we provide a detailed protocol for the isolation, characterisation and use of low-copy nuclear sequences for phylogenetic studies.

Background Douglas-fir (Pseudotsuga menziesii), one of the most economically and ecologically important tree species in the world, also has one of the largest tree breeding programs. Although the coastal and interior varieties of Douglas-fir (vars. menziesii and glauca) are native to North America, the coastal variety is also widely planted for timber production in Europe, New Zealand, Australia, and Chile. Our main goal was to develop a SNP resource large enough to facilitate genomic selection in Douglas-fir breeding programs. To accomplish this, we developed a 454-based reference transcriptome for coastal Douglas-fir, annotated and evaluated the quality of the reference, identified putative SNPs, and then validated a sample of those SNPs using the Illumina Infinium genotyping platform. Results We assembled a reference transcriptome consisting of 25,002 isogroups (unique gene models) and 102,623 singletons from 2.76 million 454 and Sanger cDNA sequences from coastal Douglas-fir. We identified 278,979 unique SNPs by mapping the 454 and Sanger sequences to the reference, and by mapping four datasets of Illumina cDNA sequences from multiple seed sources, genotypes, and tissues. The Illumina datasets represented coastal Douglas-fir (64.00 and 13.41 million reads), interior Douglas-fir (80.45 million reads), and a Yakima population similar to interior Douglas-fir (8.99 million reads). We assayed 8067 SNPs on 260 trees using an Illumina Infinium SNP genotyping array. Of these SNPs, 5847 (72.5%) were called successfully and were polymorphic. Conclusions Based on our validation efficiency, our SNP database may contain as many as ~200,000 true SNPs, and as many as ~69,000 SNPs that could be genotyped at ~20,000 gene loci using an Infinium II array—more SNPs than are needed to use genomic selection in tree breeding programs. Ultimately, these genomic resources will enhance Douglas-fir breeding and allow us to better understand landscape-scale patterns of genetic variation and potential responses to climate change.