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Species detection using eDNA is revolutionizing global capacity to monitor biodiversity. However, the lack of regional, vouchered, genomic sequence information—especially sequence information that includes intraspecific variation—creates a bottleneck for management agencies wanting to harness the complete power of eDNA to monitor taxa and implement eDNA analyses. eDNA studies depend upon regional databases of mitogenomic sequence information to evaluate the effectiveness of such data to detect and identify taxa. We created the Oregon Biodiversity Genome Project to create a database of complete, nearly error-free mitogenomic sequences for all of Oregon’s fishes. We have successfully assembled the complete mitogenomes of 313 specimens of freshwater, anadromous, and estuarine fishes representing 24 families, 55 genera, and 128 species and lineages. Comparative analyses of these sequences illustrate that many regions of the mitogenome are taxonomically informative, that the short (~150 bp) mitochondrial “barcode” regions typically used for eDNA assays do not consistently diagnose for species, and that complete single or multiple genes of the mitogenome are preferable for identifying Oregon’s fishes. This project provides a blueprint for other researchers to follow as they build regional databases, illustrates the taxonomic value and limits of complete mitogenomic sequences, and offers clues as to how current eDNA assays and environmental genomics methods of the future can best leverage this information.

Genetic diversity underpins species conservation and management goals, and ultimately determines a species’ ability to adapt. Using freshwater environmental DNA (eDNA) samples, we examined mitochondrial genetic diversity using multigene metabarcode sequence data from four Oncorhynchus species across 16 sites in Oregon and northern California. Our multigene metabarcode panel included targets commonly used in population genetic NADH dehydrogenase 2 (ND2), phylogenetic cytochrome c oxidase subunit 1 (COI) and eDNA (12S ribosomal DNA) screening. The ND2 locus showed the greatest within‐species haplotype diversity for all species, followed by COI and then 12S rDNA for all species except Oncorhynchus kisutch. Sequences recovered for O. clarkii clarkii were either identical to, or one mutation different from, previously characterized haplotypes (95.3% and 4.5% of reads, respectively). The greatest diversity in O. c. clarkii was among coastal watersheds, and subsets of this diversity were shared with fish in inland watersheds. However, coastal streams and the Umpqua River watershed appear to harbour unique haplotypes. Sequences from O. mykiss revealed a disjunction between the Willamette watershed and southern watersheds suggesting divergent histories. We also identified similarities between populations in the northern Deschutes and southern Klamath watersheds, consistent with previously hypothesized connections between the two via inland basins. Oncorhynchus kisutch was only identified in coastal streams and the Klamath River watershed, with most diversity concentrated in the coastal Coquille watershed. Oncorhynchus tshawytscha was only observed at one site, but contained multiple haplotypes at each locus. The characterization of genetic diversity at multiple loci expands the knowledge gained from eDNA sampling and provides crucial information for conservation actions and genetic management.

Species detection using eDNA is revolutionizing the global capacity to monitor biodiversity. However, the lack of regional, vouchered, genomic sequence information—especially sequence information that includes intraspecific variation—creates a bottleneck for management agencies wanting to harness the complete power of eDNA to monitor taxa and implement eDNA analyses. eDNA studies depend upon regional databases of complete mitogenomic sequence information to evaluate the effectiveness of such data to differentiate, identify and detect taxa. We created the Oregon Biodiversity Genome Project working group to utilize recent advances in sequencing technology to create a database of complete, near error-free mitogenomic sequences for all of Oregon’s resident freshwater fishes. So far, we have successfully assembled the complete mitogenomes of 313 specimens of freshwater fish representing 7 families, 55 genera, and 129 (88%) of the 146 resident species and lineages. Our comparative analyses of these sequences illustrate that the short (~150 bp) mitochondrial “barcode” regions typically used for eDNA assays are not consistently diagnostic for species-level identification and that no single region is best for metabarcoding Oregon’s fishes. However, often-overlooked intergenic regions of the mitogenome such as the D-loop have the potential to reliably diagnose and differentiate species. This project provides a blueprint for other researchers to follow as they build regional databases. It also illustrates the taxonomic value and limits of complete mitogenomic sequences, and how current eDNA assays and the “PCR-free” environmental genomics methods of the future can best leverage this information.

The plastid genomes of photosynthetic green plants have largely maintained conserved gene content and order as well as structure over hundreds of millions of years of evolution. Several plant lineages, however, have departed from this conservation and contain many plastome structural rearrangements, which have been associated with an abundance of repeated sequences both overall and near rearrangement endpoints. We sequenced the plastomes of 25 taxa of Astragalus L. (Fabaceae), a large genus in the inverted repeat-lacking clade of legumes, to gain a greater understanding of the connection between repeats and plastome inversions. We found plastome repeat structure has a strong phylogenetic signal among these closely related taxa mostly in the New World clade of Astragalus called Neo-Astragalus. Taxa without inversions also do not differ substantially in their overall repeat structure from four taxa each with one large-scale inversion. For two taxa with inversion endpoints between the same pairs of genes, differences in their exact endpoints indicate the inversions occurred independently. Our proposed mechanism for inversion formation suggests the short inverted repeats now found near the endpoints of the four inversions may be there as a result of these inversions rather than their cause. The longer inverted repeats now near endpoints may have allowed the inversions first mediated by shorter microhomologous sequences to propagate, something that should be considered in explaining how any plastome rearrangement becomes fixed regardless of the mechanism of initial formation.

Background In forest trees, genetic markers have been used to understand the genetic architecture of natural populations, identify quantitative trait loci, infer gene function, and enhance tree breeding. Recently, new, efficient technologies for genotyping thousands to millions of single nucleotide polymorphisms (SNPs) have finally made large-scale use of genetic markers widely available. These methods will be exceedingly valuable for improving tree breeding and understanding the ecological genetics of Douglas-fir, one of the most economically and ecologically important trees in the world. Results We designed SNP assays for 55,766 potential SNPs that were discovered from previous transcriptome sequencing projects. We tested the array on ~ 2300 related and unrelated coastal Douglas-fir trees (Pseudotsuga menziesii var. menziesii) from Oregon and Washington, and 13 trees of interior Douglas-fir (P. menziesii var. glauca). As many as ~ 28 K SNPs were reliably genotyped and polymorphic, depending on the selected SNP call rate. To increase the number of SNPs and improve genome coverage, we developed protocols to ‘rescue’ SNPs that did not pass the default Affymetrix quality control criteria (e.g., 97% SNP call rate). Lowering the SNP call rate threshold from 97 to 60% increased the number of successful SNPs from 20,669 to 28,094. We used a subset of 395 unrelated trees to calculate SNP population genetic statistics for coastal Douglas-fir. Over a range of call rate thresholds (97 to 60%), the median call rate for SNPs in Hardy-Weinberg equilibrium ranged from 99.2 to 99.7%, and the median minor allele frequency ranged from 0.198 to 0.233. The successful SNPs also worked well on interior Douglas-fir. Conclusions Based on the original transcriptome assemblies and comparisons to version 1.0 of the Douglas-fir reference genome, we conclude that these SNPs can be used to genotype about 10 K to 15 K loci. The Axiom genotyping array will serve as an excellent foundation for studying the population genomics of Douglas-fir and for implementing genomic selection. We are currently using the array to construct a linkage map and test genomic selection in a three-generation breeding program for coastal Douglas-fir.

Funding for this study was provided by U. S. Agency for International Development (Award 19318814Y0010-140001) to the U.S. Forest Service International Programs, the U.S.D.A. Forest Service Pacific Northwest Research Station, and the Moldenke Endowment (Botany and Plant Pathology Department, Oregon State University). The legality of wood products often depends on their origin, creating a need for forensic tools that verify claims of provenance for wood products. The neotropical tree species Cedrela odorata (Spanish cedar) is economically valuable for its wood and faces threats of overexploitation. We developed a 140 SNP assay for geographic localization of C. odorata specimens. Target capture and short-read sequencing of 46 C. odorata specimens allowed us to identify 140 spatially informative SNPs that differentiate C. odorata specimens by latitude, temperature, and precipitation. We assessed the broad applicability of these SNPs on 356 specimens from eight Cedrela species, three tissue types, and a range of DNA mass inputs. Origin prediction error was evaluated with discrete and continuous spatial assignment methods focusing on C. odorata specimens. Discrete classification with random forests readily differentiated specimens originating in Central America versus South America (5.8% error), while uncertainty increased as specimens were divided into smaller regions. Continuous spatial prediction with SPASIBA showed a median prediction error of 188.7 km. Our results demonstrate that array SNPs and resulting genotypes accurately validate C. odorata geographic origin at the continental scale and show promise for country-level verification, but that finer-scale assignment likely requires denser spatial sampling. Our study underscores the important role of herbaria for developing genomic resources, and joins a growing list of studies that highlight the role of genomic tools for conservation of threatened species. Publisher PDF

Environmental DNA (eDNA) is an emerging biological monitoring tool that can aid in assessing the effects of forestry and forest manufacturing activities on biota. Monitoring taxa across broad spatial and temporal scales is necessary to ensure forest management and forest manufacturing activities meet their environmental goals of maintaining biodiversity. Our objectives are to describe potential applications of eDNA across the wood products supply chain extending from regenerating forests, harvesting, and wood transport, to manufacturing facilities, and to review the current state of the science in this context. To meet our second objective, we summarize the taxa examined with targeted (PCR, qPCR or ddPCR) or metagenomic eDNA methods (eDNA metabarcoding), evaluate how estimated species richness compares between traditional field sampling and eDNA metabarcoding approaches, and compare the geographical representation of prior eDNA studies in freshwater ecosystems to global wood baskets. Potential applications of eDNA include evaluating the effects of forestry and forest manufacturing activities on aquatic biota, delineating fish-bearing versus non fish-bearing reaches, evaluating effectiveness of constructed road crossings for freshwater organism passage, and determining the presence of at-risk species. Studies using targeted eDNA approaches focused on fish, amphibians, and invertebrates, while metagenomic studies focused on fish, invertebrates, and microorganisms. Rare, threatened, or endangered species received the least attention in targeted eDNA research, but are arguably of greatest interest to sustainable forestry and forest manufacturing that seek to preserve freshwater biodiversity. Ultimately, using eDNA methods will enable forestry and forest manufacturing managers to have data-driven prioritization for conservation actions for all freshwater species.

Background Tree species in the genus Cedrela P. Browne are threatened by timber overexploitation across the Neotropics. Genetic identification of processed timber can be used to supplement wood anatomy to assist in the taxonomic and source validation of protected species and populations of Cedrela. However, few genetic resources exist that enable both species and source identification of Cedrela timber products. We developed several ‘omic resources including a leaf transcriptome, organelle genome (cpDNA), and diagnostic single nucleotide polymorphisms (SNPs) that may assist the classification of Cedrela specimens to species and geographic origin and enable future research on this widespread Neotropical tree genus. Results We designed hybridization capture probes to enrich for thousands of genes from both freshly preserved leaf tissue and from herbarium specimens across eight Meliaceae species. We first assembled a draft de novo transcriptome for C. odorata, and then identified putatively low-copy genes. Hybridization probes for 10,001 transcript models successfully enriched 9795 (98%) of these targets, and analysis of target capture efficiency showed that probes worked effectively for five Cedrela species, with each species showing similar mean on-target sequence yield and depth. The probes showed greater enrichment efficiency for Cedrela species relative to the other three distantly related Meliaceae species. We provide a set of candidate SNPs for species identification of four of the Cedrela species included in this analysis, and present draft chloroplast genomes for multiple individuals of eight species from four genera in the Meliaceae. Conclusions Deforestation and illegal logging threaten forest biodiversity globally, and wood screening tools offer enforcement agencies new approaches to identify illegally harvested timber. The genomic resources described here provide the foundation required to develop genetic screening methods for Cedrela species identification and source validation. Due to their transferability across the genus and family as well as demonstrated applicability for both fresh leaves and herbarium specimens, the genomic resources described here provide additional tools for studies examining the ecology and evolutionary history of Cedrela and related species in the Meliaceae. Electronic supplementary material The online version of this article (10.1186/s12864-018-5382-6) contains supplementary material, which is available to authorized users.

Summary Polyploidy, or whole‐genome duplication often with hybridization, is common in eukaryotes and is thought to drive ecological and evolutionary success, especially in plants. The mechanisms of polyploid success in ecologically relevant contexts, however, remain largely unknown.We conducted an extensive test of functional trait divergence and plasticity in conferring polyploid fitness advantage in heterogeneous environments, by growing clonal replicates of a worldwide genotype collection of six allopolyploid and five diploid wild strawberry (Fragaria) taxa in three climatically different common gardens.Among leaf functional traits, we detected divergence in trait means but not plasticities between polyploids and diploids, suggesting that increased genomic redundancy in polyploids does not necessarily translate into greater trait plasticity in response to environmental change. Across the heterogeneous garden environments, however, polyploids exhibited fitness advantage, which was conferred by both trait means and adaptive trait plasticities, supporting a ‘jack‐and‐master’ hypothesis for polyploids.Our findings elucidate essential ecological mechanisms underlying polyploid adaptation to heterogeneous environments, and provide an important insight into the prevalence and persistence of polyploid plants.

Reconstructions of vascular plant mitochondrial genomes (mt-genomes) are notoriously complicated by rampant recombination that has resulted in comparatively few plant mt-genomes being available. The dearth of plant mitochondrial resources has limited our understanding of mt-genome structural diversity, complex patterns of RNA editing, and the origins of novel mt-genome elements. Here, we use an efficient long read (PacBio) iterative assembly pipeline to generate mt-genome assemblies for Leucaena trichandra (Leguminosae: Caesalpinioideae: mimosoid clade), providing the first assessment of non-papilionoid legume mt-genome content and structure to date. The efficiency of the assembly approach facilitated the exploration of alternative structures that are common place among plant mitochondrial genomes. A compact version (729 kbp) of the recovered assemblies was used to investigate sources of mt-genome size variation among legumes and mt-genome sequence similarity to the legume associated root holoparasite Lophophytum. The genome and an associated suite of transcriptome data from select species of Leucaena permitted an in-depth exploration of RNA editing in a diverse clade of closely related species that includes hybrid lineages. RNA editing in the allotetraploid, Leucaena leucocephala, is consistent with co-option of nearly equal maternal and paternal C-to-U edit components, generating novel combinations of RNA edited sites. A preliminary investigation of L. leucocephala C-to-U edit frequencies identified the potential for a hybrid to generate unique pools of alleles from parental variation through edit frequencies shared with one parental lineage, those intermediate between parents, and transgressive patterns.

Illegal logging is a worldwide problem that degrades ecosystems, and even low-risk markets like the United States report ~1000 significant hardwood timber theft cases per year. Due to its high value, bigleaf maple (Acer macrophyllum Pursh) is a common target of timber theft in western North America. Using samples from Washington, USA, a single nucleotide polymorphism [SNP] assay was recently developed to facilitate timber poaching investigations in this species based on the single base extension Agena MassARRAY® genotyping system and 133 loci. Our study expands earlier evaluations to include the full 2000 km latitudinal range of bigleaf maple, using 1142 samples collected from California, USA to British Columbia, CAN. Populations in this study are equivalent to level 3 ecoregions, with sample sizes ranging from 28 (California Coast) to 191 (Washington Coast). Wood-derived DNA concentrations between 0.24 and 6 ng/μL yielded SNP call rates >92%, and DNA concentrations within this range did not influence genotyping call rates. DNA from different tissues showed a weak but significant difference in call rate, with DNA from wood showing a slightly higher call rate than leaves (99.4% vs 97.9%, respectively). Analysis of 108 sample replicates showed that missing genotypes occurred at 0.165% of all loci, and that allelic drop-outs and drop-ins each occurred at frequencies of 0.022% and 0.032%, respectively. Population-specific genotype profile probabilities for Pacific Northwest ecoregions were lower than 1.9×10−33; profile probabilities are higher (less powerful) in Californian trees due to lower variability across these SNP loci. Conversely, the geographic origin of trees from Southern Californian ecoregions could be predicted with lower error (8.5–11.7%) than trees from the Pacific Northwest (36–80%), based on random forest classification. This study provides a rich database for forensic individualization of bigleaf maple across its complete range in western North America.

Species delimitation in Pinaceae is often challenged by limited morphological differentiation and introgression. In Tsuga (hemlocks), species delimitation has been most challenging among northeastern Asian taxa, where the species are weakly marked morphologically and range in number from three to five in previous studies. Two low-copy nuclear four-coumarase-ligase (4CL) genes andmorphology strongly support a clade of the Japanese endemic T. diversifolia and T. sieboldii from Japan and Ulleung island (Ulleungdo) in Korea. This clade is here referred to as the oceanic hemlocks. 4CL strongly supports a sister-group relationship of the widespread northeastern Asian T. chinensis and eastern North American T. caroliniana. In contrast, chloroplast genomes, which are markedly reduced in Tsuga and relatives, strongly support Japanese T. sieboldii as sister to T. chinensis and moderately support T. caroliniana as sister to a clade of T. diversifolia and hemlocks fromUlleungdo. These divergent topologies suggest chloroplast capture of T. chinensis by Japanese T. sieboldii. Ulleungdo hemlocks are distinct from other northeastern Asian species in leaf and cone morphology and phenologically in common-garden observations. We therefore describe these hemlocks as a new species, T. ulleungensis.

Owing to high rates of introgressive hybridization, the plastid genome is poorly suited to fine-scale DNA barcoding and phylogenetic studies of the oak genus (Quercus, Fagaceae). At the tips of the oak plastome phylogeny, recent gene migration and reticulation generally cause topology to reflect geographic structure, while deeper branches reflect lineage divergence. In this study, we quantify the simple and partial effects of geographic proximity and nucleome-inferred phylogenetic history on oak plastome phylogeny at different evolutionary scales. Our study compares pairwise phylogenetic distances based on complete plastome sequences, pairwise phylogenetic distances from nuclear restriction site-associated DNA sequences (RADseq), and pairwise geographic distances for 34 individuals of the white oak clade representing 24 North American and Eurasian species. Within the North American white oak clade alone, phylogenetic history has essentially no effect on plastome variation, while geography explains 11%–21% of plastome phylogenetic variance. However, across multiple continents and clades, phylogeny predicts 30%–41% of plastome variation, geography 3%–41%. Tipwise attenuation of phylogenetic informativeness in the plastome means that in practical terms, plastome data has little use in solving phylogenetic questions, but can still be a useful barcoding or phylogenetic marker for resolving questions among major clades.

Milkweeds (Asclepias) are used in wide-ranging studies including floral development, pollination biology, plant-insect interactions and co-evolution, secondary metabolite chemistry, and rapid diversification. We present a transcriptome and draft nuclear genome assembly of the common milkweed, Asclepias syriaca. This reconstruction of the nuclear genome is augmented by linkage group information, adding to existing chloroplast and mitochondrial genomic resources for this member of the Apocynaceae subfamily Asclepiadoideae. The genome was sequenced to 80.4× depth and the draft assembly contains 54,266 scaffolds ≥1 kbp, with N50 = 3415 bp, representing 37% (156.6 Mbp) of the estimated 420 Mbp genome. A total of 14,474 protein-coding genes were identified based on transcript evidence, closely related proteins, and ab initio models, and 95% of genes were annotated. A large proportion of gene space is represented in the assembly, with 96.7% of Asclepias transcripts, 88.4% of transcripts from the related genus Calotropis, and 90.6% of proteins from Coffea mapping to the assembly. Scaffolds covering 75 Mbp of the Asclepias assembly formed eleven linkage groups. Comparisons of these groups with pseudochromosomes in Coffea found that six chromosomes show consistent stability in gene content, while one may have a long history of fragmentation and rearrangement. The progesterone 5β-reductase gene family, a key component of cardenolide production, is likely reduced in Asclepias relative to other Apocynaceae. The genome and transcriptome of common milkweed provide a rich resource for future studies of the ecology and evolution of a charismatic plant family.

During allopolyploid formation two 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 many 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 strictly 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, effectively separates homoeologues from each other and from more divergent cross-hybridizing sequences (paralogues). Each homoeologue is thereby confidently isolated from diflferent pools of size-fractionated genomic DNA. While these methods were specifically designed for isolating orthologous duplicated 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 yield strictly orthologous sequences, which are required for phylogenetic analysis. Thus, a general approach is detailed for the isolation of nuclear genes for phylogeny reconstruction.

Phylogenetics benefits from using a large number of putatively independent nuclear loci and their combination with other sources of information, such as the plastid and mitochondrial genomes. To facilitate the selection of orthologous low-copy nuclear (LCN) loci for phylogenetics in nonmodel organisms, we created an automated and interactive script to select hundreds of LCN loci by a comparison between transcriptome and genome skim data. We used our script to obtain LCN genes for southern African Oxalis (Oxalidaceae), a speciose plant lineage in the Greater Cape Floristic Region. This resulted in 1164 LCN genes greater than 600 bp. Using target enrichment combined with genome skimming (Hyb-Seq), we obtained on average 1141 LCN loci, nearly the whole plastid genome and the nrDNA cistron from 23 southern African Oxalis species. Despite a wide range of gene trees, the phylogeny based on the LCN genes was very robust, as retrieved through various gene and species tree reconstruction methods as well as concatenation. Cytonuclear discordance was strong. This indicates that organellar phylogenies alone are unlikely to represent the species tree and stresses the utility of Hyb-Seq in phylogenetics.

Premise of the study Leaf surface traits, such as trichome density and wax production, mediate important ecological processes such as anti-herbivory defense and water-use efficiency. We present a phylogenetic analysis of Asclepias plastomes as a framework for analyzing the evolution of trichome density and presence of epicuticular waxes. Methods We produced a maximum-likelihood phylogeny using plastomes of 103 species of Asclepias. We reconstructed ancestral states and used model comparisons in a likelihood framework to analyze character evolution across Asclepias. Key results We resolved the backbone of Asclepias, placing the Sonoran Desert clade and Incarnatae clade as successive sisters to the remaining species. We present novel findings about leaf surface evolution of Asclepias-the ancestor is reconstructed as waxless and sparsely hairy, a macroevolutionary optimal trichome density is supported, and the rate of evolution of trichome density has accelerated. Conclusions Increased sampling and selection of best-fitting models of evolution provide more resolved and robust estimates of phylogeny and character evolution than obtained in previous studies. Evolutionary inferences are more sensitive to character coding than model selection.

Identifying units of conservation of aquatic species is fundamental to informed natural resources science and management. We used a combination of mitochondrial and nuclear molecular methods to identify potential units of conservation of westslope cutthroat trout, a taxon native to montane river basins of the northwestern U.S. and southwestern Canada. Mitogenomic sequencing identified two major lineages composed of nine monophyletic clades, and a well-supported subclade within one of these, largely delineated by river basins. Analyses of microsatellites and single nucleotide polymorphisms corroborated most of these groupings, sometimes with less resolution but demonstrating more complex connections among clades. The mitochondrial and nuclear analyses revealed that Pleistocene glacial cycles profoundly influenced the distribution and divergence of westslope cutthroat trout, that this taxon crossed the Continental Divide in two separate events, and that genetically pure but nonindigenous fish were widely distributed. Herein, we recognize nine geographically discrete, cytonuclear lineages largely circumscribed by major river basins as potential units of conservation: 1) John Day; 2) Coeur d’Alene; 3) St. Joe; 4) North Fork Clearwater; 5) Salmon; 6) Clearwater headwaters; 7) Clearwater-eastern Cascades; 8) neoboreal, consisting of most of the Columbia upstream from central Washington, the Fraser in British Columbia, and the South Saskatchewan in Alberta; and 9) Missouri.

BackgroundPerennial growth in plants is the product of interdependent cycles of daily and annual stimuli that induce cycles of growth and dormancy. In conifers, needles are the key perennial organ that integrates daily and seasonal signals from light, temperature, and water availability. To understand the relationship between seasonal rhythms and seasonal gene expression responses in conifers, we examined diurnal and circannual needle mRNA accumulation in Douglas-fir (Pseudotsuga menziesii) needles at diurnal and circannual scales. Using mRNA sequencing, we sampled 6.1×109 microreads from 19 trees and constructed a de novo pan-transcriptome reference that includes 173,882 tree-derived transcripts. Using this reference, we mapped RNA-Seq reads from 179 samples that capture daily, seasonal, and annual variation.ResultsWe identified 12,042 diurnally-cyclic transcripts, 9,299 of which showed homology to annotated genes from other plant genomes, including angiosperm core clock genes. Annual analysis revealed 21,225 an-nually-cyclic transcripts, 17,335 of which showed homology to annotated genes from other plant genomes. The timing of maximum gene expression is associated with light quality at diurnal and photoperiod at annual scales, with two-thirds of transcripts reaching maximum expression +/− 2 hours from sunrise and sunset, and half reaching maximum expression +/− 20 days from winter and summer solstices. Comparison to published microarray-based gene expression studies in spruce (Picea) show that the rank order of expression for 760 putatively orthologous genes was significantly preserved, highlighting the generality of our findings.ConclusionsThis finding highlights the extensive annual and seasonal transcriptome variability demonstrated in conifer needles. At these temporal scales, 29% of expressed transcripts showed a significant diurnal rhythm, and 58.7% showed a significant circannual rhythm. Remarkably, thousands of genes reach their annual peak activity during winter dormancy, a time of metabolic stasis. Photoperiod appears to be a dominant driver of annual transcription patterns in Douglas-fir, and these results may be general for predicting rhythmic transcription patterns in emerging gymnosperm models.

Horizontal gene transfer (HGT) of DNA from the plastid to the nuclear and mitochondrial genomes of higher plants is a common phenomenon; however, plastid genomes (plastomes) are highly conserved and have generally been regarded as impervious to HGT. We sequenced the 158 kb plastome and the 690 kb mitochondrial genome of common milkweed (Asclepias syriaca [Apocynaceae]) and found evidence of intracellular HGT for a 2.4-kb segment of mitochondrial DNA to the rps2-rpoC2 intergenic spacer of the plastome. The transferred region contains an rpl2 pseudogene and is flanked by plastid sequence in the mitochondrial genome, including an rpoC2 pseudogene, which likely provided the mechanism for HGT back to the plastome through double-strand break repair involving homologous recombination. The plastome insertion is restricted to tribe Asclepiadeae of subfamily Asclepiadoideae, whereas the mitochondrial rpoC2 pseudogene is present throughout the subfamily, which confirms that the plastid to mitochondrial HGT event preceded the HGT to the plastome. Although the plastome insertion has been maintained in all lineages of Asclepiadoideae, it shows minimal evidence of transcription in A. syriaca and is likely nonfunctional. Furthermore, we found recent gene conversion of the mitochondrial rpoC2 pseudogene in Asclepias by the plastid gene, which reflects continued interaction of these genomes.

Summary Armillaria species display diverse ecological roles ranging from beneficial saprobe to virulent pathogen. Armillaria solidipes (formerly A. ostoyae), a causal agent of Armillaria root disease, is a virulent primary pathogen with a broad host range of woody plants across the Northern Hemisphere. This white-rot pathogen grows between trees as rhizomorphs and attacks sapwood as mycelial fans under the bark. Armillaria root disease is responsible for reduced forest productivity due to direct tree mortality and non-lethal infections that impact growth. Here, we characterize a transcriptome of a widespread, virulent genet (vegetative clone) of A. solidipes isolated from a mycelial fan on a natural grand fi r( Abies grandis) sapling in northern Idaho, USA. cDNA from polyA + -purified total RNA was sequenced using a singleend read approach on the Illumina GAIIx platform which generated 24 170 384 reads. A BLASTx search against the NCBI nr database using 39 943 de novo assembled contigs resulted in 24 442 sequences with significant hits (e-value < 1e 3 ), predominantly to fungi (85%). A filtered data set of 20 882 assembled transcripts that encoded putative homologous fungal proteins was created and used for all subsequent analyses. Signal P identified 10 668 putative signal peptides from these fungal transcripts, and 14 360 were annotated with gene ontology terms. Several sequences showed strong homology to annotated genes with functions in pathogenesis, specifically those involved in plant cell wall degradation and response to the post-infection host environment. This transcriptome contributes to the growing body of resources for studies on fungal pathogens of woody plants, and our results provide useful insights towards identifying specific genes with potential roles associated with pathogenesis and other metabolic functions.

Whitebark pine (Pinus albicaulis) inhabits an expansive range in western North America, and it is a keystone species of subalpine environments. Whitebark is susceptible to multiple threats – climate change, white pine blister rust, mountain pine beetle, and fire exclusion – and it is suffering significant mortality range-wide, prompting the tree to be listed as ‘globally endangered’ by the International Union for Conservation of Nature (IUCN) and ‘endangered’ by the Canadian government. Conservation collections (in situ and ex situ) are being initiated to preserve the genetic legacy of the species. Reliable, transferrable, and highly variable genetic markers are essential for quantifying the genetic profiles of seed collections relative to natural stands, and ensuring the completeness of conservation collections. We evaluated the use of hybridization-based target capture to enrich specific genomic regions from the 30+ GB genome of whitebark pine, and to evaluate genetic variation across loci, trees, and geography. Probes were designed to capture 7,849 distinct genes, and screening was performed on 48 trees. Despite the inclusion of repetitive elements in the probe pool, the resulting dataset provided information on 4,452 genes and 32% of targeted positions (528,873 bp), and we were able to identify 12,390 segregating sites from 47 trees. Variations reveal strong geographic trends in heterozygosity and allelic richness, with trees from the southern Cascade and Sierra Range showing the greatest distinctiveness and differentiation. Our results show that even under non-optimal conditions (low enrichment efficiency; inclusion of repetitive elements in baits), targeted enrichment produces high quality, codominant genotypes from large genomes. The resulting data can be readily integrated into management and gene conservation activities for whitebark pine, and have the potential to be applied to other members of 5-needle pine group (Pinus subsect. Quinquefoliae) due to their limited genetic divergence.

Native plant restoration policy calls for use of “genetically appropriate” native plant material on USDI Bureau of Land Management (BLM) and USDA Forest Service (USFS) lands. In this article, we summarize experimental evidence showing that local adaptation is widespread in all kingdoms of life, and how this “home-field advantage” has been exploited in forest restoration activities to develop and promote regionally adapted, genetically diverse restoration materials. The importance of such materials is highlighted in the context of changing environmental conditions, such as those predicted in future climate change scenarios. Although the adaptive properties of most restoration species remains unexamined, numerous tools exist for identifying similar environments and ecosystems, and these can be used to develop first-generation seed zones. Finally, general recommendations for establishing foundation populations are outlined so that pre-adapted populations retain sufficient genetic diversity to acclimate to new evolutionary challenges, while minimizing the likelihood of deleterious genetic outcomes (for example, inadvertent selection of undesirable traits; inbreeding depression).

A method to sequence multiple chloroplast genomes using ultra high throughput sequencing technologies was recently described. Complete chloroplast genome sequences can resolve phylogenetic relationships at low taxonomic levels and identify informative point mutations and indels. The objective of this research was to sequence multiple Fragaria chloroplast genomes using the Illumina Genome Analyzer. Sixty-three PCR fragments from 22 species were sequenced in four multiplex sequencing runs. Plastome sequences were assembled using a combination of de novo and reference guided assembly with F. vesca 'Hawaii 4' providing the reference. De novo assembly resulted in plastome coverage of 43-74%, and reference guided assembly increased the plastome coverage to 76-82%. The alignment of sequenced chloroplast genomes was 96,438 bp and contained 319 parsimony-informative sites which will be useful in identifying chloroplast genome regions of high sequence variation for testing evolutionary relationships.

Massively parallel sequencing technologies (MPST) offer unprecedented opportunities for novel sequencing projects. MPST, while offering tremendous sequencing capacity, are typically most effective in resequencing projects (as opposed to the sequencing of novel genomes) due to the fact that sequence is returned in relatively short reads. Nonetheless, there is great interest in applying MPST to genome sequencing in non-model organisms. We have developed a bioinformatics pipeline to assemble short read sequence data into nearly complete chloroplast genomes using a combination of de novo and reference-guided assembly, while decreasing reliance on a reference genome. Initially, short read sequences are assembled into larger contigs using de novo assembly. De novo contigs are then aligned to the corresponding reference genome of the most closely related taxon available and merged to form a consensus sequence. The consensus sequence and reference are in turn 'merged' such that aligned de novo sequence remains unaffected while missing sequence is filled in using the reference sequence. This chimeric reference is then utilized in reference-guided assembly to align the original short read data, resulting in a draft plastome. Using two established Pinus reference plastomes, our method has been effective in the assembly of 33 chloroplast genomes within the genus Pinus, and results with four species representing other genera of Pinaceae suggest the method will be of general use in land plants, particularly once limitations of PCR-based chloroplast enrichment are overcome.

Critical to conservation efforts and other investigations at low taxonomic levels, DNA sequence data offer important insights into the distinctiveness, biogeographic partitioning and evolutionary histories of species. The resolving power of DNA sequences is often limited by insufficient variability at the intraspecific level. This is particularly true of studies involving plant organelles, as the conservative mutation rate of chloroplasts and mitochondria makes it difficult to detect polymorphisms necessary to track genealogical relationships among individuals, populations and closely related taxa, through space and time. Massively parallel sequencing (MPS) makes it possible to acquire entire organelle genome sequences to identify cryptic variation that would be difficult to detect otherwise. We are using MPS to evaluate intraspecific chloroplast-level divergence across biogeographic boundaries in narrowly endemic and widespread species of Pinus. We focus on one of the world's rarest pines - Torrey pine (Pinus torreyana) - due to its conservation interest and because it provides a marked contrast to more widespread pine species. Detailed analysis of nearly 90% ( approximately 105 000 bp each) of these chloroplast genomes shows that mainland and island populations of Torrey pine differ at five sites in their plastome, with the differences fixed between populations. This is an exceptionally low level of divergence (1 polymorphism/ approximately 21 kb), yet it is comparable to intraspecific divergence present in widespread pine species and species complexes. Population-level organelle genome sequencing offers new vistas into the timing and magnitude of divergence within species, and is certain to provide greater insight into pollen dispersal, migration patterns and evolutionary dynamics in plants.

Interspecific gene flow via hybridization may play a major role in evolution by creating reticulate rather than hierarchical lineages in plant species. Occasional diploid pine hybrids indicate the potential for introgression, but reticulation is hard to detect because ancestral polymorphism is still shared across many groups of pine species. Nucleotide sequences for 53 accessions from 17 species in subsection Ponderosae (Pinus) provide evidence for reticulate evolution. Two discordant patterns among independent low-copy nuclear gene trees and a chloroplast haplotype are better explained by introgression than incomplete lineage sorting or other causes of incongruence. Conflicting resolution of three monophyletic Pinus coulteri accessions is best explained by ancient introgression followed by a genetic bottleneck. More recent hybridization transferred a chloroplast from P. jeffreyi to a sympatric P. washoensis individual. We conclude that incomplete lineage sorting could account for other examples of non-monophyly, and caution against any analysis based on single-accession or single-locus sampling in Pinus.

Antelope bitterbrush ( Purshia tridentata Pursh DC; Rosaceae) is an arid-land shrub that occupies an important ecological niche in various fire-dominated communities across much of the western United States. Because of its importance as a browse for large mammals and a food source for granivores, P. tridentata is frequently planted by Federal agencies in arid-land revegetation. We are currently analyzing the range-wide genetic diversity of this species as part of a larger effort to develop seed movement guidelines. In this study, we describe the development of eight novel nuclear microsatellite loci and characterize the amount and apportionment of range-wide nuclear genetic diversity. The eight microsatellite loci exhibited a high level of polymorphism (13–33 alleles per locus) and in general, observed levels of heterozygosity did not deviate form Hardy–Weinberg expectations. An initial screen of 196 individuals from 12 widely distributed populations revealed a moderate amount of genetic differentiation ( F ST = 0.09, p Purshia species. These results demonstrate the utility of these markers and provide useful measures of neutral genetic diversity and population differentiation.

To detect and avoid illegal logging of valuable tree species, identification methods for the origin of timber are necessary. We used next-generation sequencing to identify chloroplast genome regions that differentiate the origin of white oaks from the three continents; Asia, Europe, and North America. By using the chloroplast genome of Asian Q. mongolica as a reference, we identified 861 variant sites (672 single nucleotide polymorphisms (SNPs); 189 insertion/deletion (indel) polymorphism) from representative species of three continents (Q. mongolica from Asia; Q. petraea and Q. robur from Europe; Q. alba from North America), and we identified additional chloroplast polymorphisms in pools of 20 individuals each from Q. mongolica (789 variant sites) and Q. robur (346 variant sites). Genome sequences were screened for insertion/deletion (indel) polymorphisms to develop markers that identify continental origin of oak species, and that can be easily evaluated using a variety of detection methods. We identified five indel and one SNP that reliably identify continent-of-origin, based on evaluations of up to 1078 individuals representing 13 white oak species and three continents. Due to the size of length polymorphisms revealed, this marker set can be visualized using capillary electrophoresis or high resolution gel (acrylamide or agarose) electrophoresis. With these markers, we provide the wood trading market with an instrument to comply with the U.S. and European laws that require timber companies to avoid the trade of illegally harvested timber.

Phylogenetic relationships among Pinus species from subgenus Strobus remain unresolved despite combined efforts based on nrFTS and cpDNA. To provide greater resolution among these taxa, a 900-bp intron from a late embryogenesis abundant (LEA)-like gene (IFG8612) was sequenced from 39 pine species, with two or more alleles representing 33 species. Nineteen of 33 species exhibited allelic nonmonphyly in the strict consensus tree, and 10 deviated significantly from allelic monophyly based on topology incongruence tests. Intraspecific nucleotide diversity ranged from 0.0 to 0.0211, and analysis of variance shows that nucleotide diversity was strongly associated (P < 0.0001) with the degree of species monophyly. Although species nonmonophyly complicates phylogenetic interpretations, this nuclear locus offers greater topological support than previously observed for cpDNA or nrlTS. Lacking evidence for hybridization, recombination, or imperfect taxonomy, we feel that incomplete lineage sorting remains the best explanation for the polymorphisms shared among species. Depending on the species, coalescent expectations indicate that reciprocal monophyly will be more likely than paraphyly in 1.71 to 24.0 x 10 6 years, and that complete genome-wide coalescence in these species may require up to 76.3 x 10 6 years. The absence of allelic coalescence is a severe constraint in the application of phylogenetic methods in Pinus, and taxa sharing similar life history traits with Pinus are likely to show species nonmonophyly using nuclear markers. (Lineage sorting; monophyly; nonmonophyly; nuclear genes; pinaceae; Pinus; phylogeny.) Whenever a phylogenetic study uses a single individ- ual to represent a species, an implicit assumption is made that the species is monophyletic (Funk and Omland, 2003; Shaw and Small, 2005). To the extent that complicat- ing factors (e.g., reticulation) are rare in the divergence history of terminal taxa, this assumption offers a con- venient simplification for sampling. However, sampling a single individual per species provides no opportunity to test the hypothesis of allelic monophyly (coalescence) within species. In molecular phylogenetics, this issue can become acute, because gene trees are used to infer or- ganismal phylogenies. These gene trees are subject to processes that can result in the nonmonophyly of se- quences sampled from a single species (lineage sorting, reticulate evolution; Nei, 1987; Wendel and Doyle, 1998). Errors in phylogenetic estimation can occur when gene tree/species tree incongruence exists, but species sam- pling is insufficient to detect the responsible phenomena. Many species remain poorly known, and the presence of cryptic taxa or an inadequate taxonomic treatment can be recognized when nonmonophyletic species are dis- covered in a phylogenetic analysis.

16 pages, 3 figures, 2 tables.-- Full-text version available Open Access at: http://www.rjb.csic.es/jardinbotanico/ficheros/documentos/pdf/pubinv/IAF/19.pdf American diploid cottons (Gossypium L., subgenus Houzingenia Fryxell) form a monophyletic group of 13 species distributed mainly in western Mexico, extending into Arizona, Baja California, and with one disjunct species each in the Galapagos Islands and Peru. Prior phylogenetic analyses based on an alcohol dehydrogenase gene (AdhA) and nuclear ribosomal DNA indicated the need for additional data from other molecular markers to resolve phylogenetic relationships within this subgenus. Toward this end, we sequenced three nuclear genes, the anonymous locus A1341, an alcohol dehydrogenase gene (AdhC), and a cellulose synthase gene (CesA1b). Independent and combined analyses resolved clades that are congruent with current taxonomy and previous phylogenies. Our analyses diagnose at least two long distance dispersal events from the Mexican mainland to Baja California, following a rapid radiation of the primary lineages early in the diversification of the subgenus. Molecular data support the proposed recognition of a new species closely related to Gossypium laxum that was recently collected in Mexico. Research support was provided by the National Science Foundation and by the Spanish Ministry of Science, Education and Sports.

Seed movement guidelines for restoration activities are lacking for most native grasses, forbs, and shrubs. The forestry community has decades of experience in establishing seed zones and seed movement guidelines that may be of value to restoration managers. We review the history of seed zone development in forest trees, with emphasis on the Pacific Northwest, and make some suggestions concerning seed transfer guidelines for other native plants.

Summary It has long been recognized that interspecific hybridization is common in plants, enhancing processes of diversification and speciation. With the widespread utilization of molecular tools, interspecific hybridization – as revealed through incongruence among two or more phylogenetic data sets – is now inferred to be even more prevalent than indicated by morphological and cytogenetic evidence. Using Gossypium as an example, we show how multiple molecular markers have implicated a high frequency of historical hybridization between lineages whose modern descendants are strongly isolated by geography and intrinsic genetic barriers. For example, transoceanic dispersal of propagules from Africa to the New World led to the creation of a novel allotetraploid lineage, as well as the introgression of African repetitive elements into a Mexican diploid species. By mechanisms that remain obscure, fully one-quarter of modern Gossypium species appear to have experienced historical interspecific cytoplasmic and possibly nuclear introgression. These remarkable observations of interspecific genetic exchange emerge from a genus for which such contact would appear improbable, implying that historical hybridization is a more creative force than suspected in angiosperm evolution.

The Mexican cotton Gossypium gossypioides is a perplexing entity, with conflicting morphological, cytogenetic, and molecular evidence of its phylogenetic affinity to other American cottons. We reevaluated the evolutionary history of this enigmatic species using 16.4 kb of DNA sequence. Phylogenetic analyses show that chloroplast DNA (7.3 kb), nuclear ribosomal internal transcribed spacers (ITS; 0.69 kb), and unique nuclear genes (8.4 kb) yield conflicting resolutions for G. gossypioides. Eight low-copy nuclear genes provide a nearly unanimous resolution of G. gossypioides as the basalmost American diploid cotton, whereas cpDNA sequences resolve G. gossypioides deeply nested within the American diploid clade sister to Peruvian G. raimondii, and ITS places G. gossypioides in an African (rather than an American) clade. These data, in conjunction with previous evidence from the repetitive fraction of the genome, implicate a complex history for G. gossypioides possibly involving temporally separated introgression events from genetically divergent cottons that are presently restricted to different hemispheres. Based on repetitive nuclear DNA, it appears that G. gossypioides experienced nuclear introgression from an African species shortly after divergence from the remainder of the American assemblage. More recently, hybridization with a Mexican species may have resulted in cpDNA introgression, and possibly a second round of cryptic nuclear introgression. Gossypium gossypioides provides a striking example of the previously unsuspected chimeric nature of some plant genomes and the resulting phylogenetic complexity produced by multiple historical reticulation events.

Repeated rounds of polyploidy have been commonplace in the lineages leading to modern eukaryotic genomes, giving rise to widespread gene duplication. Genes duplicated by polyploidy, or “homoeologs,” may continue to be expressed at equal levels following polyploidization or their expression may be dramatically altered. In this report, we describe how SSCP analysis of RT-PCR products can be used to evaluate the expression status (presence and relative quantity) of highly similar homoeologous gene pairs from an allotetraploid genome. This cDNA-SSCP approach was used to evaluate transcript abundance in “synthetic tetraploid” mRNA pools (i.e., mixtures of diploid products) and three natural homoeologous gene pairs expressed in tetraploid cotton (Gossypium hirsutum) ovules. Results from replicated tests show that cDNA-SSCP reliably separates duplicated transcripts with 99% sequence identity. Most significantly, the method yields quantitative estimates of transcript ratios in template pools that range from equimolar to approximately 100:1.

Most eukaryotes have genomes that exhibit high levels of gene redundancy, much of which seems to have arisen from one or more cycles of genome doubling. Polyploidy has been particularly prominent during flowering plant evolution, yielding duplicated genes (homoeologs) whose expression may be retained or lost either as an immediate consequence of polyploidization or on an evolutionary timescale. Expression of 40 homoeologous gene pairs was assayed by cDNA-single-stranded conformation polymorphism in natural (1- to 2-million-yr-old) and synthetic tetraploid cotton ( Gossypium ) to determine whether homoeologous gene pairs are expressed at equal levels after polyploid formation. Silencing or unequal expression of one homoeolog was documented for 10 of 40 genes examined in ovules of Gossypium hirsutum. Assays of homoeolog expression in 10 organs revealed variable expression levels and silencing, depending on the gene and organ examined. Remarkably, silencing and biased expression of some gene pairs are reciprocal and developmentally regulated, with one homoeolog showing silencing in some organs and the other being silenced in other organs, suggesting rapid subfunctionalization. Duplicate gene expression was examined in additional natural polyploids to characterize the pace at which expression alteration evolves. Analysis of a synthetic tetraploid revealed homoeolog expression and silencing patterns that sometimes mirrored those of the natural tetraploid. Both long-term and immediate responses to polyploidization were implicated. Data suggest that some silencing events are epigenetically induced during the allopolyploidization process.

Forest health issues are on the rise in the United States, resulting from introduction of alien pests and diseases, coupled with abiotic stresses related to climate change. Increasingly, forest scientists are finding genetic/genomic resources valuable in addressing forest health issues. For a set of ten ecologically and economically important native hardwood tree species representing a broad phylogenetic spectrum, we used low coverage whole genome sequencing from multiplex Illumina paired ends to economically profile their genomic content. For six species, the genome content was further analyzed by flow cytometry in order to determine the nuclear genome size. Sequencing yielded a depth of 0.8X to 7.5X, from which in silico analysis yielded preliminary estimates of gene and repetitive sequence content in the genome for each species. Thousands of genomic SSRs were identified, with a clear predisposition toward dinucleotide repeats and AT-rich repeat motifs. Flanking primers were designed for SSR loci for all ten species, ranging from 891 loci in sugar maple to 18,167 in redbay. In summary, we have demonstrated that useful preliminary genome information including repeat content, gene content and useful SSR markers can be obtained at low cost and time input from a single lane of Illumina multiplex sequence.

It has long been known that genomes vary over a remarkable range of sizes in both plants (Bennett, Cox, and Leitch 1997) and animals (Gregory 2001). It also has become evident that across the broad phylogenetic sweep, genome size may be correlated with intron size (Deutsch and Long 1999; Vinogradov 1999; McLysaght et al. 2000), suggesting that some component of genome size evolution takes place within genes. Examples include humans and pufferfish (Fugu), where comparisons of 199 introns in 22 orthologous genes showed that introns in Fugu were on average eight times as small as those in humans, consistent with their ratio of genome sizes (McLysaght et al. 2000). Similarly, Deutsch and Long (1999) tabulated intron sizes across a broad phylogenetic spectrum of eukaryotes and noted a general but weak correlation with genome size, with humans having the most and longest introns (mean of 3.4 kbp) among the 10 taxa studied. Intron size is also correlated with genome size in Drosophila (Moriyama, Petrov, and Hartl 1998), showing that the correlation may extend to more recent divergences. At present there is little information on the correlation between genome and intron sizes in plants, although there are suggestions that plants with small genomes have smaller introns (Deutsch and Long 1999; Vinogradov 1999). Whereas broad comparisons across widely divergent taxa are now possible given completed draft sequences for the rice (Goff et al. 2002; Yu et al. 2002) and Arabidopsis (Arabidopsis Genome Initiative 2000) genomes, the divergence time between Poaceae and Brassicaceae is so large that the influence of genome size on intron size may be confounded by numerous other, unstudied covariables. It seems likely that more informative studies will involve closely related taxa that vary significantly in genome size but which share recent evolutionary history and a broad suite of life-history features. An additional advantage of comparing close relatives is that orthology among genes, and hence introns, may be more readily established. This latter point may be especially important, given the relative lability of copy-number for many gene families (Small and Wendel 2000). To exemplify this approach, we studied the relationship between intron size and genome size for or