Ultraconserved elements anchor thousands of genetic markers spanning multiple evolutionary timescales

Although massively parallel sequencing has facilitated large-scale DNA sequencing, comparisons among distantly related species rely upon small portions of the genome that are easily aligned. Methods are needed to efficiently obtain comparable DNA fragments prior to massively parallel sequencing, particularly for biologists working with non-model organisms. We introduce a new class of molecular marker, anchored by ultraconserved genomic elements (UCEs), that universally enable target enrichment and sequencing of thousands of orthologous loci across species separated by hundreds of millions of years of evolution. Our analyses here focus on use of UCE markers in Amniota because UCEs and phylogenetic relationships are well-known in some amniotes. We perform an in silico experiment to demonstrate that sequence flanking 2030 UCEs contains information sufficient to enable unambiguous recovery of the established primate phylogeny. We extend this experiment by performing an in vitro enrichment of 2386 UCE-anchored loci from nine, non-model avian species. We then use alignments of 854 of these loci to unambiguously recover the established evolutionary relationships within and among three ancient bird lineages. Because many organismal lineages have UCEs, this type of genetic marker and the analytical framework we outline can be applied across the tree of life, potentially reshaping our understanding of phylogeny at many taxonomic levels.     

Solution-based targeted genomic enrichment for precious DNA samples

ABSTRACT: BACKGROUND: Solution-based targeted genomic enrichment (TGE) protocols permit selective sequencing of genomic regions of interest on a massively parallel scale. These protocols could be improved by: 1) modifying or eliminating time consuming steps; 2) increasing yield to reduce input DNA and excessive PCR cycling; and 3) enhancing reproducible. RESULTS: We developed a solution-based TGE method for downstream Illumina sequencing in a non-automated workflow, adding standard Illumina barcode indexes during the post-hybridization amplification to allow for sample pooling prior to sequencing. The method utilizes Agilent SureSelect baits, primers and hybridization reagents for the capture, off-the-shelf reagents for the library preparation steps, and adaptor oligonucleotides for Illumina paired-end sequencing purchased directly from an oligonucleotide manufacturing company. CONCLUSIONS: This solution-based TGE method for Illumina sequencing is optimized for small- or medium-sized laboratories and addresses the weaknesses of standard protocols by reducing the amount of input DNA required, increasing capture yield, optimizing efficiency, and improving reproducibility.     

Utility of plastid psaB gene sequences for investigating intrafamilial relationships within Orchidaceae

DNA sequences of the plastid gene psaB were completed for 182 species of Orchidaceae (representing 150 different genera) and outgroup families in Asparagales. These data were analyzed using parsimony, and resulting trees were compared to a rbcL phylogeny of Orchidaceae for the same set of taxa after an additional 30 new rbcL sequences were added to a previously published matrix. The psaB tree topology is similar to the rbcL tree, although the psaB data contain less homoplasy and provide greater bootstrap support than rbcL alone. In combination, the two-gene tree recovers the five monophyletic subfamilial clades currently recognized in Orchidaceae, but fails to resolve the positions of Cypripedioideae and Vanilloideae. These new topologies help to clarify some of the anomalous results recovered when rbcL is analyzed alone. Both genes appear to be absent from the plastid genome of several achlorophyllous orchids, but are present in the form of presumably non-functional pseudogenes in Cyrtosia. This study is the first to document the utility of psaB sequences for phylogenetic studies of plants below the family level.     

Origins and introgression of polyploid species in Mentzelia section Trachyphytum (Loasaceae)

? Premise of the study: Polyploid speciation has been important in plant evolution. However, the conditions that favor the origination and persistence of polyploids are still not well understood. Here, we examine origins of 16 polyploid species in Mentzelia section Trachyphytum. ? Methods: We used phylogeny reconstructions based on DNA sequences from plastid regions and the nuclear gene isocitrate dehydrogenase (idh) to construct hypotheses of introgression and polyploidization. ? Key results: Molecular data suggest that homoploid hybridization has been surprisingly common in Trachyphytum. Diploid species had unequal involvement in polyploid origins, but most polyploid taxa had allopolyploid origins from extant progenitors. A few polyploids with extreme phenotypes did not appear to have extant progenitors. We infer that the progenitors of these species were derived from extinct diploid lineages or ancestral lineages of multiple extant diploids. In agreement with other recent studies, we recovered molecular evidence of multiple phylogenetically distinct origins for several polyploid taxa, including the widespread octoploid M. albicaulis. ? Conclusions: Evidence of high levels of introgression and allopolyploidy suggests that hybridization has played an important role in the evolution of Trachyphytum. Although idh sequences exhibited complicated evolution, including gene duplication, deletion, and recombination, they provided a higher percentage of informative characters for phylogeny reconstruction than the most variable plastid regions, allowing tests of hypotheses regarding polyploid origins. Given the necessity for rapidly evolving low-copy nuclear genes, researchers studying hybridization and polyploidy may increasingly turn to complex sequence data.     

Ultra-barcoding in cacao (Theobroma spp.; Malvaceae) using whole chloroplast genomes and nuclear ribosomal DNA

? Premise of study: To reliably identify lineages below the species level such as subspecies or varieties, we propose an extension to DNA-barcoding using next-generation sequencing to produce whole organellar genomes and substantial nuclear ribosomal sequence. Because this method uses much longer versions of the traditional DNA-barcoding loci in the plastid and ribosomal DNA, we call our approach ultra-barcoding (UBC). ? Methods: We used high-throughput next-generation sequencing to scan the genome and generate reliable sequence of high copy number regions. Using this method, we examined whole plastid genomes as well as nearly 6000 bases of nuclear ribosomal DNA sequences for nine genotypes of Theobroma cacao and an individual of the related species T. grandiflorum, as well as an additional publicly available whole plastid genome of T. cacao. ? Key results: All individuals of T. cacao examined were uniquely distinguished, and evidence of reticulation and gene flow was observed. Sequence variation was observed in some of the canonical barcoding regions between species, but other regions of the chloroplast were more variable both within species and between species, as were ribosomal spacers. Furthermore, no single region provides the level of data available using the complete plastid genome and rDNA. ? Conclusions: Our data demonstrate that UBC is a viable, increasingly cost-effective approach for reliably distinguishing varieties and even individual genotypes of T. cacao. This approach shows great promise for applications where very closely related or interbreeding taxa must be distinguished.     

Multiplex sequencing of bacterial artificial chromosomes for assembling complex plant genomes

Hierarchical shotgun sequencing remains the method of choice for assembling high‐quality reference sequences of complex plant genomes. The efficient exploitation of current high‐throughput technologies and powerful computational facilities for large‐insert clone sequencing necessitates the sequencing and assembly of a large number of clones in parallel. We developed a multiplexed pipeline for shotgun sequencing and assembling individual bacterial artificial chromosomes (BACs) using the Illumina sequencing platform. We illustrate our approach by sequencing 668 barley BACs (Hordeum vulgare L.) in a single Illumina HiSeq 2000 lane. Using a newly designed parallelized computational pipeline, we obtained sequence assemblies of individual BACs that consist, on average, of eight sequence scaffolds and represent >98% of the genomic inserts. Our BAC assemblies are clearly superior to a whole‐genome shotgun assembly regarding contiguity, completeness and the representation of the gene space. Our methods may be employed to rapidly obtain high‐quality assemblies of a large number of clones to assemble map‐based reference sequences of plant and animal species with complex genomes by sequencing along a minimum tiling path.     

A comparison of solution and membrane-bound DNA × DNA hybridization,as used to infer phylogeny

A new method of membrane-bound DNA × DNA hybridization was devised to accommodate the study of small quantities of DNA obtained from museum specimens for phylogeny reconstruction. Membranebound, single-stranded target genomic DNAs were competitively hybridized with a total genomic DNA probe to form hybrid duplexes required for the DNA dissociation experiments. We compared the thermal elution profiles derived from dissociating duplexes made with probes of whole genomic, single-copy, and repetitive DNA, as well as solution DNA × DNA hybridization using sc tracer. Quantitatively, pairwise indices of genetic distance derived from duplexes made with genomic probes depended entirely on hybridization of repetitive sequences, but a parallel set of experiments using repetitive and sc probes produced qualitatively similar results. The indices of genetic distance generated by the membrane-bound hybrids form an internally consistent, resolved tree which is in agreement with the solution DNA × DNA hybridization trials and traditional views of the phylogeny of the taxa under study.Correspondence to: P. Houde     

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

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

Evolutionary processes in the genus Coreocarpus: insights from molecular phylogenetics

A molecular phylogenetic study of the plant genus Coreocarpus was conducted using nuclear (ITS) and plastid (rpl16 intron) DNA sequences, with phylogenies of the nuclear and plastid sequences highly congruent in defining a monophyletic group of six species (core Coreocarpus), although three other species often placed within the genus were excluded. Relationships within the genus are largely but not totally concordant with prior biosystematic studies. Despite strong molecular support, no morphological characters uniting the six species of core Coreocarpus have been identified; retention of plesiomorphic characters and the genetic lability of characters are two probable factors contributing to lack of consistent defining characters. The age of the core Coreocarpus is estimated at 1 million years because the basal species is endemic to a volcanic island that emerged in the past million years. Mapping the results of earlier breeding studies on the molecular phylogeny showed that use of cross-compatibility as a criterion for species delimitation would result in the recognition of paraphyletic species. Prior field, morphological, and biosystematic studies provided no indication of past hybridization in the evolution of Coreocarpus, and species in the genus appeared to be well defined morphologically. However, three instances of incongruence were observed. Two of these were between the nuclear and plastid partitions, and the third was between the morphological species assignment of one accession and the molecular data. If hybridization accounts for incongruence between the nuclear and plastid data, it occurred between species that now appear to be cross-incompatible and allopatric. The incongruence between morphological species assignment and the molecular data could be the result of parallel fixation of characters that have a simple genetic basis. This study suggests that the evolutionary history of Coreocarpus is much more complex than indicated from prior biosystematic investigations and that biosystematic and molecular phylogenetic studies may complement each other for elucidating the evolution and phylogeny of a group.     

Whole Mitochondrial and Plastid Genome SNP Analysis of Nine Date Palm Cultivars Reveals Plastid Heteroplasmy and Close Phylogenetic Relationships among Cultivars

Date palm is a very important crop in western Asia and northern Africa, and it is the oldest domesticated fruit tree with archaeological records dating back 5000 years. The huge economic value of this crop has generated considerable interest in breeding programs to enhance production of dates. One of the major limitations of these efforts is the uncertainty regarding the number of date palm cultivars, which are currently based on fruit shape, size, color, and taste. Whole mitochondrial and plastid genome sequences were utilized to examine single nucleotide polymorphisms (SNPs) of date palms to evaluate the efficacy of this approach for molecular characterization of cultivars. Mitochondrial and plastid genomes of nine Saudi Arabian cultivars were sequenced. For each species about 60 million 100 bp paired-end reads were generated from total genomic DNA using the Illumina HiSeq 2000 platform. For each cultivar, sequences were aligned separately to the published date palm plastid and mitochondrial reference genomes, and SNPs were identified. The results identified cultivar-specific SNPs for eight of the nine cultivars. Two previous SNP analyses of mitochondrial and plastid genomes identified substantial intra-cultivar ( = intra-varietal) polymorphisms in organellar genomes but these studies did not properly take into account the fact that nearly half of the plastid genome has been integrated into the mitochondrial genome. Filtering all sequencing reads that mapped to both organellar genomes nearly eliminated mitochondrial heteroplasmy but all plastid SNPs remained heteroplasmic. This investigation provides valuable insights into how to deal with interorganellar DNA transfer in performing SNP analyses from total genomic DNA. The results confirm recent suggestions that plastid heteroplasmy is much more common than previously thought. Finally, low levels of sequence variation in plastid and mitochondrial genomes argue for using nuclear SNPs for molecular characterization of date palm cultivars.     

A Phylogenomic Perspective on the Radiation of Ray-Finned Fishes Based upon Targeted Sequencing of Ultraconserved Elements (UCEs)

Ray-finned fishes constitute the dominant radiation of vertebrates with over 32,000 species. Although molecular phylogenetics has begun to disentangle major evolutionary relationships within this vast section of the Tree of Life, there is no widely available approach for efficiently collecting phylogenomic data within fishes, leaving much of the enormous potential of massively parallel sequencing technologies for resolving major radiations in ray-finned fishes unrealized. Here, we provide a genomic perspective on longstanding questions regarding the diversification of major groups of ray-finned fishes through targeted enrichment of ultraconserved nuclear DNA elements (UCEs) and their flanking sequence. Our workflow efficiently and economically generates data sets that are orders of magnitude larger than those produced by traditional approaches and is well-suited to working with museum specimens. Analysis of the UCE data set recovers a well-supported phylogeny at both shallow and deep time-scales that supports a monophyletic relationship between Amia and Lepisosteus (Holostei) and reveals elopomorphs and then osteoglossomorphs to be the earliest diverging teleost lineages. Our approach additionally reveals that sequence capture of UCE regions and their flanking sequence offers enormous potential for resolving phylogenetic relationships within ray-finned fishes.     

Networks in a Large-Scale Phylogenetic Analysis: Reconstructing Evolutionary History of Asparagales (Lilianae) Based on Four Plastid Genes

Phylogenetic analysis aims to produce a bifurcating tree, which disregards conflicting signals and displays only those that are present in a large proportion of the data. However, any character (or tree) conflict in a dataset allows the exploration of support for various evolutionary hypotheses. Although data-display network approaches exist, biologists cannot easily and routinely use them to compute rooted phylogenetic networks on real datasets containing hundreds of taxa. Here, we constructed an original neighbour-net for a large dataset of Asparagales to highlight the aspects of the resulting network that will be important for interpreting phylogeny. The analyses were largely conducted with new data collected for the same loci as in previous studies, but from different species accessions and greater sampling in many cases than in published analyses. The network tree summarised the majority data pattern in the characters of plastid sequences before tree building, which largely confirmed the currently recognised phylogenetic relationships. Most conflicting signals are at the base of each group along the Asparagales backbone, which helps us to establish the expectancy and advance our understanding of some difficult taxa relationships and their phylogeny. The network method should play a greater role in phylogenetic analyses than it has in the past. To advance the understanding of evolutionary history of the largest order of monocots Asparagales, absolute diversification times were estimated for family-level clades using relaxed molecular clock analyses.     

Time-calibrated phylogeny of the woody Australian genus Hakea (Proteaceae) supports multiple origins of insect-pollination among bird-pollinated ancestors

? Premise of the study: A past study based on morphological data alone showed that the means by which plants of the Australian genus Hakea reduce florivory is related to the evolution of bird pollination. For example, bird pollination was shown to have arisen only in insect-pollinated lineages that already produced greater amounts of floral cyanide, a feature that reduces florivory. We examine a central conclusion of that study, and a common assumption in the literature, that bird pollination arose in insect-pollinated lineages, rather than the reverse. ? Methods: We combined morphological and DNA data to infer the phylogeny and age of the Australian genus Hakea, using 9.2 kilobases of plastid and nuclear DNA and 46 morphological characters from a taxonomically even sampling of 55 of the 149 species. ? Key results: Hakea is rooted confidently in a position that has not been suggested before. The phylogeny implies that bird pollination is primitive in Hakea and that multiple shifts to insect pollination have occurred. The unexpectedly young age of Hakea (a crown age of ca. 10 Ma) makes it coincident with its primary bird pollinators (honeyeaters) throughout its history. ? Conclusions: Our study demonstrates that Hakea is an exception to the more commonly described shift from insect to bird pollination. However, we note that only one previous phylogenetic study involved Australian plants and their honeyeater pollinators and that our finding might prove to be more common on that continent.     

Inferring phylogeny at low taxonomic levels: utility of rapidly evolving cpDNA and nuclear ITS loci

Plant molecular systematic studies of closely related taxa have relied heavily on sequence data from nuclear ITS and cpDNA. Positive attributes of using ITS sequence data include the rapid rate of evolution compared to most plastid loci and availability of universal primers for amplification and sequencing. On the other hand, ITS sequence data may not adequately track organismal phylogeny if concerted evolution and high rDNA array copy number do not permit identification of orthologous copies. Shaw et al. (American Journal of Botany 92: 142-166) recently identified nine plastid regions that appear to provide more potentially informative characters than many other plastid loci. In the present study, sequences of these loci and ITS were obtained for six taxonomic groups in which phylogenetic relationships have been difficult to establish using other data. The relative utility of these regions was compared by assessing the number of parsimony informative characters, character congruence, resolution of inferred trees, clade support, and accuracy. No single locus emerged as the best in all lineages for any of these measures of utility. Results further indicated that in preliminary studies, sampling strategy should include at least four exemplar taxa. The importance of sampling data from independent distributions is also discussed.     

Ultraconserved Elements Sequencing as a Low-Cost Source of Complete Mitochondrial Genomes and Microsatellite Markers in Non-Model Amniotes

Sequence capture of ultraconserved elements (UCEs) associated with massively parallel sequencing has become a common source of nuclear data for studies of animal systematics and phylogeography. However, mitochondrial and microsatellite variation are still commonly used in various kinds of molecular studies, and probably will complement genomic data in years to come. Here we show that besides providing abundant genomic data, UCE sequencing is an excellent source of both sequences for microsatellite loci design and complete mitochondrial genomes with high sequencing depth. Identification of dozens of microsatellite loci and assembly of complete mitogenomes is exemplified here using three species of Poospiza warbling finches from southern and southeastern Brazil. This strategy opens exciting opportunities to simultaneously analyze genome-wide nuclear datasets and traditionally used mtDNA and microsatellite markers in non-model amniotes at no additional cost.     

The complex history of the genus Ilex L. (Aquifoliaceae): evidence from the comparison of plastid and nuclear DNA sequences and from fossil data

 A plastid phylogeny of the genus Ilex based on three different loci (the atpB-rbcL spacer, trnL-trnF and rbcL) is compared with its nuclear phylogeny based on two different loci (the ribosomal ITS and the 5S RNA spacer). These two sets of molecular data are then compared to geographical and temporal data from the fossil record. The plastid phylogeny is strongly correlated with the geographic distribution of extant species. However, the nuclear phylogeny is strongly incongruent with the plastid phylogeny, suggesting frequent interlineage hybridizations. Moreover, the comparison of the ribosomal ITS tree and the 5S RNA spacer tree indicates also possible lineage sorting. Particularly interesting is the finding of two different Ilex lineages in the plastid American clade showing different biogeographic patterns in South America. One of them has a simple North American/South American biogeographical relationship. The other has complex biogeographical relationships, some species showing direct Asian/South American biogeographical relationships. During its history, the genus Ilex probably experienced frequent lineage sorting and interlineage hybridization with subsequent nuclear or cytoplasmic introgression, making the study of its history very complex. Received September 24, 2001; accepted August 19, 2002 Published online: November 28, 2002 Addresses of the authors: Jean-Fran?ois Manen (e-mail: manen@cjb.ville-ge.ch), Yamama Naciri-Graven, Conservatoire et Jardin Botaniques, Impératrice 1, CH-1292 Chambésy/Genève, Switzerland. Michael C. Boulter, Palaeobiology Research Unit, University of East London, Romford Road, London E15 4LZ, UK.     

Using partial genomic fosmid libraries for sequencing complete organellar genomes

Organellar genome sequences provide numerous phylogenetic markers and yield insight into organellar function and molecular evolution. These genomes are much smaller in size than their nuclear counterparts; thus, their complete sequencing is much less expensive than total nuclear genome sequencing, making broader phylogenetic sampling feasible. However; for some organisms, it is challenging to isolate plastid DNA for sequencing using standard methods. To overcome these difficulties, we constructed partial genomic libraries from total DNA preparations of two heterotrophic and two autotrophic angiosperm species using fosmid vectors. We then used macroarray screening to isolate clones containing large fragments of plastid DNA. A minimum tiling path of clones comprising the entire genome sequence of each plastid was selected, and these clones were shotgun-sequenced and assembled into complete genomes. Although this method worked well for both heterotrophic and autotrophic plants, nuclear genome size had a dramatic effect on the proportion of screened clones containing plastid DNA and, consequently, the overall number of clones that must be screened to ensure full plastid genome coverage. This technique makes it possible to determine complete plastid genome sequences for organisms that defy other available organellar genome sequencing methods, especially those for which limited amounts of tissue are available.