Quality and quantity of data recovered from massively parallel sequencing: Examples in Asparagales and Poaceae.

PREMISE OF THE STUDY: Genome survey sequences (GSS) from massively parallel sequencing have potential to provide large, cost-effective data sets for phylogenetic inference, replace single gene or spacer regions as DNA barcodes, and provide a plethora of data for other comparative molecular evolution studies. Here we report on the application of this method to estimating the molecular phylogeny of core Asparagales, investigating plastid gene losses, assembling complete plastid genomes, and determining the type and quality of assembled genomic data attainable from Illumina 80-120-bp reads. METHODS: We sequenced total genomic DNA from samples in two lineages of monocotyledonous plants, Poaceae and Asparagales, on the Illumina platform in a multiplex arrangement. We compared reference-based assemblies to de novo contigs, evaluated consistency of assemblies resulting from use of various references sequences, and assessed our methods to obtain sequence assemblies in nonmodel taxa. KEY RESULTS: Our method returned reliable, robust organellar and nrDNA sequences in a variety of plant lineages. High quality assemblies are not dependent on genome size, amount of plastid present in the total genomic DNA template, or relatedness of available reference sequences for assembly. Phylogenetic results revealed familial and subfamilial relationships within Asparagales with high bootstrap support, although placement of the monotypic genus Aphyllanthes was placed with moderate confidence. CONCLUSIONS: The well-supported molecular phylogeny provides evidence for delineation of subfamilies within core Asparagales. With advances in technology and bioinformatics tools, the use of massively parallel sequencing will continue to become easier and more affordable for phylogenomic and molecular evolutionary biology investigations.

Biodiversity assessment: state-of-the-art techniques in phylogenomics and species identification.

PREMISE OF THE STUDY: Biodiversity assessment is the first step in protecting the complete range of morphological and genetic diversity of species on Earth, and in reaching the goals of conservation biology. Assessment begins with identifying organisms that make up biological communities and understanding evolutionary histories. Scientific advancements in molecular sequencing can help clarify and provide support for identifications. Massively parallel DNA sequencing technologies are being used to sequence complete genomes of model organisms; however, this resource has not been fully used for species identifications. Animal researchers commonly use one mitochondrial region, and groups of plant scientists have proposed numerous combinations of two or three chloroplast markers as genomic identifiers. Yet, nearly as many studies have reported that the proposed regions are uninformative in some plant groups and at various taxonomic levels. METHODS: We propose a combination of whole (or nearly whole) chloroplast genomes, mitochondrial genes, and nuclear repeat regions for both species identifications and phylogenetic analyses, obtained from a simple total DNA extraction and one run on massively parallel DNA sequencing machines. KEY RESULTS: We have recovered both coding and noncoding sequences from multiple genetic sources, providing genomic information for comparisons within and between multiple taxonomic levels. CONCLUSIONS: In combination with morphological and other data, this abundance of genomic information will have a broad range of applications, including not only helping conservation biologists understand ecosystem biodiversity, but also understanding the evolutionary histories of organisms, mending damaged landscapes, and investigating interactions of plants with pollinators and pests.

Molecular systematics of the neotropical genus Psiguria (Cucurbitaceae): Implications for phylogeny and species identification.

Varying morphological features in many groups of tropical vines confound identification, requiring molecular tools for distinguishing species. Confusion is amplified in Psiguria, a small genus found in Central and South America and the Caribbean, because male and female flowers of these monoecious plants are widely separated by time and position on a branch. We present the first phylogeny of Psiguria utilizing a combination of eight chloroplast intergenic spacers, the internal transcribed spacer (ITS) regions of the nuclear ribosomal DNA repeat, and the intron of the low-copy nuclear gene serine/threonine phosphatase, for a total aligned length of 9456 base pairs. Analyses include multiple accessions of all species in the genus. The data support the monophyly of Psiguria and elucidate several species boundaries. Also presented are Psiguria-specific DNA barcodes, which include the chloroplast regions: ndhC-trnV, rps16-trnQ, rpoB-trnC, ndhF-rpl32, and psbZ-trnM. For the first time, systematists, ecologists, and evolutionary biologists will have the tools to confidently identify species of Psiguria with DNA barcodes that may be useful in other genera of Cucurbitaceae.

Phylogenetic utility of 141 low-copy nuclear regions in taxa at different taxonomic levels in two distantly related families of rosids.

Angiosperm systematics has progressed to the point where it is now expected that multiple, independent markers be used in phylogenetic studies. Universal primers for amplifying informative regions of the chloroplast genome are readily available, but in the faster-evolving nuclear genome it is challenging to discover priming sites that are conserved across distantly related taxa. With goals including the identification of informative markers in rosids, and perhaps other angiosperms, we screened 141 nuclear primer combinations for phylogenetic utility in two distinct groups of rosids at different taxonomic levels-Psiguria (Cucurbitaceae) and Geraniaceae. We discovered three phylogenetically informative regions in Psiguria and two in Geraniaceae, but none that were useful in both groups. Extending beyond rosids, we combined our findings with those of another recent effort testing these primer pairs in Asteraceae, Brassicaceae, and Orchidaceae. From this comparison, we identified 32 primer combinations that amplified regions in representative species of at least two of the five distantly related angiosperm families, giving some prior indication about phylogenetic usefulness of these markers in other flowering plants. This reduced set of primer pairs for amplifying low-copy nuclear markers along with a recommended experimental strategy provide a framework for identifying phylogenetically informative regions in angiosperms.