Peter M. Hollingsworth

A DNA barcode for land plants

DNA barcoding involves sequencing a standard region of DNA as a tool for species identification. However, there has been no agreement on which region(s) should be used for barcoding land plants. To provide a community recommendation on a standard plant barcode, we have compared the performance of 7 leading candidate plastid DNA regions (atpF–atpH spacer, matK gene, rbcL gene, rpoB gene, rpoC1 gene, psbK–psbI spacer, and trnH–psbA spacer). Based on assessments of recoverability, sequence quality, and levels of species discrimination, we recommend the 2-locus combination of rbcL+matK as the plant barcode. This core 2-locus barcode will provide a universal framework for the routine use of DNA sequence data to identify specimens and contribute toward the discovery of overlooked species of land plants.

Choosing and using a plant DNA barcode.

The main aim of DNA barcoding is to establish a shared community resource of DNA sequences that can be used for organismal identification and taxonomic clarification. This approach was successfully pioneered in animals using a portion of the cytochrome oxidase 1 (CO1) mitochondrial gene. In plants, establishing a standardized DNA barcoding system has been more challenging. In this paper, we review the process of selecting and refining a plant barcode; evaluate the factors which influence the discriminatory power of the approach; describe some early applications of plant barcoding and summarise major emerging projects; and outline tool development that will be necessary for plant DNA barcoding to advance.

Chloroplast microsatellites: new tools for studies in plant ecology and evolution

The nonrecombinant, uniparentally inherited nature of organelle genomes makes them useful tools for evolutionary studies. However, in plants, detecting useful polymorphism at the population level is often difficult because of the low level of substitutions in the chloroplast genome, and because of the slow substitution rates and intramolecular recombination of mtDNA. Chloroplast microsatellites represent potentially useful markers to circumvent this problem and, to date, studies have demonstrated high levels of intraspecific variability. Here, we discuss the use of these markers in ecological and evolutionary studies of plants, as well as highlighting some of the potential problems associated with such use.

How much effort is required to isolate nuclear microsatellites from plants

The attributes of codominance, reproducibility and high resolution have all contributed towards the current popularity of nuclear microsatellites as genetic markers in molecular ecological studies. One of their major drawbacks, however, is the development phase required to obtain working primers for a given study species. To facilitate project planning, we have reviewed the literature to quantify the workload involved in isolating nuclear microsatellites from plants. We highlight the attrition of loci at each stage in the process, and the average effort required to obtain 10 working microsatellite primer pairs.

Molecular Systematics and Plant Evolution

1. Using Organelle Markers to Elucidate the History, Ecology and Evolution of Plant Populations R.A. Ennos, W.T. Sinclair, X.-S. Hu and A. Langdon 2. Isolation Within Species and the History of Glacial Refugia C. Ferris, R.A King and G.M. Hewitt 3. The Use of Uniparentally Inherited Simple Sequence Repeat Markers in Plant Population Studies and Systematics J. Provan, N. Soranzo, N.J. McNicol, M. Morgante and W. Powell 4. The Use of RAPD Data in the Analysis of Population Genetic Structure: Case-studies of Alkanna (Boraginaceae) and Plantago (Plantaginaceae) K. Wolff and M. Morgan-Richards 5. Metapopulation Dynamics and Maring-system Evolution in Plants S.C.H. Barrett, J.R. Pannell 6. Identifying Multiple Origins in Polyploid Homosporous Pteridophytes J.C. Vogel, J.A. Barrett, F.J. Rumsey and M. Gibby 7. Population Genetic Structure in Agamospermous Plants R.J. Gornall 8. Monophyly, Populations and Species J.I. Davis 9. Reticulate Evolution in the Mediterranean Species Complex of Senecio Sect. Senecio: Uniting Phylogenetic and Population-level Approaches H.P. Comes and R.J. Abbott 10. The Value of Genomic In Situ Hybridization (GISH) in Plant Taxonomic and Evolutionary Studies C.A. Stace and J.P. Bailey 11. RAPDs in Systematics - a Useful Methodology? S.A. Harris 12. Nuclear Protein-coding Genes in Phylogeny Reconstruction and Homology Assessment: Some Examples From Leguminosae J.J. Doyle and J.L. Doyle 13. Spectral Analysis - a Brief Introduction M.A. Charleston and R.D.M. Page 14. Ribosomal DNA Sequences and Angiosperm Systematics M.A. Hershkovitz, E.A. Zimmer and W.J. Hahn 15. Proteins Encoded in Sequenced Chloroplast Genomes: an Overview of Gene Content, Phylogenetic Information and Endosymbiotic Transfer to the Nucleus B. Stoebe, S. Hansmann, V. Goremykin, K.V. Kowallik and W. Martin 16. Phylogenetics and Diversification in Pelargonium F.T. Bakker, A. Culham and M. Gibby 17. Integrating Molecular Phylogenies and Developmental Genetics: a Gesneriaceae Case Study M. Moller, M. Clokie, P. Cubas and Q.C.B. Cronk 18. Inferior Ovaries and Angiosperm Diversification M.H.G. Gustafasson and V.A. Albert 19. Intergrating Molecular and Morphological Evidence of Evolutionary Radiations R.M. Bateman Albert, The New York Botanical Garden, UK, J.P. Bailey, University of Leicester, UK, F.T. Bakker, University of Reading, UK, J.A. Barrett, University of Cambridge, UK, S.C.H. Barrett, University of Toronto, UK, R.M. Bateman, Royal Botanic Garden, Edinburgh, UK, M.A. Charleston, University of Oxford, UK, M. Clokie, University of Leicester, UK, H.P. Comes, Institut fur Spezielle Botanik und Botanisher Garten, Germany, Q.C.B. Cronk, University of Edinburgh, UK, P. Cubas, Institut Nacional de Investigaciones Agrarias, Spain, A. Culham, University of Reading, UK, J.I. Davis, Cornell University, USA, J.J. Doyle, Cornell University, USA, J.L. Doyle, Cornell University, USA, R.A. Ennos, University of Edinburgh, UK, C. Ferris, University of Leicester, UK, M. Gibby, The Natural History Museum, UK, V. Goremykin, Hans-Knoll-Institut fur Naturstoff-Forschung, Germany, R.J. Gornall, University of Leicester, UK, M.H.G. Gustafsson, The New York Botanical Garden, USA, W.J. Hahn, Columbia University, USA, S. Hansmann, Technische Universitat Braunschwieg, Germany, S.A. Harris, University of Oxford, UK, M.A. Hershovitz, Laboratory of Molecular Systematics, UK, G.M. Hewitt, University of East Anglia, UK, X-S. Hu, University of Edinburgh, UK, R.A. King, University of Leicester, UK, K.V. Kowallik, Heinrich-Heine-Universitat Dusseldorf, Germany, A. Langdon, University of Edinburgh, UK, W. Martin, Technische Universitat Braunschwieg, Germany, J.W. McNicol, Scottish Crop Research Institute, UK, M. Moller, Royal Botanic Gardens Edinburgh, UK, M. Morgan-Richards, Otago University, New Zealand, M. Morgante, Universita di Udine, Italy, R.D.M. Page, University of Glasgow, UK, J.R. Pannell, University of Oxford, UK, W. Powell, DuPont Agricultural Biotech, USA, J. Provan, Scottish Crop Research Institute, UK, F.J. Rumsey, The Natural History Museum, UK, W.T. Sincalir, The Scottish Agricultural College, UK, N. Soranzo, Scottish Crop Research Institute, UK, C.A. Stace, University of Leicester, UK, B. Stoebe, Heinrich-Heine-Universitat Dusseldorf, Germany, J.C. Vogel, The Natural History Museum, UK, N.J. Wilson, Scottish Crop Research Institute, UK, K. Wolff, The University of Newcastle upon Tyne, UK, E.A. Zimmer, Laboratory of Molecular Systematics, USA.

Selecting barcoding loci for plants: evaluation of seven candidate loci with species-level sampling in three divergent groups of land plants

There has been considerable debate, but little consensus regarding locus choice for DNA barcoding land plants. This is partly attributable to a shortage of comparable data from all proposed candidate loci on a common set of samples. In this study, we evaluated the seven main candidate plastid regions (rpoC1, rpoB, rbcL, matK, trnH‐psbA, atpF‐atpH, psbK‐psbI) in three divergent groups of land plants [Inga (angiosperm); Araucaria (gymnosperm); Asterella s.l. (liverwort)]. Across these groups, no single locus showed high levels of universality and resolvability. Interspecific sharing of sequences from individual loci was common. However, when multiple loci were combined, fewer barcodes were shared among species. Evaluation of the performance of previously published suggestions of particular multilocus barcode combinations showed broadly equivalent performance. Minor improvements on these were obtained by various new three‐locus combinations involving rpoC1, rbcL, matK and trnH‐psbA, but no single combination clearly outperformed all others. In terms of absolute discriminatory power, promising results occurred in liverworts (e.g. c. 90% species discrimination based on rbcL alone). However, Inga (rapid radiation) and Araucaria (slow rates of substitution) represent challenging groups for DNA barcoding, and their corresponding levels of species discrimination reflect this (upper estimate of species discrimination = 69% in Inga and only 32% in Araucaria; mean = 60% averaging all three groups).

Comparative analysis of population genetic structure in Athyrium distentifolium (Pteridophyta) using AFLPs and SSRs from anonymous and transcribed gene regions

To examine the performance and information content of different marker systems, comparative assessment of population genetic diversity was undertaken in nine populations of Athyrium distentifolium using nine genomic and 10 expressed sequence tag (EST) microsatellite (SSR) loci, and 265 amplified fragment length polymorphism (AFLP) loci from two primer combinations. In range‐wide comparisons (European vs. North American populations), the EST‐SSR loci showed more reliable amplification and produced more easily scorable bands than genomic simple sequence repeats (SSRs). Genomic SSRs showed significantly higher levels of allelic diversity than EST‐SSRs, but there was a significant correlation in the rank order of population diversities revealed by both marker types. When AFLPs, genomic SSRs, and EST‐SSRs are considered, comparisons of different population diversity metrics/markers revealed a mixture of significant and nonsignificant rank–order correlations. However, no hard incongruence was detected (in no pairwise comparison of populations did different marker systems or metrics detect opposingly significant different amounts of variation). Comparable population pairwise estimates of FST were obtained for all marker types, but whilst absolute values for genomic and EST‐SSRs were very similar (FST = 0.355 and 0.342, respectively), differentiation was consistently higher for AFLPs in pairwise and global comparisons (global AFLP FST = 0.496). The two AFLP primer combinations outperformed 18 SSR loci in assignment tests and discriminatory power in phenetic cluster analyses. The results from marker comparisons on A. distentifolium are discussed in the context of the few other studies on natural plant populations comparing microsatellite and AFLP variability.

Refining the DNA barcode for land plants

The goal of DNA barcoding is conceptually simple: Find one or a few regions of DNA that will distinguish among the majority of the worlds species, and sequence these from diverse sample sets to produce a large-scale reference library of life on earth (1). This approach can then be used as a tool for species identification and to help in the discovery of new species (2). Since the first DNA barcoding study in 2003 (1), the “animal barcode,” a portion of the mitochondrial gene Cytochrome Oxidase 1, has proved remarkably effective at discriminating among species in diverse groups such as birds, fishes, and insects. In contrast, finding a robust and effective barcode for plants has been more difficult. In 2009, a large consortium of researchers, the “Consortium for the Barcode of Life (CBOL) Plant Working Group,” proposed portions of two coding regions from the plastid (chloroplast) genome—rbcL and matK—as a “core barcode” for plants, to be supplemented with additional regions as required (3). This recommendation was accepted by the international Consortium for the Barcode of Life, but with the important qualifier that further sequencing of additional markers should be undertaken during a “trial period” (4). This trial period was driven by concerns that routine use of a third (or even a fourth) marker may be necessary to obtain adequate discriminatory power and to guard against sequencing failure for one of the markers (matK can be difficult to amplify and sequence). In PNAS, the China Plant Barcode of Life (BOL) Group provides an impressive dataset tackling this question (5) and assesses the potential benefits of supplementing the core barcode for land plants.

Population genetic structure in European populations of Spiranthes romanzoffiana set in the context of other genetic studies on orchids

Spiranthes romanzoffiana Cham. is restricted in Europe to the British Isles, where it is recognised as a conservation priority species due to frequent extirpation of populations along with no evidence of seed set; vegetative reproduction has been invoked as the sole means of perpetuation and dispersal. To investigate the reproductive ecology of this species, 17 populations have been sampled for chloroplast microsatellites and amplified fragment length polymorphisms (AFLPs). These markers revealed a previously unsuspected genetic–geographic split in the species, which correlates with differences in patterns of within-population variation. Northern populations were fixed for one chloroplast haplotype but showed high levels of AFLP genotypic diversity consistent with sexual reproduction (proportion of genotypes distinguishable, PD=0.98). More southerly populations showed fixed differences from the northern populations in their chloroplast haplotype and for 10 AFLP markers. They harboured only 12 unique multilocus genotypes among 113 individuals from six populations (PD=0.11). These genotypes differed mostly by single bands, and none by more than 4/138 loci, with identical multilocus genotypes occurring in widely separated populations. This uniformity in southern populations is consistent with agamospermous or autogamous reproduction, and/or an extreme population bottleneck. Finally, the observed patterns of population differentiation in S. romanzoffiana are compared with other studies of orchids, revealing a wide range of values that belie recent contrasting published generalisations that claim that orchids show either higher, or lower, levels of population differentiation than other plant families.

Partitioning and diversity of nuclear and organelle markers in native and introduced populations of Epipactis helleborine (Orchidaceae)

Variability of allozymes (1170 individuals, 47 populations) and chloroplast DNA (692 individuals, 29 populations) was examined in native European and introduced North American populations of Epipactis helleborine (Orchidaceae). At the species level, the percentage of allozyme loci that were polymorphic (P(99)) was 67%, with a mean of 2.11 alleles (A) per locus, and an expected heterozygosity (H(exp)) of 0.294. At the population level, mean P(99) = 56%, mean A = 1.81, and mean H(exp) = 0.231. Although field observations suggest that self-pollination occurs frequently, populations had a genetic structure consistent with Hardy-Weinberg expectations and random mating (mean F(IS) = 0.002). There was significant deviation from panmixia associated with population differentiation (mean F(ST) = 0.206). The distribution of two chloroplast haplotypes showed that 15 of the 29 populations were polymorphic. Using both nuclear and organelle F(ST) estimates, a pollen to seed flow ratio of 1.43 : 1 was calculated. This is very low compared with published estimates for other plant groups, consistent with the high dispersability of orchid seeds. Finally, there was no evidence for a genetic bottleneck associated with the introduction of E. helleborine to North America.