Steven G. Newmaster

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.

Multiple Multilocus DNA Barcodes from the Plastid Genome Discriminate Plant Species Equally Well

A universal barcode system for land plants would be a valuable resource, with potential utility in fields as diverse as ecology, floristics, law enforcement and industry. However, the application of plant barcoding has been constrained by a lack of consensus regarding the most variable and technically practical DNA region(s). We compared eight candidate plant barcoding regions from the plastome and one from the mitochondrial genome for how well they discriminated the monophyly of 92 species in 32 diverse genera of land plants (N = 251 samples). The plastid markers comprise portions of five coding (rpoB, rpoC1, rbcL, matK and 23S rDNA) and three non-coding (trnH-psbA, atpF–atpH, and psbK–psbI) loci. Our survey included several taxonomically complex groups, and in all cases we examined multiple populations and species. The regions differed in their ability to discriminate species, and in ease of retrieval, in terms of amplification and sequencing success. Single locus resolution ranged from 7% (23S rDNA) to 59% (trnH-psbA) of species with well-supported monophyly. Sequence recovery rates were related primarily to amplification success (85–100% for plastid loci), with matK requiring the greatest effort to achieve reasonable recovery (88% using 10 primer pairs). Several loci (matK, psbK–psbI, trnH-psbA) were problematic for generating fully bidirectional sequences. Setting aside technical issues related to amplification and sequencing, combining the more variable plastid markers provided clear benefits for resolving species, although with diminishing returns, as all combinations assessed using four to seven regions had only marginally different success rates (69–71%; values that were approached by several two- and three-region combinations). This performance plateau may indicate fundamental upper limits on the precision of species discrimination that is possible with DNA barcoding systems that include moderate numbers of plastid markers. Resolution to the contentious debate on plant barcoding should therefore involve increased attention to practical issues related to the ease of sequence recovery, global alignability, and marker redundancy in multilocus plant DNA barcoding systems.

Testing candidate plant barcode regions in the Myristicaceae

The concept and practice of DNA barcoding have been designed as a system to facilitate species identification and recognition. The primary challenge for barcoding plants has been to identify a suitable region on which to focus the effort. The slow relative nucleotide substitution rates of plant mitochondria and the technical issues with the use of nuclear regions have focused attention on several proposed regions in the plastid genome. One of the challenges for barcoding is to discriminate closely related or recently evolved species. The Myristicaceae, or nutmeg family, is an older group within the angiosperms that contains some recently evolved species providing a challenging test for barcoding plants. The goal of this study is to determine the relative utility of six coding (Universal Plastid Amplicon — UPA, rpoB, rpoc1, accD, rbcL, matK) and one noncoding (trnH‐psbA) chloroplast loci for barcoding in the genus Compsoneura using both single region and multiregion approaches. Five of the regions we tested were predominantly invariant across species (UPA, rpoB, rpoC1, accD, rbcL). Two of the regions (matK and trnH‐psbA) had significant variation and show promise for barcoding in nutmegs. We demonstrate that a two‐gene approach utilizing a moderately variable region (matK) and a more variable region (trnH‐psbA) provides resolution among all the Compsonuera species we sampled including the recently evolved C. sprucei and C. mexicana. Our classification analyses based on nonmetric multidimensional scaling ordination, suggest that the use of two regions results in a decreased range of intraspecific variation relative to the distribution of interspecific divergence with 95% of the samples correctly identified in a sequence identification analysis.

Are plant species inherently harder to discriminate than animal species using DNA barcoding markers

The ability to discriminate between species using barcoding loci has proved more difficult in plants than animals, raising the possibility that plant species boundaries are less well defined. Here, we review a selection of published barcoding data sets to compare species discrimination in plants vs. animals. Although the use of different genetic markers, analytical methods and depths of taxon sampling may complicate comparisons, our results using common metrics demonstrate that the number of species supported as monophyletic using barcoding markers is higher in animals (> 90%) than plants (~70%), even after controlling for the amount of parsimony‐informative information per species. This suggests that more than a simple lack of variability limits species discrimination in plants. Both animal and plant species pairs have variable size gaps between intra‐ and interspecific genetic distances, but animal species tend to have larger gaps than plants, even in relatively densely sampled genera. An analysis of 12 plant genera suggests that hybridization contributes significantly to variation in genetic discontinuity in plants. Barcoding success may be improved in some plant groups by careful choice of markers and appropriate sampling; however, overall fine‐scale species discrimination in plants relative to animals may be inherently more difficult because of greater levels of gene‐tree paraphyly.

Testing plant barcoding in a sister species complex of pantropical Acacia (Mimosoideae, Fabaceae)

Acacia species are quite difficult to differentiate using morphological characters. Routine identification of Acacia samples is important in order to distinguish invasive species from rare species or those of economic importance, particularly in the forest industry. The genus Acacia is quite abundant and diverse comprising approximately 1355 species, which is currently divided into three subgenera: subg. Acacia (c. 161 species), subg. Aculiferum (c. 235 species), and subg. Phyllodineae (c. 960 species). It would be prudent to utilize DNA barcoding in the accurate and efficient identification of acacias. The objective of this research is to test barcoding in discriminating multiple populations among a sister‐species complex in pantropical Acacia subg. Acacia, across three continents. Based on previous research, we chose three cpDNA regions (rbcL, trnH‐psbA and matK). Our results show that all three regions (rbcL, matK and trnH‐psbA) can distinguish and support the newly proposed genera of Vachellia Wight & Arn. from Acacia Mill., discriminate sister species within either genera and differentiate biogeographical patterns among populations from India, Africa and Australia. A morphometric analysis confirmed the cryptic nature of these sister species and the limitations of a classification based on phenetic data. These results support the claim that DNA barcoding is a powerful tool for taxonomy and biogeography with utility for identifying cryptic species, biogeograhic patterns and resolving classifications at the rank of genera and species.

Improving sequencing quality from PCR products containing long mononucleotide repeats

Stutter products are a common artifact in the PCR amplification of frequently used genetic markers that contain mononucleotide simple sequence repeats. Despite the importance of accurate determination of nucleotide sequence and allele size, there has been little progress toward decreasing the formation of stutter products during PCR. In this study, we tested the effects of lowered extension temperatures, inclusion of co-solutes in PCR, PCR cycle number, and the use of different polymerases on sequence quality for a set of sequences containing mononucleotide A/T repeats of 10-17 bp. Our analyses showed that sequence quality of mononucleotide repeats <or=15 bp is greatly improved with the use of proofreading DNA polymerases fused to nonspecific dsDNA binding domains. Our findings also suggest that the number of nucleotides with which the DNA polymerase interacts may be the most important factor in the reduction of slipped-strand mispairings in vitro.

Spatial patterns of plant diversity below-ground as revealed by DNA barcoding.

Our understanding of the spatial organization of root diversity in plant communities and of the mechanisms of community assembly has been limited by our ability to identify plants based on root tissue, especially in diverse communities. Here, we test the effectiveness of the plastid gene rbcL, a core plant DNA barcoding marker, for investigating spatial patterns of root diversity, and relate observed patterns to above‐ground community structure. We collected 3800 root fragments from four randomly positioned, 1‐m‐deep soil profiles (two vertical transects per plot), located in an old‐field community in southern Ontario, Canada, and extracted and sequenced DNA from 1531 subsampled fragments. We identified species by comparing sequences with a DNA barcode reference library developed previously for the local flora. Nearly 85% of sampled root fragments were successfully sequenced and identified as belonging to 29 plant species or species groups. Root abundance and species richness varied in horizontal space and were negatively correlated with soil depth. The relative abundance of taxa below‐ground was correlated with their frequency above‐ground (r = 0.73, P = 0.0001), but several species detected in root tissue were not observed in above‐ground quadrats. Multivariate analyses indicated that diversity was highly structured below‐ground, and associated with depth, root morphology, soil chemistry and soil texture, whereas little structure was evident above‐ground. Furthermore, analyses of species co‐occurrence indicates strong species segregation overall but random co‐occurrence among confamilials. Our results provide insights into the role of environmental filtering and competitive interactions in the organization of plant diversity below‐ground, and also demonstrate the utility of barcoding for the identification of plant roots.

DNA barcoding discriminates a new cryptic grass species revealed in an ethnobotany study by the hill tribes of the Western Ghats in southern India

Our research brought together traditional aboriginal knowledge (TK) and scientific knowledge (SK) to explore the relationship between scientific and aboriginal systems of botanical classification and the corresponding valorization(s) of biological diversity in the Western Ghats of southern India. We worked with two aboriginal cultures namely ‘Irulas’ and ‘Malasars’ of the Nilgiri Biosphere Reserve with an objective of evaluating the ability of different knowledge systems (SK and TK) to distinguish grass species belonging to the genus Tripogon, and assess the ability of DNA barcoding to discriminate a new cryptic species ‘Tripogon cope’ as deciphered by the hill tribes. We discovered that the aboriginal informants identified a common ethnotaxa ‘Sunai pul’, which is a cryptic species of grass not recognized by the SK classification.sunai pul’ is very important to both aboriginal cultures with ritualistic and economic utility. Morphometric analysis confirms the cryptic nature of this new species, which was validated using DNA barcoding. DNA barcode regions matK and trnH‐psbA showed distinct sequence variations among the closely related ethnotaxa. Given the cryptic nature of ethnotaxa, we propose that a DNA barcode may be a reliable tool to identify ethnotaxa. We have initiated further studies in other cultures to develop theoretically sophisticated insights concerning the encounter between ‘local’ and ‘scientific’ approaches to the use of biodiversity knowledge. Furthermore, the research will add to a unifying global effort to speed up the documentation and understanding of the planets natural diversity, while simultaneously respecting the cultural heterogeneity as a vital component of biological diversity.

Understanding the spectacular failure of DNA barcoding in willows (Salix): does this result from a trans-specific selective sweep?

Willows (Salix: Salicaceae) form a major ecological component of Holarctic floras and consequently are an obvious target for a DNA‐based identification system. We surveyed two to seven plastid genome regions (~3.8 kb; ~3% of the genome) from 71 Salix species across all five subgenera, to assess their performance as DNA barcode markers. Although Salix has a relatively high level of interspecific hybridization, this may not sufficiently explain the near complete failure of barcoding that we observed: only one species had a unique barcode. We recovered 39 unique haplotypes, from more than 500 specimens, that could be partitioned into six major haplotype groups. A unique variant of group I (haplotype 1*) was shared by 53 species in three of five Salix subgenera. This unusual pattern of haplotype sharing across infrageneric taxa is suggestive of either a massive nonrandom coalescence failure (incomplete lineage sorting), or of repeated plastid capture events, possibly including a historical selective sweep of haplotype 1* across taxonomic sections. The former is unlikely as molecular dating indicates that haplotype 1* originated recently and is nested in the oldest major haplotype group in the genus. Further, we detected significant non‐neutrality in the frequency spectrum of mutations in group I, but not outside group I, and demonstrated a striking absence of geographical (isolation by distance) effects in the haplotype distributions of this group. The most likely explanation for the patterns we observed involves recent repeated plastid capture events, aided by widespread hybridization and long‐range seed dispersal, but primarily propelled by one or more trans‐species selective sweeps.

Valorizing the 'Irulas' traditional knowledge of medicinal plants in the Kodiakkarai Reserve Forest, India

A mounting body of critical research is raising the credibility of Traditional Knowledge (TK) in scientific studies. These studies have gained credibility because their claims are supported by methods that are repeatable and provide data for quantitative analyses that can be used to assess confidence in the results. The theoretical importance of our study is to test consensus (reliability/replicable) of TK within one ancient culture; the Irulas of the Kodiakkarai Reserve Forest (KRF), India. We calculated relative frequency (RF) and consensus factor (Fic) of TK from 120 Irulas informants knowledgeable of medicinal plants. Our research indicates a high consensus of the Irulas TK concerning medicinal plants. The Irulas revealed a diversity of plants that have medicinal and nutritional utility in their culture and specific ethnotaxa used to treat a variety of illnesses and promote general good health in their communities. Throughout history aboriginal people have been the custodians of bio-diversity and have sustained healthy life-styles in an environmentally sustainable manner. However this knowledge has not been transferred to modern society. We suggest this may be due to the asymmetry between scientific and TK, which demands a new approach that considers the assemblage of TK and scientific knowledge. A greater understanding of TK is beginning to emerge based on our research with both the Irulas and Malasars; they believe that a healthy lifestyle is founded on a healthy environment. These aboriginal groups chose to share this knowledge with society-at-large in order to promote a global lifestyle of health and environmental sustainability.