Diana M. Percy

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.

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.

Plant–Insect Interactions: Double-Dating Associated Insect and Plant Lineages Reveals Asynchronous Radiations

An increasing number of plant-insect studies using phylogenetic analysis suggest that cospeciation events are rare in plant-insect systems. Instead, nonrandom patterns of phylogenetic congruence are produced by phylogenetically conserved host switching (to related plants) or tracking of particular resources or traits (e.g., chemical). The dominance of host switching in many phytophagous insect groups may make the detection of genuine cospeciation events difficult. One important test of putative cospeciation events is to verify whether reciprocal speciation is temporally plausible. We explored techniques for double-dating of both plant and insect phylogenies. We use dated molecular phylogenies of a psyllid (Hemiptera)-Genisteae (Fabaceae) system, a predominantly monophagous insect-plant association widespread on the Atlantic Macaronesian islands. Phylogenetic reconciliation analysis suggests high levels of parallel cladogenesis between legumes and psyllids. However, dating using molecular clocks calibrated on known geological ages of the Macaronesian islands revealed that the legume and psyllid radiations were not contemporaneous but sequential. Whereas the main plant radiation occurred some 8 million years ago, the insect radiation occurred about 3 million years ago. We estimated that >60% of the psyllid speciation has resulted from host switching between related hosts. The only evidence for true cospeciation is in the much more recent and localized radiation of genistoid legumes in the Canary Islands, where the psyllid and legume radiations have been partially contemporaneous. The identification of specific cospeciation events over this time period, however, is hindered by the phylogenetic uncertainty in both legume and psyllid phylogenies due to the apparent rapidity of the species radiations.

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.

RADIATION, DIVERSITY, AND HOST-PLANT INTERACTIONS AMONG ISLAND AND CONTINENTAL LEGUME-FEEDING PSYLLIDS

Abstract Island archipelagos and insect‐plant associations have both independently provided many useful systems for evolutionary study. The arytainine psyllid (Sternorrhyncha: Hemiptera) radiation on broom (Fabaceae: Genisteae) in the Canary Island archipelago provides a discrete system for examining the speciation of highly host‐specific phytophagous insects in an island context. Phylogenetic reconstructions based on three datasets (adult and nymph morphological characters, and two mitochondrial DNA regions: part of the small subunit rRNA, and part of cytochrome oxidase I, cytochrome oxidase II and the intervening tRNA leucine) are generally consistent. The combined molecular tree provides a well‐supported estimate of psyllid relationships and shows that there have been several colonizations of the Macaronesian islands but that only one has resulted in a significant radiation. Psyllid diversification has apparently been constrained by the presence of suitable host groups within the genistoid legumes, and the diversity, distribution, and abundance of those groups. The phylogeny, by indicating pairs of sister species, allows putative mechanisms of speciation to be assessed. The most common conditions associated with psyllid speciation are geographical allopatry with a host switch to closely related hosts (six examples), or geographical allopatry on the same host (four examples). Where allopatric speciation involves a host switch, these have all been to related hosts. There is some evidence that switches between unrelated host plants may be more likely in sympatry. Only one sister pair (Arytainilla cytisi and A. telonicola) and the putative host races of Arytinnis modica are sympatric but on unrelated hosts, which may be a necessary condition for sympatric speciation in these insects. Where several psyllids share the same host, resources appear to be partitioned by ecological specialization and differing psyllid phenology.

Psyllid communication: acoustic diversity, mate recognition and phylogenetic signal

Acoustic signals play an important role in mate selection and speciation in diverse groups of insects. We report reciprocal acoustic mate signalling, often as highly synchronised duetting, for several species of psyllid (Hemiptera:Psylloidea). We reveal that considerable acoustic diversity is present in Australian psyllids belonging to the family Triozidae. The acoustic signals are species and gender specific. Our acoustic analysis and observations suggest that acoustic signals are important in both species recognition and mate selection in psyllids. We found a significant level of phylogenetic signal in the acoustic data when we compared divergence in genetic data (obtained from mitochondrial DNA sequences of the small subunit rRNA) with divergence in acoustic signals in two groups of Australian psyllids. Phylogenetic reconstruction based on DNA sequence data supports the monophyly of the Eucalyptus-feeding genus Schedotrioza Tuthill & Taylor, 1955, whereas a diverse but little known group on Casuarinaceae hosts appears to be paraphyletic. These two psyllid groups also differ in amounts of geographical and ecological sympatry. We found a significant positive correlation between acoustic distance and genetic distance using pairwise comparisons for all taxa, but the trends within the two groups differ due to a negative association between acoustic and genetic divergence among the sympatric taxa. Phylogenetic information in acoustic data may be greatest in recently speciating and allopatric groups because of increased acoustic divergence in sympatric taxa and greater acoustic convergence in more distantly related species. Additional keywords: acoustic communication, Allocasuarina, Casuarinaceae, Eucalyptus, Psylloidea, speciation, substrate vibration, Triozidae.

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.

Psyllid Host-Plants (Hemiptera: Psylloidea): Resolving a Semantic Problem

Abstract Evolutionary and biological patterns can be obscured by inadequate or ill-defined terminology. An example is the generally very specific relationship between the sap-feeding hemipteran group, psyllids, and their breeding plants, commonly called host-plants. The literature is clogged with references to so called ‘hosts’, which are often merely plants on which psyllids were found accidentally, and no immature development was detected. Recently the term host has also been applied by some authors to any plant on which immature or adults feed. Here we propose a terminology to clarify associated plant definitions, and we suggest restricting the use of the term host-plant to plants on which a psyllid species completes its immature to adult life cycle. For the other plant associations we suggest the terms overwintering or shelter plant (plants on which adult psyllids overwinter and on which they may feed), food plant (plants on which adult psyllids feed, but do not breed and do not spend an extended period of time) and casual plant (plants on which adult psyllids land but do not feed).

Invasion of French Polynesia by the Glassy-Winged Sharpshooter, Homalodisca coagulata (Hemiptera: Cicadellidae): A New Threat to the South Pacific

ABSTRACT The glassy-winged sharpshooter, Homalodisca coagulata (Say), is a major pest of agricultural, ornamental, and native plants. It is native to the southeastern United States and northeastern Mexico. Homalodisca coagulata was first recorded in Tahiti (French Polynesia) in 1999. It reproduced and spread rapidly in French Polynesia and is currently found in almost all islands in the Society Islands group (Tahiti, Moorea, Raiatea, Huahine, Bora Bora, Tahaa, Maupiti), in Nuku Hiva in the Marquesas, and in Tubuai and Rurutu in the Australs. Tahiti and Moorea are the most heavily infested islands, where H. coagulata populations have reached densities far exceeding those observed in California (this pest invaded California in the late 1980s) or in its native range. Major factors responsible for high population densities of H. coagulata in French Polynesia are permissive environmental conditions (mild climate and abundant year-round feeding and oviposition substrates), absence of host-specific natural enemies, intoxication of generalist predators that attack nymphal and adult stages, and limited competition for resources. Homalodisca coagulata causes several problems in French Polynesia: dripping excreta from high densities of feeding adults and nymphs affect outdoor recreation under trees and create a social nuisance, attraction of large numbers of flying adults to lights at night and collisions with people are severe annoyances, and large numbers of H. coagulata feeding on plants can cause impaired growth. The major concern for French Polynesia is the possibility of this pest acquiring and vectoring the lethal plant bacterium Xylella fastidiosa, which could have a disastrous impact on ornamental, agricultural, and native plants. Surveys are currently under way to detect X. fastidiosa in French Polynesia. Presence of large populations of H. coagulata in French Polynesia presents a major threat to agriculture and the biodiversity of South Pacific countries because this insect has clearly demonstrated a high invasion potential.