DNA barcoding insect-host plant associations

Abstract

Short-sequence fragments ('DNA barcodes') used widely for plant identification and inventorying remain to be applied to complex biological problems. Host-herbivore interactions are fundamental to coevolutionary relationships of a large proportion of species on the Earth, but their study is frequently hampered by limited or unreliable host records. Here we demonstrate that DNA barcodes can greatly improve this situation as they (i) provide a secure identification of host plant species and (ii) establish the authenticity of the trophic association. Host plants of leaf beetles (subfamily Chrysomelinae) from Australia were identified using the chloroplast trnL(UAA) intron as barcodes amplified from beetle DNA extracts. Sequence similarity and phylogenetic analyses provided precise identifications of each host species at tribal, generic and specific levels, depending on the available database coverage in various plant lineages. The 76 species of Chrysomelinae included-more than 10 per cent of the known Australian fauna-feed on 13 plant families, with preference for Australian radiations of Myrtaceae (eucalypts) and Fabaceae (acacias). Phylogenetic analysis of beetles shows general conservation of host association but with rare host shifts between distant plant lineages, including a few cases where barcodes supported two phylogenetically distant host plants. The study demonstrates that plant barcoding is already feasible with the current publicly available data. By sequencing plant barcodes directly from DNA extractions made from herbivorous beetles, strong physical evidence for the host association is provided. Thus, molecular identification using short DNA fragments brings together the detection of species and the analysis of their interactions.

Figure 1

Identification of host plants against GenBank entries. trnL intron sequences obtained from beetle tissue were subjected to phylogenetic analysis together with their respective GenBank top hits. In each case, sequence divergence was estimated by pairwise comparisons with all sequences in this clade; minimum, mean and standard deviation measures (p-distance) are given here. Details on each clade are in fig. S1 in the electronic supplementary material. Similarity levels in each individual case are compared with the mean trnL divergences and standard deviations for all sequences available on GenBank for the identified family, shown by coloured bands to illustrate divergences at the level of the entire family and the average divergence for tribes and genera within each family.

Figure 2

Bayesian phylogenetic tree of trnL intron sequences obtained from beetle tissue. Terminals are given as the taxa of Chrysomelinae yielding each sequence; those in bold represent individuals used for reconstructing the genus-level phylogeny of the beetles (see figure 3). The topology is broadly congruent with plant systematics and major plant lineages are labelled. Only PP≥0.70 are shown.

Figure 3

Phylogeny of hosts and herbivores. The tree shows the relationships of beetle genera from combined cox1 and EF1a sequences. Each genus is represented by one species, with further species added where a genus was feeding on more than one plant family (terminals in boldface in figure 2). Inferred host associations are plotted on the tree for (a) the Gonioctenini and (b) the Phyllocharitini chrysomelines showing a high degree of conservatism with few host shifts (dashed lines) between major plant lineages. (d) Simplified eudicot phylogeny for relevant plant orders, consistent with the tree obtained here from trnL intron sequences; redrawn from Soltis et al. 2005). Beetles: (c) Paropsis maculata Marsham on Myrtaceae; (e) Johannica gemellata (Westwood) on Bignoniaceae (photographs: J. A. Jurado).