Evaluation of bias on the assessment of diet breadth of herbivorous insects using molecular methods.

The interactions between herbivores and their host plants play a key role in ecological processes. Understanding the width and nature of these interactions is fundamental to ecology and conservation. Recent research on DNA-based inference of trophic associations suggests that the host range of phytophagous insects in the tropics may be wider than previously thought based on traditional observation. However, the reliability of molecular inference of ecological associations, still strongly dependent on PCR and thus exposed to the risk of contamination with environmental DNA, is under debate. Here, we explored alternative procedures to reduce the chance of amplification of external, nondiet DNA, including surface decontamination and analysis of mid/hind guts, comparing the results with those obtained using the standard protocol. We studied 261 specimens in eight species of Neotropical Chrysomelidae that yielded 316 psbA-trnH intergenic spacer sequences (cpDNA marker of putative diets) from unique and multiple-band PCR results. The taxonomic identity of these sequences was inferred using the automated pipeline BAGpipe, yielding results consistent with 31 plant families. Regardless of the protocol used, a wide taxonomic spectrum of food was inferred for all chrysomelid species. Canonical Correspondence Analysis using these data revealed significant differences attributed mainly to species (expectedly, since they represent different ecologies), but also to treatment (untreated vs. cleaned/gut samples) and PCR results (single vs. multiple bands). Molecular identification of diets is not straightforward and, regardless of the species' niche breadth, combining approaches that reduce external contamination and studying multiple individuals per species may help increasing confidence in results.

High-throughput biodiversity analysis: Rapid assessment of species richness and ecological interactions of Chrysomelidae (Coleoptera) in the tropics.

Biodiversity assessment has been the focus of intense debate and conceptual and methodological advances in recent years. The cultural, academic and aesthetic impulses to recognise and catalogue the diversity in our surroundings, in this case of living objects, is furthermore propelled by the urgency of understanding that we may be responsible for a dramatic reduction of biodiversity, comparable in magnitude to geological mass extinctions. One of the most important advances in this attempt to characterise biodiversity has been incorporating DNA-based characters and molecular taxonomy tools to achieve faster and more efficient species delimitation and identification, even in hyperdiverse tropical biomes. In this assay we advocate for a broad understanding of Biodiversity as the inventory of species in a given environment, but also the diversity of their interactions, with both aspects being attainable using molecular markers and phylogenetic approaches. We exemplify the suitability and utility of this framework for large-scale biodiversity assessment with the results of our ongoing projects trying to characterise the communities of leaf beetles and their host plants in several tropical setups. Moreover, we propose that approaches similar to ours, establishing the inventories of two ecologically inter-related and species-rich groups of organisms, such as insect herbivores and their angiosperm host-plants, can serve as the foundational stone to anchor a comprehensive assessment of diversity, also in tropical environments, by subsequent addition of trophic levels.

Automated DNA-based plant identification for large-scale biodiversity assessment.

Rapid degradation of tropical forests urges to improve our efficiency in large-scale biodiversity assessment. DNA barcoding can assist greatly in this task, but commonly used phenetic approaches for DNA-based identifications rely on the existence of comprehensive reference databases, which are infeasible for hyperdiverse tropical ecosystems. Alternatively, phylogenetic methods are more robust to sparse taxon sampling but time-consuming, while multiple alignment of species-diagnostic, typically length-variable, markers can be problematic across divergent taxa. We advocate the combination of phylogenetic and phenetic methods for taxonomic assignment of DNA-barcode sequences against incomplete reference databases such as GenBank, and we developed a pipeline to implement this approach on large-scale plant diversity projects. The pipeline workflow includes several steps: database construction and curation, query sequence clustering, sequence retrieval, distance calculation, multiple alignment and phylogenetic inference. We describe the strategies used to establish these steps and the optimization of parameters to fit the selected psbA-trnH marker. We tested the pipeline using infertile plant samples and herbivore diet sequences from the highly threatened Nicaraguan seasonally dry forest and exploiting a valuable purpose-built resource: a partial local reference database of plant psbA-trnH. The selected methodology proved efficient and reliable for high-throughput taxonomic assignment, and our results corroborate the advantage of applying 'strict' tree-based criteria to avoid false positives. The pipeline tools are distributed as the scripts suite 'BAGpipe' (pipeline for Biodiversity Assessment using GenBank data), which can be readily adjusted to the purposes of other projects and applied to sequence-based identification for any marker or taxon.