New universal ITS2 primers for high-resolution herbivory analyses using DNA metabarcoding in both tropical and temperate zones

DNA metabarcoding is a rapidly growing technique for obtaining detailed dietary information. Current metabarcoding methods for herbivory, using a single locus, can lack taxonomic resolution for some applications. We present novel primers for the second internal transcribed spacer of nuclear ribosomal DNA (ITS2) designed for dietary studies in Mauritius and the UK, which have the potential to give unrivalled taxonomic coverage and resolution from a short-amplicon barcode. In silico testing used three databases of plant ITS2 sequences from UK and Mauritian floras (native and introduced) totalling 6561 sequences from 1790 species across 174 families. Our primers were well-matched in silico to 88% of species, providing taxonomic resolution of 86.1%, 99.4% and 99.9% at the species, genus and family levels, respectively. In vitro, the primers amplified 99% of Mauritian (n = 169) and 100% of UK (n = 33) species, and co-amplified multiple plant species from degraded faecal DNA from reptiles and birds in two case studies. For the ITS2 region, we advocate taxonomic assignment based on best sequence match instead of a clustering approach. With short amplicons of 187–387 bp, these primers are suitable for metabarcoding plant DNA from faecal samples, across a broad geographic range, whilst delivering unparalleled taxonomic resolution

Species separation within, and preliminary phylogeny for, the leafhopper genus Anoscopus with particular reference to the putative British endemic Anoscopus duffieldi (Hemiptera: Cicadellidae)

The subfamily Aphrodinae (Hemiptera: Cicadellidae) contains ~33 species in Europe within four genera. Species in two genera in particular, Aphrodes and Anoscopus, have proved to be difficult to distinguish morphologically. Our aim was to determine the status of the putative species Anoscopus duffieldi, found only on the RSPB Nature Reserve at Dungeness, Kent, a possible rare UK endemic. DNA from samples of all seven UK Anoscopus species (plus Anoscopusalpinus from the Czech Republic) were sequenced using parts of the mitochondrial cytochrome oxidase I and 16S rRNA genes. Bayesian inference phylogenies were created. Specimens of each species clustered into monophyletic groups, except for Anoscopusalbifrons, A. duffieldi and Anoscopuslimicola. Two A. albifrons specimens grouped with A. duffieldi repeatedly with strong support, and the remaining A. albifrons clustered within A. limicola. Genetic distances suggest that A. albifrons and A. limicola are a single interbreeding population (0% divergence), while A. albifrons and A. duffieldi diverged by only 0.28%. Shared haplotypes between A. albifrons, A. limicola and A. duffieldi strongly suggest interbreeding, although misidentification may also explain these topologies. However, all A. duffieldi clustered together in the trees. A conservative approach might be to treat A. duffieldi, until other evidence is forthcoming, as a possible endemic subspecies

Impacts of herbivory by ecological replacements on an island ecosystem

The use of ecological replacements (analogue species to replace extinct taxa) to restore ecosystem functioning is a promising conservation tool. However, this approach is controversial, in part due to a paucity of data on interactions between analogue species and established taxa in the ecosystem. We conducted ecological surveys, comprehensively DNA barcoded an ecosystem's flora and inferred the diet of the introduced Aldabra giant tortoise, acting as an ecological replacement, to understand how it might have modified island plant communities on a Mauritian islet. Through further dietary analyses, we investigated consequential effects on the threatened endemic Telfair's skink. Dietary overlap between tortoises and skinks was greater than expected by chance. However, there was a negative correlation between tortoise and skink preferences in herbivory and minimal overlap in the plants most frequently consumed by the reptiles. Changes in the plant community associated with 7 years of tortoise grazing were characterised by a decrease in the percentage cover of native herbs and creepers, and an increase in the cover of an invasive herb when compared to areas without tortoises. However, tortoise dietary preferences themselves did not directly drive changes in the plant community. Tortoises successfully dispersed the seeds of an endemic palm, which in time may increase the extent of unique palm-rich habitat. We found no evidence that tortoises have increased the extent of plant species hypothesised to be part of a lost Mauritian tortoise grazed community. Synthesis and applications. Due to a negative correlation in tortoise and skink dietary preferences and minimal overlap in the most frequently consumed taxa, the presence of tortoises is unlikely to have detrimental impacts on Telfair's skinks. Tortoise presence is likely to be beneficial to skinks in the long term by increasing the extent of palm-rich habitat. Although tortoises are likely to play a role in controlling invasive plants, they are not a panacea for this challenge. After 7 years, tortoises have not resurrected a lost tortoise grazed community that we hypothesise might have existed in limited areas on the islet, indicating that further interventions may be required to restore this plant community